@phdthesis{Hampe2018, author = {Hampe, Irene Aurelia Ida}, title = {Analysis of the mechanism and the regulation of histatin 5 resistance in \(Candida\) \(albicans\)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-159634}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2018}, abstract = {Antimycotics such as fluconazole are frequently used to treat C. albicans infections of the oral mucosa. Prolonged treatment of the fungal infection with fluconazole pose a risk to resistance development. C. albicans can adapt to these stressful environmental changes by regulation of gene expression or by producing genetically altered variants that arise in the population. Adapted variants frequently carry activating mutations in zinc cluster transcription factors, which cause the upregulation of their target genes, including genes encoding efflux pumps that confer drug resistance. MDR1, regulated by the zinc cluster transcription factor Mrr1, as well as CDR1 and CDR2, regulated by the zinc cluster transcription factor Tac1, are well-known examples of genes encoding efflux pumps that extrude the antimycotic fluconazole from the fungal cell and thus contribute to the survival of the fungus. In this study, it was investigated if C. albicans can develop resistance to the antimicrobial peptide histatin 5, which serves as the first line of defence in the oral cavity of the human host. Recently, it was shown that C. albicans transports histatin 5 outside of the Candia cell via the efflux pump Flu1. As efflux pumps are often regulated by zinc cluster transcription factors, the Flu1 efflux pump could also be regulated by a zinc cluster transcription factor which could in a hyperactive form upregulate the expression of the efflux pump, resulting in increased export of histatin 5 and consequently in histatin 5 resistance. In order to find a zinc cluster transcription factor that upregulates FLU1 expression, a comprehensive library of C. albicans strains containing artificially activated forms of zinc cluster transcription factors was screened for suitable candidates. The screening was conducted on medium containing mycophenolic acid because mycophenolic acid is also a substrate of Flu1 and a strain expressing a hyperactive zinc cluster transcription factor that upregulates FLU1 expression should exhibit an easily recognisable mycophenolic acid-resistant phenotype. Further, FACS analysis, quantitative real-time RT-PCR analysis, broth microdilution assays as well as histatin 5 assays were conducted to analyse the mechanism and the regulation of histatin 5 resistance. Several zinc cluster transcription factors caused mycophenolic acid resistance and upregulated FLU1 expression. Of those, only hyperactive Mrr1 was able to confer increased histatin 5 resistance. Finding Mrr1 to confer histatin 5 resistance was highly interesting as fluconazole-resistant strains with naturally occurring Mrr1 gain of function mutations exist, which were isolated from HIV-infected patients with oral candidiasis. These Mrr1 gain of function mutations as well as artificially activated Mrr1 cause fluconazole resistance by upregulation of the efflux pump MDR1 and other target genes. In the course of the study, it was found that expression of different naturally occurring MRR1 gain-of-function mutations in the SC5314 wild type background caused increased FLU1 expression and increased histatin 5 resistance. The same was true for fluconazole-resistant clinical isolates with Mrr1 gain of function mutations, which also caused the overexpression of FLU1. Those cells were less efficiently killed by histatin 5 dependent on Mrr1. Surprisingly, FLU1 contributed only little to histatin 5 resistance, rather, overexpression of MDR1 mainly contributed to the Mrr1-mediated histatin 5 resistance, but also additional Mrr1-target genes were involved. These target genes are yet to be uncovered. Moreover, if a link between the yet unknown Mrr1-target genes contributing to fluconazole resistance and increased histatin 5 resistance can be drawn remains to be discovered upon finding of the responsible target genes. Collectively, this study contributes to the understanding of the impact of prolonged antifungal exposure on the interaction between host and fungus. Drug therapy can give rise to resistance evolution resulting in strains that have not only developed resistance to fluconazole but also to an innate host mechanism, which allows adaption to the host niche even in the absence of the drug.}, subject = {Histatin 5}, language = {en} } @article{HampeFriedmanEdgertonetal.2017, author = {Hampe, Irene A. I. and Friedman, Justin and Edgerton, Mira and Morschh{\"a}user, Joachim}, title = {An acquired mechanism of antifungal drug resistance simultaneously enables Candida albicans to escape from intrinsic host defenses}, series = {PLoS Pathogens}, volume = {13}, journal = {PLoS Pathogens}, number = {9}, doi = {10.1371/journal.ppat.1006655}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-158883}, pages = {e1006655}, year = {2017}, abstract = {The opportunistic fungal pathogen Candida albicans frequently produces genetically altered variants to adapt to environmental changes and new host niches in the course of its life-long association with the human host. Gain-of-function mutations in zinc cluster transcription factors, which result in the constitutive upregulation of their target genes, are a common cause of acquired resistance to the widely used antifungal drug fluconazole, especially during long-term therapy of oropharyngeal candidiasis. In this study, we investigated if C. albicans also can develop resistance to the antimicrobial peptide histatin 5, which is secreted in the saliva of humans to protect the oral mucosa from pathogenic microbes. As histatin 5 has been shown to be transported out of C. albicans cells by the Flu1 efflux pump, we screened a library of C. albicans strains that contain artificially activated forms of all zinc cluster transcription factors of this fungus for increased FLU1 expression. We found that a hyperactive Mrr1, which confers fluconazole resistance by upregulating the multidrug efflux pump MDR1 and other genes, also causes FLU1 overexpression. Similarly to the artificially activated Mrr1, naturally occurring gain-of-function mutations in this transcription factor also caused FLU1 upregulation and increased histatin 5 resistance. Surprisingly, however, Mrr1-mediated histatin 5 resistance was mainly caused by the upregulation of MDR1 instead of FLU1, revealing a previously unrecognized function of the Mdr1 efflux pump. Fluconazole-resistant clinical C. albicans isolates with different Mrr1 gain-of-function mutations were less efficiently killed by histatin 5, and this phenotype was reverted when MRR1 was deleted. Therefore, antimycotic therapy can promote the evolution of strains that, as a consequence of drug resistance mutations, simultaneously have acquired increased resistance against an innate host defense mechanism and are thereby better adapted to certain host niches.}, language = {en} } @phdthesis{HagmanngebKischkies2016, author = {Hagmann [geb. Kischkies], Laura Violetta}, title = {Stringent response regulation and its impact on ex vivo survival in the commensal pathogen \(Neisseria\) \(meningitidis\)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-144352}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2016}, abstract = {Neisseria meningitidis is a commensal bacterium which sometimes causes serious disease in humans. Recent studies in numerous human pathogenic bacteria have shown that the stringent response contributes to bacterial virulence. Therefore, this study analyzed the regulation of the stringent response in meningococci and in particular of RelA as well as its contribution to ex vivo fitness in a strain- and condition- dependent manner by using the carriage strain α522 and the hyperinvasive strain MC58 in different in vitro and ex vivo conditions. Growth experiments revealed that both wild-type strains were almost indistinguishable in their ex vivo phenotypes. However, quantitative real time PCR (qRT-PCR) found differences in the gene expression of relA between both strains. Furthermore, in contrast to the MC58 RelA mutant strain α522 deficient in RelA was unable to survive in human whole blood, although both strains showed the same ex vivo phenotypes in saliva and cerebrospinal fluid. Moreover, strain α522 was depended on a short non-coding AT-rich repeat element (ATRrelA) in the promoter region of relA to survive in human blood. Furthermore, cell culture experiments with human epithelial cells revealed that in both strains the deletion of relA resulted in a significantly decreased invasion rate while not significantly affecting adhesion. In order to better understand the conditional lethality of the relA deletion, computational and experimental analyses were carried out to unravel differences in amino acid biosynthetic pathways between both strains. Whereas strain MC58 is able to synthesize all 20 amino acids, strain α522 has an auxotrophy for cysteine and glutamine. In addition, the in vitro growth experiments found that RelA is required for growth in the absence of external amino acids in both strains. Furthermore, the mutant strain MC58 harboring an ATRrelA in its relA promoter region showed improved growth in minimal medium supplemented with L-cysteine and/or L-glutamine compared to the wild-type strain. Contrary, in strain α522 no differences between the wild-type and the ATRrelA deletion mutant were observed. Together this indicates that ATRrelA interferes with the complex regulatory interplay between the stringent response pathway and L-cysteine as well as L-glutamine metabolism. It further suggests that meningococcal virulence is linked to relA in a strain- and condition- depended manner. In conclusion, this work highlighted the role of the stringent response and of non-coding regulatory elements for bacterial virulence and indicates that virulence might be related to the way how meningococci accomplish growth within the host environments.}, subject = {Neisseria meningitidis}, language = {en} } @article{HackerUlmerFasskeetal.1987, author = {Hacker, J{\"o}rg and Ulmer, E. and Fasske, E. and Schmidt, G.}, title = {Isolation and characterization of coliphage Omega18A specific for Escherichia coli O18ac strains}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73001}, year = {1987}, abstract = {The bactedophage Q18A, specific for Escherichia coli 018ac srrains, was isolated frorn sewage. The results of host range and conjugation experiments showed that the sensitivity of bacteria to the phage is associated with rhe presence of 018ac antigens. With sorne of rhe 018 strains rhe phage Q18A produces clear Iysis on bacterial lawns only when applied at a high multiplicity and moreover the phage does not multiply. With rhe help of the phage Ql8A, E. coli 0 18ac strains could be divided inro rwo serologically clistinct subgroups called 018A and 018A1• E. coli strains belanging to the sugroup 0 ISAare sensitive to phage Q t8A wheteas bacteria of subgroup A1 are resistanr.}, subject = {Escherichia coli}, language = {en} } @article{HackerSchrettenbrunnerSchroeteretal.1986, author = {Hacker, J{\"o}rg and Schrettenbrunner, A. and Schr{\"o}ter, G. and Schmidt, G. and D{\"u}vel, H. and Goebel, W.}, title = {Characterization of Escherichia coli wild-type strains by means of agglutination with antisera raised against cloned P-, S- and MS-fimbriae antigens, hemagglutination, serotyping and hemolysin-production}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72992}, year = {1986}, abstract = {E. coli stcains isolated from patients with urinary tcact infecrions (UTn very often possess mannose"sensitive (MS) and mannose-resistant (MR) adherence facmrs (fimbriae). According to their receptor specificity the mannose-resistant adhesins can be divided inm several types, P, S, M and X. We have cloned rhe determinants of rhree groups of UTI E. coli adhesins, MS, p and S, and prepared specific aorisera against the fimbriae antigens. 189 hernagglutination (HA+) -positive stcains, 96 fecal isolates and 93 strains isoJated from UTI . have been tesred with rhese specific antisera and further characterized by receptor specific : HA, HA parteras and further of rhe "common 0 serogroups" 01, 02, 04, 06, 07, 08, 018, ' 025, 075, most prevalenr in UTI, and hemolysin production. · 68 (73 \%) of the UTI srrains a.nd 50 (52\%) of the fecal isolates showed P-receptor specificiry; 16 (17\%) of the uropathogenic bacteria and 33 (34\%) of the fecal strains exhibited S, M or X-fimbriae antigens. 24\% of rhe P-hemagglutinating (P+) strains reacted wirb P (F8)-specific antiserum. In contrast, more than three quaner of the s+-srrains were agglutinated by S-specific antiserum. HA-pattern VJ and 018 amigen were found to be associared with P-fimbriae strains, wbereas HA-pattern V and VII and the 0 anrigens 02 (M-type), 06 and 018 (5-type) occurred most frequently in p- -strains. A high percentage of P-fimbriated strains showed mannose-sensitive hemagglurination and hemolysin production.}, subject = {Escherichia coli}, language = {en} } @article{HackerSchmidtHughesetal.1985, author = {Hacker, J{\"o}rg and Schmidt, G. and Hughes, C. and Knapp, S. and Marget, M. and Goebel, W.}, title = {Cloning and characterization of genes involved in the production of mannose-resistant, neuraminidase-susceptible (X) fimbriae from an uropathogenic O6:K15:K31 Escherichia coli strain}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59353}, year = {1985}, abstract = {The Qropathogenic Escherichia coli strain 536 (06:K15:H31) exhibits a mannose-resistant hemagglutination phenotype (Mrh) with bovine erythrocytes and delayed Mrh with human and guinea pig erythrocytes. Neuraminidase treatment of the erythrocytes abolishes mannose resistant hemagglutination, which is typical for X fimbriae. E. coli strain 536 synthesizes two different fimbriae (Fim phenotype) prQtein subunits, 16.5 and 22 kilodaltons in size. In addition the strain shows mannose-sensitive hemagglutination and common type I (Fl) fimbriae. The cosmid clone E. coli K-12(pANN801) and another nine independently isolated Mrh+ cosmid clones derived from a cosmid gene bank of strain 536 express the 16.5-kilodalton protein band, bot not the 22-kilodalton protein, indicating an association of the Mrh+ property with the "16.5-kilodalton fimbriae." All cosmid clones were fimbriated, and they reacted with antiserum produced against Mrh+ fimbriae of the E. coli strain HB101(pANN801) and lacked mannose-sensitive hemagglutination (Fl) funbriae. From the Mrh fim cosmid DNA pANN801, several subclones coding for hemagglutination and X fimbriae were constructed. Subclones that express both hemagglutination and fimbriae and subclones that only code for the hemagglutination antigen were isolated; subclones that only produce fimbriae were not detected. By transposon Tn5 mutagenesis we demonstrated that about 6.5 kilobases of DNA is required for the Mrh+ Fim+ phenotype, and the 1.5- to 2-kilobase DNA region coding for the structural proteiil of the fimbriae has been mapped adjacent to the region responsible for the Mrh+ phenotype. Two different regions can thus be distinguished in the adhesion determinant, one coding for hemagglutination and the other coding for fimbria formation. Transformation of plasmid DNA from these subclones into a Mrh- Fim- mutant of E. coli 536 and into a galE (rough) strain of Salmonella typhimurium yielded transformants that expressed both hemagglutination and fimbria production.}, subject = {Infektionsbiologie}, language = {en} } @article{HackerRdestWintermeyeretal.1991, author = {Hacker, J{\"o}rg and Rdest, Ursula and Wintermeyer, E. and Ludwig, B.}, title = {Legiolysin, a New Hemolysin from L. pneumophila}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73070}, year = {1991}, abstract = {Legionella pneumophila generares exotoxins, cytolysins, proteases oc hemolysins that darnage host cells llke erythrocytes or rissue cu lrure cells. The gene for a new L. pneumophila hemolysin withour a proteolytic activiry was idemified, cloned in E. coli and sequenced. The gene producr was analysed by SDS-Polyacrylamide-gel-electrophoresis.}, subject = {H{\"a}molysin}, language = {en} } @article{HackerOttWintermeyeretal.1993, author = {Hacker, J{\"o}rg and Ott, Manfred and Wintermeyer, Eva and Ludwig, Birgit and Fischer, Gunter}, title = {Analysis of virulence factors of Legionella pneumophila.}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-70620}, year = {1993}, abstract = {Legionella pneumophila, the causative agent of Legionnaires' disease is a facultative intracellular bacterium, which in the course of human infection multiplies in lung macrophages predominantly manifesting as pneumonia. The natural habitat of Legionella is found in sweet water reservoirs and man-made water systems. Virulent L. pneumophila spontaneously convert to an avirulent status at a high frequency. Genetic approaches have led to the identification of various L. pneumophila genes. The mip (macrophage infectivity potentiator) determinant remains at present the sole established virulence factor. The Mip protein exhibits activity of a peptidyl prolyl cis trans isomerase (PPiase), an enzyme which is able to bind the immunosuppressant FK506 and is involved in protein folding. The recently cloned major outer membrane protein (MOMP) could play a role in the uptake of legionellae by macrophages. Cellular models are useful in studying the intracellular replication of legionellae in eukaryotic cells. Human celllines and protozoan models are appropriate for this purpose. By using U 937 macrophage-like cells and Acanthamoeba castellanii as hosts, we could discriminate virulent and avirulent L. pneumophila variants since only the virulent strain was capable of intracellular growth at 37 oc. By using these systems we further demonstrated that a hemolytic factor cloned and characterized in our laboratory, legiolysin (lly), had no influence on the intracellular growth of L. pneumophila.}, subject = {Legionella pneumophila}, language = {en} } @article{HackerOttBlumetal.1992, author = {Hacker, J{\"o}rg and Ott, Manfred and Blum, Gabriele and Marre, Reinhard and Heesemann, J{\"u}rgen and Tsch{\"a}pe, Helmut and Goebel, Werner}, title = {Genetics of Escherichia coli uropathogenicity: Analysis of the O6:K15:H31 isolate 536}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-71578}, year = {1992}, abstract = {E. coli strain 536 (06: K15: H31) isolated from a case of acute pyelonephritis, expresses S-fimbrial adhesins, P-related fimbriae, common type I fimbriae, and hemolysins. The respective chromosomally encoded determinants were cloned by constructing a genomic library of this strain. Furthermore, the strain produces the iron uptake substance, enterocheline, damages HeLa cells, and behaves in a serum-resistant mode. Genetic analysis of spontaneously arising non-hemolytic variants revealed that some of the virulence genes were physically linked to large unstable DNA regions, termed "pathogenicity islands", which were mapped in the respective positions on the E. coli K-12linkage map. By comparing the wild type strain and mutants in in vitro and in vivo assays, virulence features have been evaluated. In addition, a regulatory cross talk between adhesin determinants was found for the wild-type isolate. This particular mode of virulence regulation is missing in the mutant strain.}, subject = {Escherichia coli}, language = {en} } @article{HackerOttSchmidtetal.1986, author = {Hacker, J{\"o}rg and Ott, M. and Schmidt, G. and Hull, R. and Goebel, W.}, title = {Molecular cloning of the F8 fimbrial antigen from Escherichia coli}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59391}, year = {1986}, abstract = {The genetic determinant coding for the Pspecific F8 fimbriae was cloned from · the chromosome of the Escherichia coli wild-type strain 2980 (018: K5: H5: FlC, F8). The F8 determinant was further subcloned into the Pstl site of pBR322 and a restriction map was established. In a Southern hybridization experiment identity between the chromosomally encoded F8 determinant of 2980 and its cloned Counterpart was demonstrated. The cloned F8 fimbri{\"a}e and those of the wild type strain consist of a protein subunit of nearly 20 kDa. F8 fimbriated strains were agglutinated by an F8 polyclonal antiserum, caused mannose-resistant hemagglutination and attached to human uroepi thellal cells. The cloned F8 determinant was weil expressed in a variety of host strains.}, subject = {Infektionsbiologie}, language = {en} } @article{HackerOttLudwigetal.1991, author = {Hacker, J{\"o}rg and Ott, M. and Ludwig, B. and Rdest, U.}, title = {Intracellular survival and expression of virulence determinants of Legionella pneumophila}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59681}, year = {1991}, abstract = {Legionella pneumophila, the causative agent of Legionnaires' disease is able to live and multiply within macrophages as weil as within protozoan organisms. Legionella strains inhibit phagosome-lysosome fusion and phagosome acidification. By using two different cell culture systems, one derived from human macrophages and the other from human.embryo lung fibro:blastic cells, it is demonstrated that Legionella strains lose their virulence following cultivation in the laboratory. In order to study the mechanisms involved in intracellular survival of Legionella a genomic library of strain Legionella pneumophila Philadelphia I was established in Escherichia coli K-12. By cosmid cloning technique we were able to clone five putative virulence factors, two of which exhibit hemolytic activities and three of which represent membrane-associated proteins of 19, 26 and 60 kilodalton. One of the hemolytic proteins, termed legiolysin, represents a new toxin which specifically lyses human erythrocytes. The other hemolysin exhibits proteolytic properties in addition and is cytolytic for Vero and CHO cells. Further sturlies will be necessary to determine the exact role of the cloned proteins in the pathogenesis of Legionella. Zusammenfassung: Intrazellul{\"a}res {\"U}berleben}, subject = {Infektionsbiologie}, language = {en} } @article{HackerOttHof1993, author = {Hacker, J{\"o}rg and Ott, M. and Hof, H.}, title = {Effects of low, subinhibitory concentrations of antibiotics on expression of a virulence gene cluster of pathogenic E. coli by using a wild-type gene fusion}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59874}, year = {1993}, abstract = {No abstract available}, subject = {Infektionsbiologie}, language = {en} } @article{HackerKnappGoebel1983, author = {Hacker, J{\"o}rg and Knapp, S. and Goebel, W.}, title = {Spontaneous deletions and flanking regions of the chromosomal inherited hemolysin determinant of an Escherichia coli 06 strain}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-40260}, year = {1983}, abstract = {The hemolytic Escherichia coli strain 536 (06) propagates spontaneous hemolysin- negative mutants at relatively high rates (10-3 to 10-4 ). One type of mutant (type I) lacks both secreted (external) and periplasmic (internal) hemolysin activity (HlYex - IHlYin -) and in addition shows no mannose-resistant hemagglutination (Mrh -), whereas the other type (type II) is HlYex -IHIYin + and Mrh +. The genetic determinants for hemolysin production (hly) and for mannose-resistant hemagglutination (mrh) of this strain are located on the chromosome. Hybridization experiments with DNA probes specific for various parts of the hly determinant reveal that mutants of type I have lost the total hly determinant, whereas those of type 11 lack only part of the hlyB that is essential for transport of hemolysin across the outer membrane. Using a probe that contains the end sequence of the plasmid pHly152-encoded hly determinant (adjacent to hlyB), we determined that a related sequence flanks also the hlyB-distal end of the chromosomal hly determinant of E. coli 536. In addition several other similar or even identical sequences are found in the vicinity of the hlyC- and the hlyB-distal ends of both the chromosomal and the plasmid hly determinants.}, language = {en} } @article{HackerKestlerHoschuetzkyetal.1993, author = {Hacker, J{\"o}rg and Kestler, H. and Hosch{\"u}tzky, H. and Jann, K. and Lottspeich, F. and Korhonen, T. K.}, title = {Cloning and characterization of the S fimbrial adhesin (SfaII) complex of an Escherichia coli O18:K1 meningitis isolate}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59853}, year = {1993}, abstract = {S fimbrial adbesins (Sfa), which are able to recognize sialic acid-containing receptors on eukaryotic cells, are produced by Escherichia coli strains causing urinary tract infections or newbom meningitis. We recently described tbe cloning and molecular cbaracterization of a determinant, termed sftJI, from the chromosome of an E. coli urinary tract infection strain. Herewe present data conceming a S fimbria-specific gene duster, designated sfall, of an E. coli newbom meningitis strain. Like tbe Sfal complex, Sfall consists of tbe major subunit protein SfaA (16 kDa) and the minor subunit proteins SfaG (17 kDa), SfaS (15 kDa), and SfaH (29 kDa). The genes encoding tbe subunit proteins of Sfall were identified and sequenced. Their protein sequences were calculated from the DNA sequences and compared with tbose of the Sfal complex subunits. Altbough the sequences ofthe two major SfaA subunits ditf'ered markedly, tbe sequences ofthe minor subunits sbowed only a few amino acid exchanges (SfaG, SfaH) or were completely identical (SfaS). The introduction of a site-specific mutation into the gene sfaSII and subsequent analysis of an SfaS-negative clone indicated that sfaSII codes for the sialic acid-specific adhesin of tbe meninigitis isolate. These data were confirmed by tbe isolation and characterization of tbe SfaSII protein and the determination of its N-terminal amino acid sequence. The identity between the sialic acid-specific adhesins of Sfal and Sfall revealed that difl'erences between the two Sfa complexes with respect to tbeir capacities to agglutinate erythrocytes must result from sequence alterations of subunit proteins other tban SfaS.}, subject = {Infektionsbiologie}, language = {en} } @article{HackerHughesHofetal.1983, author = {Hacker, J{\"o}rg and Hughes, C. and Hof, H. and Goebel, W.}, title = {Cloned hemolysin genes from Escherichia coli that cause urinary tract infection determine different levels of toxicity in mice}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59330}, year = {1983}, abstract = {After intraperitoneal injection of mice with Escherichia coli strains isolated from patients with urinary tract infections, the mortality due to hemolytic (Hly+) and nonhemolytic (Hiy-) isolates was 77 and 40\%, respectively. Deletion of the chromosomal hemolysin (h/y) determinant in an E. co/i 06:K15:H31 urinary tract infection strain led to a significant reduction in toxicity for mice, and its reintroduction on a recombinant plasmid partially restored the original toxicity. Although introduction of the cloned plasmid pHiy152-encoded hly determinant into the Hly- E. coli 06 mutant strain increased toxicity by only a marginal degree, transformation with the cloned chromosomal hly determinants from two E. coli strains of serotypes 018ac:K5:H- and 075:K95:H? resulted in markedly greater toxicity, even exceeding that of the original Hly+ E. coli 06 wild-type strain.}, subject = {Infektionsbiologie}, language = {en} } @article{HackerHofHughesetal.1985, author = {Hacker, J{\"o}rg and Hof, H. and Hughes, C. and Goebel, W.}, title = {Salmonella typhimurium strains carrying hemolysin plasmids and cloned hemolysin. genes from Escherichia coli}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-40309}, year = {1985}, abstract = {Like all other Salmonella typhimurium strains examined, the smooth variants SF1397 (L T2) and 1366 and also their semi-rough and rough derivatives are non-haemolytic. Nevertheless, two haemolysin (Hly) plasmids of E. coli belonging to the inc groups incFllI,lv (pSU316) and incIz (pHly152) were able to be introduced into these strains by conjugation and stably maintained. A considerable percentage of the Hly+ transconjugants obtained had lost parts of their O-side chains, a result of selection for the better recipient capability of « semi-rough» variants rather than the direct influence of the Hly+ plasmids themselves. In contrast to the incF1lI1V plasmid pSU316, which exhibited higher conjugation rates with rough recipients, the incIz plasmid pHly152 was accepted best by smooth strains. Transformation with cloned E. coli haemolysin (hly) determinant was inefficient ( <10-8) for smooth strains, but 102-103 times higher for rough recipients, and was increased by the use of Salmonella-modified DNA. The transform ants and transconjugants were relatively stable and showed the same haemolytic activity as the E. coli donor strains. The virulence of the Hly+ smooth, semi-rough and rough S. typhimurium strains was tested in two mouse models, and neither the mortality rate nor the ability to multiply within the mouse spleen was influenced by the hly determinants.}, language = {en} } @article{HackerHofEmoedyetal.1986, author = {Hacker, J{\"o}rg and Hof, H. and Em{\"o}dy, L. and Goebel, W.}, title = {Influence of cloned Escherichia coli hemolysin genes, S fimbriae and serum resistance on pathogenicity in different animal models}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59423}, year = {1986}, abstract = {The virulence of the uropathogenic E. coli strain 536 (06: K 1 5: H31) which produces the S-fimbrial adhesin (Sfa•), is serum-resistant (Sre+) and hemolytic (Hiy+) and its derivatives were assessed in five different animal models. Cloned hemolysin (h/y) determinants from the Chromosomes of 06,018 and 075 E. colistrains and from the plasmid pHiy152 were introduced into the spontaneaus Sfa-, Sre-, Hly- mutant 536-21 and its Sfa+, Sre+, Hly- variant 536-31. As already demonstrated for the 536-21 strains {lnfect. Immun. 42: 57-63) the 018-hly determinant but not the plasmid-encoded hly determinant of pHiy 1 52 transformed into 536-31 contribute to lethality in a mouse peritonitis modal. Similar results were obtained with both Hlyhost strains and their Hly+ transformants in a chicken embryo test and in a mouse nephropathogenicity assay in which the renal bacterial counts were measured 1 5 min to 8 hours after i.v. infection. S-fimbriae and serum resistance had only a marginal influence in these three in vivo systems. ln centrast all three factors, S-fimbriae, serum resistance and hemolysin, were necessary for full virulence in a respiratory mouse infection assay. ln a subcutaneously-induced sepsis model in the mouse restoration of S-fimbriae and serum resistance and separately chromosomally-encoded hemolysis increased virulence to a Ievel comparable to that of the parental 536 strain.}, subject = {Infektionsbiologie}, language = {en} } @article{HackerGadebergOrskov1989, author = {Hacker, J{\"o}rg and Gadeberg, Ole V. and Orskov, Ida}, title = {Role of alpha-Hemolysin for the in vitro Phagocytosis and intracellular killing of Escherichia coli}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73019}, year = {1989}, abstract = {The_role of a-hemolysin for the elimination of Eschericbia coli by phagocyres in vitro was investigated using sets of isogenic strains which included wild-type a -hemolyric srrains, derived strains with a reduced production of a-hemolysin and derived nonhemolytic strains. Phagocyrosis and intracellular killing of the bacteria by human blood granulocytes or monocytes were measured using growth inhibition rechniques. a-hemolytic strains were phagocytosed and killed ro a Jesser extent than isogenic strains with a reduced production of o:hemoJysin and isogenic nonhemolytic strains. The results obrained with granulocyres were similar to rhose obtained with monocyres although the elimination of bacteria by monocytes was less than that by granulocytes. These resulcs strongJy suggest that production of ahemolysin is a means by which E. coli counteracrs the activity of phagocytes by injuring these cells with the toxin.}, subject = {Escherichia coli}, language = {en} } @article{HackerBenderOttetal.1990, author = {Hacker, J{\"o}rg and Bender, L. and Ott, M. and Wingeder, J. and Lund, B. and Marre, R. and Goebel, W.}, title = {Deletions of chromosomal regions coding for fimbriae and hemolysins occur in vivo and in vitro in various extraintestinal Escherichia coli isolates}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59608}, year = {1990}, abstract = {No abstract available}, subject = {Infektionsbiologie}, language = {en} } @article{GuptaSrivastavaOsmanogluetal.2021, author = {Gupta, Shishir K. and Srivastava, Mugdha and Osmanoglu, {\"O}zge and Xu, Zhuofei and Brakhage, Axel A. and Dandekar, Thomas}, title = {Aspergillus fumigatus versus genus Aspergillus: conservation, adaptive evolution and specific virulence genes}, series = {Microorganisms}, volume = {9}, journal = {Microorganisms}, number = {10}, issn = {2076-2607}, doi = {10.3390/microorganisms9102014}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-246318}, year = {2021}, abstract = {Aspergillus is an important fungal genus containing economically important species, as well as pathogenic species of animals and plants. Using eighteen fungal species of the genus Aspergillus, we conducted a comprehensive investigation of conserved genes and their evolution. This also allows us to investigate the selection pressure driving the adaptive evolution in the pathogenic species A. fumigatus. Among single-copy orthologs (SCOs) for A. fumigatus and the closely related species A. fischeri, we identified 122 versus 50 positively selected genes (PSGs), respectively. Moreover, twenty conserved genes of unknown function were established to be positively selected and thus important for adaption. A. fumigatus PSGs interacting with human host proteins show over-representation of adaptive, symbiosis-related, immunomodulatory and virulence-related pathways, such as the TGF-β pathway, insulin receptor signaling, IL1 pathway and interfering with phagosomal GTPase signaling. Additionally, among the virulence factor coding genes, secretory and membrane protein-coding genes in multi-copy gene families, 212 genes underwent positive selection and also suggest increased adaptation, such as fungal immune evasion mechanisms (aspf2), siderophore biosynthesis (sidD), fumarylalanine production (sidE), stress tolerance (atfA) and thermotolerance (sodA). These genes presumably contribute to host adaptation strategies. Genes for the biosynthesis of gliotoxin are shared among all the close relatives of A. fumigatus as an ancient defense mechanism. Positive selection plays a crucial role in the adaptive evolution of A. fumigatus. The genome-wide profile of PSGs provides valuable targets for further research on the mechanisms of immune evasion, antimycotic targeting and understanding fundamental virulence processes.}, language = {en} } @article{GuptaSrivastavaMinochaetal.2021, author = {Gupta, Shishir K. and Srivastava, Mugdha and Minocha, Rashmi and Akash, Aman and Dangwal, Seema and Dandekar, Thomas}, title = {Alveolar regeneration in COVID-19 patients: a network perspective}, series = {International Journal of Molecular Sciences}, volume = {22}, journal = {International Journal of Molecular Sciences}, number = {20}, issn = {1422-0067}, doi = {10.3390/ijms222011279}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-284307}, year = {2021}, abstract = {A viral infection involves entry and replication of viral nucleic acid in a host organism, subsequently leading to biochemical and structural alterations in the host cell. In the case of SARS-CoV-2 viral infection, over-activation of the host immune system may lead to lung damage. Albeit the regeneration and fibrotic repair processes being the two protective host responses, prolonged injury may lead to excessive fibrosis, a pathological state that can result in lung collapse. In this review, we discuss regeneration and fibrosis processes in response to SARS-CoV-2 and provide our viewpoint on the triggering of alveolar regeneration in coronavirus disease 2019 (COVID-19) patients.}, language = {en} } @article{GuptaMinochaThapaetal.2022, author = {Gupta, Shishir K. and Minocha, Rashmi and Thapa, Prithivi Jung and Srivastava, Mugdha and Dandekar, Thomas}, title = {Role of the pangolin in origin of SARS-CoV-2: an evolutionary perspective}, series = {International Journal of Molecular Sciences}, volume = {23}, journal = {International Journal of Molecular Sciences}, number = {16}, issn = {1422-0067}, doi = {10.3390/ijms23169115}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-285995}, year = {2022}, abstract = {After the recent emergence of SARS-CoV-2 infection, unanswered questions remain related to its evolutionary history, path of transmission or divergence and role of recombination. There is emerging evidence on amino acid substitutions occurring in key residues of the receptor-binding domain of the spike glycoprotein in coronavirus isolates from bat and pangolins. In this article, we summarize our current knowledge on the origin of SARS-CoV-2. We also analyze the host ACE2-interacting residues of the receptor-binding domain of spike glycoprotein in SARS-CoV-2 isolates from bats, and compare it to pangolin SARS-CoV-2 isolates collected from Guangdong province (GD Pangolin-CoV) and Guangxi autonomous regions (GX Pangolin-CoV) of South China. Based on our comparative analysis, we support the view that the Guangdong Pangolins are the intermediate hosts that adapted the SARS-CoV-2 and represented a significant evolutionary link in the path of transmission of SARS-CoV-2 virus. We also discuss the role of intermediate hosts in the origin of Omicron.}, language = {en} } @phdthesis{Grozdanov2004, author = {Grozdanov, Lubomir Assenov}, title = {Analysis of the genome organization and fitness traits of non-pathogenic Escherichia coli strain Nissle 1917 (O6:K5:H1)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-9304}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2004}, abstract = {In the last years more than one hundred microbial genomes have been sequenced, many of them from pathogenic bacteria. The availability of this huge amount of sequence data enormously increases our knowledge on the genome structure and plasticity, as well as on the microbial diversity and evolution. In parallel, these data are the basis for the scientific "revolution" in the field of industrial and environmental biotechnology and medical microbiology - diagnostics and therapy, development of new drugs and vaccines against infectious agents. Together with the genomic approach, other molecular biological methods such as PCR, DNA-chip technology, subtractive hybridization, transcriptomics and proteomics are of increasing importance for research on infectious diseases and public health. The aim of this work was to characterize the genome structure and -content of the probiotic Escherichia coli strain Nissle 1917 (O6:K5:H31) and to compare these data with publicly available data on the genomes of different pathogenic and non-pathogenic E. coli strains and other closely related species. A cosmid genomic library of strain Nissle 1917 was screened for clones containing the genetic determinants contributing to the successful survival in and colonization of the human body, as well as to mediate this strain's probiotic effect as part of the intestinal microflora. Four genomic islands (GEI I-IVNissle 1917) were identifed and characterized. They contain many known fitness determinants (mch/mcm, foc, iuc, kps, ybt), as well as novel genes of unknown function, mobile genetic elements or newly identified putative fitness-contributing factors (Sat, Iha, ShiA-homologue, Ag43-homologues). All islands were found to be integrated next to tRNA genes (serX, pheV, argW and asnT, respectively). Their structure and chromosomal localization closely resembles those of analogous islands in the genome of uropathogenic E. coli strain CFT073 (O6:K2(?):H1), but they lack important virulence genes of uropathogenic E. coli (hly, cnf, prf/pap). Evidence for instability of GEI IINissle 1917 was given, since a deletion event in which IS2 elements play a role was detected. This event results in loss of a 30 kb DNA region, containing important fitness determinants (iuc, sat, iha), and therefore probably might influence the colonization capacity of Nissle 1917 strain. In addition, a screening of the sequence context of tRNA-encoding genes in the genome of Nissle 1917 was performed to identify genome wide potential integration sites of "foreign" DNA. As a result, similar "tRNA screening patterns" have been observed for strain Nissle 1917 and for the uropathogenic E. coli O6 strains (UPEC) 536 and CFT073. I. Summary 4 The molecular reason for the semi-rough phenotype and serum sensitivity of strain Nissle 1917 was analyzed. The O6-antigen polymerase-encoding gene wzy was identified, and it was shown that the reason for the semi-rough phenotype is a frame shift mutation in wzy, due to the presence of a premature stop codon. It was shown that the restoration of the O side-chain LPS polymerization by complementation with a functional wzy gene increased serumresistance of strain Nissle 1917. The results of this study show that despite the genome similarity of the E. coli strain Nissle 1917 with the UPEC strain CFT073, the strain Nissle 1917 exhibits a specific set of geno- and phenotypic features which contribute to its probiotic action. By comparison with the available data on the genomics of different species of Enterobacteriaceae, this study contributes to our understanding of the important processes such as horizontal gene transfer, deletions and rearrangements which contribute to genome diversity and -plasticity, and which are driving forces for the evolution of bacterial variants. At last, the fim, bcs and rfaH determinats whose expression contributes to the mutlicellular behaviour and biofilm formation of E. coli strain Nissle 1917 have been characterized.}, subject = {Escherichia coli}, language = {en} } @article{GrohAbdelwahabKattimanietal.2023, author = {Groh, Janos and Abdelwahab, Tassnim and Kattimani, Yogita and H{\"o}rner, Michaela and Loserth, Silke and Gudi, Viktoria and Adalbert, Robert and Imdahl, Fabian and Saliba, Antoine-Emmanuel and Coleman, Michael and Stangel, Martin and Simons, Mikael and Martini, Rudolf}, title = {Microglia-mediated demyelination protects against CD8\(^+\) T cell-driven axon degeneration in mice carrying PLP defects}, series = {Nature Communications}, volume = {14}, journal = {Nature Communications}, doi = {10.1038/s41467-023-42570-2}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-357641}, year = {2023}, abstract = {Axon degeneration and functional decline in myelin diseases are often attributed to loss of myelin but their relation is not fully understood. Perturbed myelinating glia can instigate chronic neuroinflammation and contribute to demyelination and axonal damage. Here we study mice with distinct defects in the proteolipid protein 1 gene that develop axonal damage which is driven by cytotoxic T cells targeting myelinating oligodendrocytes. We show that persistent ensheathment with perturbed myelin poses a risk for axon degeneration, neuron loss, and behavioral decline. We demonstrate that CD8\(^+\) T cell-driven axonal damage is less likely to progress towards degeneration when axons are efficiently demyelinated by activated microglia. Mechanistically, we show that cytotoxic T cell effector molecules induce cytoskeletal alterations within myelinating glia and aberrant actomyosin constriction of axons at paranodal domains. Our study identifies detrimental axon-glia-immune interactions which promote neurodegeneration and possible therapeutic targets for disorders associated with myelin defects and neuroinflammation.}, language = {en} } @phdthesis{Grimm2000, author = {Grimm, Dorothee}, title = {Entwicklung von neuen Nachweismethoden f{\"u}r Legionellen und Am{\"o}ben und ihre Anwendung in {\"o}kologischen Studien}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-1351}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2000}, abstract = {Legionella pneumophila wurde 1976 als Erreger der Legionellose, einer schweren Form von Lungenentz{\"u}ndung, identifiziert. Die inzwischen 42 Arten umfassende Gattung ist weltweit in aquatischen Biotopen verbreitet. Die Bakterien leben vergesellschaftet mit anderen Mikroorganismen in Biofilmen oder intrazellul{\"a}r in Protozoen. Sie haben ein duales Wirtssystem, das heißt, sie sind in der Lage, sich sowohl in Einzellern als auch in humanen Phagozyten zu vermehren. F{\"u}r die Erweiterung ihrer Habitate spielen verschiedene Umweltfaktoren eine Rolle. Eine exakte und schnelle Detektion und Identifikation der humanpathogenen Keime ist sowohl f{\"u}r die Lokalisierung der Infektionsquelle als auch f{\"u}r eine rechtzeitige Therapie der Patienten von großer Bedeutung. Die Technik der fluoreszierenden in situ Hybridisierung (FISH) basiert auf der Bindung einer spezifischen, mit einem Fluoreszenzfarbstoff markierten Oligonukleotidsonde an eine komplement{\"a}re Zielsequenz der ribosomalen RNA. In der vorliegenden Arbeit wurde f{\"u}r die in situ Hybridisierung eine neue 16S rRNA-gerichtete Sonde LEGPNE1 entwickelt, die spezifisch ist f{\"u}r L. pneumophila. In Versuchsreihen mit verschiedenen Bakterienkulturen wurden die Spezifit{\"a}t und Sensitivit{\"a}t der Gensonde ermittelt. LEGPNE1 erwies sich als artspezifisch und erkannte alle getesteten L. pneumophila-St{\"a}mme, ungeachtet ihrer Serogruppe. Nicht-pneumophila-Referenzst{\"a}mme hybridisierten nicht mit der Sonde, ein einziger Basenaus-tausch in der Sequenz war f{\"u}r diese Unterscheidung ausreichend. Die Anwendung der Sonde wurde auch in Am{\"o}ben-Infektionsassays und Umweltproben erfolgreich durchgef{\"u}hrt. Unterschiedliche, intrazellul{\"a}r vorliegende Bakterien wurden von der Sonde spezifisch erkannt. Eine in situ Dokumentation der Infektions- und Vermehrungsrate war damit m{\"o}glich. Durch die meist fakultativ intrazellul{\"a}re Lebensweise der Legionellen ist es wichtig, auch die Wirtszellen der Keime qualitativ zu detektieren und zu identifizieren. Die Entwicklung neuer Gensonden wurde daher auf die beiden bekannten Wirtsam{\"o}ben Hartmannella und Naegleria ausgedehnt. Basierend auf Sequenzvergleichen wurden die gattungsspezifischen 18S rRNA-gerichteten Sonden HART498 und NAEG1088 konstruiert und in Versuchsreihen mit Referenzst{\"a}mmen bei steigender Stringenz etabliert. Mit ihrer Hilfe konnten die Ergebnisse der zeit- und arbeitsaufwendigen Determination unbekannter Am{\"o}ben anhand morphologischer Merkmale best{\"a}tigt werden. In situ Hybridisierungen mit einer Kombination von 16S und 18S rRNA-gerichteten Sonden wurden in Am{\"o}ben-Infektionsassays mit Hartmannella vermiformis und L. pneumophila erfolgreich durchgef{\"u}hrt. Eine Interferenz der Sonden fand nicht statt. Die in situ Untersuchung der Struktur und Funktion komplexer mikrobieller Lebensgemeinschaften erfordert eine kultivierungsunabh{\"a}ngige und hochaufl{\"o}sende Methode, wie sie die fluoreszierende in situ Hybridisierung darstellt. Mit dem Ziel, m{\"o}gliche Pr{\"a}ferenzen der Legionellen f{\"u}r bestimmte Parameter, wie pH, Temperatur, elektrische Leitf{\"a}higkeit, Str{\"o}mungsverh{\"a}ltnisse, zu erkennen und zu definieren, wurden mit Hilfe der neu entwickelten 16S rRNA-gerichteten Sonde 21 verschiedene Kaltwasserhabitate auf die Verbreitung von Legionella untersucht. Die Bakterien zeigten jedoch ein breites Toleranzspektrum gegen{\"u}ber den gemessenen Parametern. Sie waren in nahezu allen beprobten Gew{\"a}ssern zu finden und ließen sich unabh{\"a}ngig von der Jahreszeit nachweisen. Die neue Sonde LEGPNE1 zeigte sich in in situ Hybridisierungen der Umweltproben als hochspezifisch. Mit ihr konnten auch nicht kultivierbare Legionellen detektiert werden. In drei Legionella-positiven Gew{\"a}ssern wurde außerdem das Vorkommen von Am{\"o}ben untersucht. Es konnten insgesamt acht Am{\"o}bengattungen isoliert, kultiviert und bestimmt werden. Dominierend waren St{\"a}mme des nicht humanpathogenen Naegleria gruberi-Komplexes, Echinamoeba spp. und Echinamoeba-like Am{\"o}ben. In einzelnen Proben wurden Acanthamoeba spp. Gruppe II, Hartmannella spp., Platyamoeba placida, Saccamoeba spp., Thecamoeba quadrilineata und Vexillifera spp. gefunden. Durch in situ Hybridisierung mit den neuen 18S rRNA-gerichteten Sonden HART498 und NAEG1088 konnten die Ergebnisse der morphologischen Bestimmung der Am{\"o}ben best{\"a}tigt werden. Auch die Am{\"o}ben zeigten keine Pr{\"a}ferenzen bez{\"u}glich der in den Standorten gemessenen Wasserparameter. Die in situ Hybridisierung mit rRNA-gerichteten Gensonden erlaubt eine Analyse der Struktur und Dynamik von Bioz{\"o}nosen, erm{\"o}glicht aber keine Aussage {\"u}ber die speziellen Aktivit{\"a}ten der nachgewiesenen Bakterien. Eine L{\"o}sung hierf{\"u}r k{\"o}nnte der spezifische in situ Nachweis von mRNA-Molek{\"u}len darstellen. Ein Problem hierbei stellt ihre, im Vergleich zu anderen Molek{\"u}len wie rRNA, sehr kurze Halbwertszeit und das Vorhandensein von nur wenigen Kopien pro Zelle dar. Daher ist im Anschluss an die in situ Hybridisierung in den meisten F{\"a}llen eine Signalamplifikation n{\"o}tig, um ein detektierbares Signal zu erhalten. In dieser Arbeit sollte die f{\"u}r das iap-Gen in Listeria monocytogenes entwickelte Methode zum Nachweis der mip-mRNA in L. pneumophila etabliert werden. Erste Anwendungen in Dot blot- und in situ Hybridisierungen mit mehrfach DIG-markierten Polyribonukleotidsonden bzw. mit simultan eingesetzten, einfach DIG-markierten Oligonukleotiden zeigten noch nicht die gew{\"u}nschte Spezifit{\"a}t. Diese Ergebnisse stellen jedoch eine wichtige Grundlage f{\"u}r zuk{\"u}nftige Experimente dar.}, subject = {Legionella pneumophila}, language = {de} } @article{GorskiVogelSalibaetal.2014, author = {Gorski, Stanislaw A. and Vogel, J{\"o}rg and Saliba, Antoine-Emmanuel and Westermann, Alexander J.}, title = {Single-cell RNA-seq: advances and future challenges}, doi = {10.1093/nar/gku555}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-110993}, year = {2014}, abstract = {Phenotypically identical cells can dramatically vary with respect to behavior during their lifespan and this variation is reflected in their molecular composition such as the transcriptomic landscape. Singlecell transcriptomics using next-generation transcript sequencing (RNA-seq) is now emerging as a powerful tool to profile cell-to-cell variability on a genomic scale. Its application has already greatly impacted our conceptual understanding of diverse biological processes with broad implications for both basic and clinical research. Different single-cell RNAseq protocols have been introduced and are reviewed here - each one with its own strengths and current limitations. We further provide an overview of the biological questions single-cell RNA-seq has been used to address, the major findings obtained from such studies, and current challenges and expected future developments in this booming field.}, subject = {RNS}, language = {en} } @article{GomesWestermannSauerweinetal.2019, author = {Gomes, Sara F. Martins and Westermann, Alexander J. and Sauerwein, Till and Hertlein, Tobias and F{\"o}rstner, Konrad U. and Ohlsen, Knut and Metzger, Marco and Shusta, Eric V. and Kim, Brandon J. and Appelt-Menzel, Antje and Schubert-Unkmeir, Alexandra}, title = {Induced pluripotent stem cell-derived brain endothelial cells as a cellular model to study Neisseria meningitidis infection}, series = {Frontiers in Microbiology}, volume = {10}, journal = {Frontiers in Microbiology}, number = {1181}, doi = {10.3389/fmicb.2019.01181}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-201562}, year = {2019}, abstract = {Meningococcal meningitis is a severe central nervous system infection that occurs when Neisseria meningitidis (Nm) penetrates brain endothelial cells (BECs) of the meningeal blood-cerebrospinal fluid barrier. As a human-specific pathogen, in vivo models are greatly limited and pose a significant challenge. In vitro cell models have been developed, however, most lack critical BEC phenotypes limiting their usefulness. Human BECs generated from induced pluripotent stem cells (iPSCs) retain BEC properties and offer the prospect of modeling the human-specific Nm interaction with BECs. Here, we exploit iPSC-BECs as a novel cellular model to study Nm host-pathogen interactions, and provide an overview of host responses to Nm infection. Using iPSC-BECs, we first confirmed that multiple Nm strains and mutants follow similar phenotypes to previously described models. The recruitment of the recently published pilus adhesin receptor CD147 underneath meningococcal microcolonies could be verified in iPSC-BECs. Nm was also observed to significantly increase the expression of pro-inflammatory and neutrophil-specific chemokines IL6, CXCL1, CXCL2, CXCL8, and CCL20, and the secretion of IFN-γ and RANTES. For the first time, we directly observe that Nm disrupts the three tight junction proteins ZO-1, Occludin, and Claudin-5, which become frayed and/or discontinuous in BECs upon Nm challenge. In accordance with tight junction loss, a sharp loss in trans-endothelial electrical resistance, and an increase in sodium fluorescein permeability and in bacterial transmigration, was observed. Finally, we established RNA-Seq of sorted, infected iPSC-BECs, providing expression data of Nm-responsive host genes. Altogether, this model provides novel insights into Nm pathogenesis, including an impact of Nm on barrier properties and tight junction complexes, and suggests that the paracellular route may contribute to Nm traversal of BECs.}, language = {en} } @phdthesis{Goebel2000, author = {Goebel, Stefan}, title = {Mechanismen der Toxoplasma-gondii-vermittelten Inhibierung der Apoptose in humanen Wirtszelllinien}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-1325}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2000}, abstract = {Als obligat intrazellul{\"a}rer Parasit und Erreger von lebenslang persistierenden Infektionen in Mensch und Tier d{\"u}rfte Toxoplasma gondii von der Integrit{\"a}t seiner Wirtszelle in besonderem Maße abh{\"a}ngig sein. Ziel der Arbeit war es, den Einfluss des Parasiten auf die Wirtszellapoptose zu untersuchen. T. gondii inhibiert die in vitro induzierte Apoptose in humanen HL-60- und U937-Zellen. Dabei muss der Parasit aktiv in die Zelle eindringen, jedoch nicht in dieser replizieren k{\"o}nnen. Er interferiert dabei mit mindestens zwei Komponenten der Apoptose-Signalkaskade: Erstens vermindert T. gondii die Herunterregulation der Mcl-1-Expression nach Apoptoseinduktion. Das f{\"u}hrt dazu, dass trotz Apoptoseinduktion die Translokation von Cytochrom c aus den Mitochondrien in das Zytoplasma inhibiert wird und daraufhin die Caspasen 9 und 3 sowie deren Substrate weniger stark aktiviert werden. Zweitens wird die Expression der Poly-(ADP-Ribose)Polymerase (PARP) durch T. gondii inhibiert. Beide Mechanismen k{\"o}nnten an der Inhibierung der Wirtszellapoptose durch T. gondii beteiligt sein und dem Parasiten damit sein intrazellul{\"a}res {\"U}berleben sichern.}, subject = {Toxoplasmase gondii}, language = {de} } @article{GlaserSchurigtSuzukietal.2015, author = {Glaser, Jan and Schurigt, Uta and Suzuki, Brian M. and Caffrey, Connor R. and Holzgrabe, Ulrike}, title = {Anti-Schistosomal Activity of Cinnamic Acid Esters: Eugenyl}, series = {Molecules}, volume = {20}, journal = {Molecules}, doi = {10.3390/molecules200610873}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-125712}, pages = {10873-10883}, year = {2015}, abstract = {Bornyl caffeate (1) was previously isolated by us from Valeriana (V.) wallichii rhizomes and identified as an anti-leishmanial substance. Here, we screened a small compound library of synthesized derivatives 1-30 for activity against schistosomula of Schistosoma (S.) mansoni. Compound 1 did not show any anti-schistosomal activity. However, strong phenotypic changes, including the formation of vacuoles, degeneration and death were observed after in vitro treatment with compounds 23 (thymyl cinnamate) and 27 (eugenyl cinnamate). Electron microscopy analysis of the induced vacuoles in the dying parasites suggests that 23 and 27 interfere with autophagy.}, language = {en} } @article{GlaserSchultheisHazraetal.2014, author = {Glaser, Jan and Schultheis, Martina and Hazra, Sudipta and Hazra, Banazri and Moll, Heidrun and Schurigt, Uta and Holzgrabe, Ulrike}, title = {Antileishmanial Lead Structures from Nature: Analysis of Structure-Activity Relationships of a Compound Library Derived from Caffeic Acid Bornyl Ester}, doi = {10.3390/molecules19021394}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-112835}, year = {2014}, abstract = {Bioassay-guided fractionation of a chloroform extract of Valeriana wallichii (V. wallichii) rhizomes lead to the isolation and identification of caffeic acid bornyl ester (1) as the active component against Leishmania major (L. major) promastigotes (IC50 = 48.8 µM). To investigate the structure-activity relationship (SAR), a library of compounds based on 1 was synthesized and tested in vitro against L. major and L. donovani promastigotes, and L. major amastigotes. Cytotoxicity was determined using a murine J774.1 cell line and bone marrow derived macrophages (BMDM). Some compounds showed antileishmanial activity in the concentration range of pentamidine and miltefosine which are the standard drugs in use. In the L. major amastigote assay compounds 15, 19 and 20 showed good activity with relatively low cytotoxicity against BMDM, resulting in acceptable selectivity indices. Molecules with adjacent phenolic hydroxyl groups exhibited elevated cytotoxicity against murine cell lines J774.1 and BMDM. The Michael system seems not to be essential for antileishmanial activity. Based on the results compound 27 can be regarded as new lead structure for further structure optimization}, language = {en} } @article{GillitzerMoll1993, author = {Gillitzer, Reinhard and Moll, Heidrun}, title = {Simultaneous demonstration of two antigens with immunogold-silver staining and immunoenzymatic labeling}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-33019}, year = {1993}, abstract = {A novel technique for independent and simultaneous labeling of two antigens expressed on individual cells (referred to as mixed labeling) is presented. The staining procedure combined three-step (streptavidin-biotin) immunogold-silver staining with three-step immunoenzymatic labeling. To ensure both high specificity and high sensitivity, particular emphasis was placed on designing a protocol that avoids immunological crossreactivity between the antibody reagents and overlapping of the final color products. Two examples for usage of this mixed labeling technique are described: lymphocyte subpopulations were identified in inflammatory lesions of human skin and infected host cells were characterized in the skin of mice infected with the obligatory intracellular parasite Leishmania major, a cause of human cutaneous leishmaniasis.}, language = {en} } @misc{GillitzerBergerMoll1990, author = {Gillitzer, Reinhard and Berger, Rudolf and Moll, Heidrun}, title = {A reliable method for simultaneous demonstration of two antigens using a novel combination of immunogold-silver staining with immunoenzymatic labeling}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-31092}, year = {1990}, abstract = {We have developed a reliable and sensitive immunohistochemical staining technique which allows the simultaneous demonstration of two different antigens expressed in or on the same cell (referred to as mixed labeling), together with the evaluation of the general histopathological appearance of the tissue. The staining procedure combines a three-step (streptavidin-biotin) immunogold-silver staining (IGSS) with a three-step immunoenzymatic labeling. For this purpose, we investigated the compatibility ofIGSS with various substrates of peroxidase or alkaline phosphatase (AP). Highly reliable and discernible mixed labeling was achieved only after iniriallabeling with IGSS followed by AP labeling using the substrates naphthol AS-MX phosphate/Fast Blue or naphthol AS-HI phosphate/New Fuchsin, respectively. To ensure utmost specificity, we applied FlTC-conjugated mouse monoclonal antibodies and rabbit anti-FlTC immunoglobulins visualized by AP-labeled immunoglobulins and the respective substrate in a final step. This novel approach provides an excellent means for demonstration of immunocompetent cells and unequivocal determination of the percentage of specific cell subsets in infiltrated tissue. The advantages of this method, as compared with double immunofluorescence or double immunoenzymatic labeling, were investigated and are discussed. (J Histochem Cytochem 38:307-313, 1990)}, language = {en} } @article{GholamiChenBelinetal.2013, author = {Gholami, Sepideh and Chen, Chun-Hao and Belin, Laurence J. and Lou, Emil and Fujisawa, Sho and Antonacci, Caroline and Carew, Amanda and Chen, Nanhai G. and De Brot, Marina and Zanzonico, Pat B. and Szalay, Aladar A. and Fong, Yuman}, title = {Vaccinia virus GLV-1h153 is a novel agent for detection and effective local control of positive surgical margins for breast cancer}, series = {Breast Cancer Research}, volume = {15}, journal = {Breast Cancer Research}, number = {R26}, doi = {10.1186/bcr3404}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-122140}, year = {2013}, abstract = {Introduction: Surgery is currently the definitive treatment for early-stage breast cancer. However, the rate of positive surgical margins remains unacceptably high. The human sodium iodide symporter (hNIS) is a naturally occurring protein in human thyroid tissue, which enables cells to concentrate radionuclides. The hNIS has been exploited to image and treat thyroid cancer. We therefore investigated the potential of a novel oncolytic vaccinia virus GLV1h-153 engineered to express the hNIS gene for identifying positive surgical margins after tumor resection via positron emission tomography (PET). Furthermore, we studied its role as an adjuvant therapeutic agent in achieving local control of remaining tumors in an orthotopic breast cancer model. Methods: GLV-1h153, a replication-competent vaccinia virus, was tested against breast cancer cell lines at various multiplicities of infection (MOIs). Cytotoxicity and viral replication were determined. Mammary fat pad tumors were generated in athymic nude mice. To determine the utility of GLV-1h153 in identifying positive surgical margins, 90\% of the mammary fat pad tumors were surgically resected and subsequently injected with GLV-1h153 or phosphate buffered saline (PBS) in the surgical wound. Serial Focus 120 microPET images were obtained six hours post-tail vein injection of approximately 600 mu Ci of I-124-iodide. Results: Viral infectivity, measured by green fluorescent protein (GFP) expression, was time-and concentrationdependent. All cell lines showed less than 10\% of cell survival five days after treatment at an MOI of 5. GLV-1h153 replicated efficiently in all cell lines with a peak titer of 27 million viral plaque forming units (PFU) ( < 10,000-fold increase from the initial viral dose) by Day 4. Administration of GLV-1h153 into the surgical wound allowed positive surgical margins to be identified via PET scanning. In vivo, mean volume of infected surgically resected residual tumors four weeks after treatment was 14 mm(3) versus 168 mm(3) in untreated controls (P < 0.05). Conclusions: This is the first study to our knowledge to demonstrate a novel vaccinia virus carrying hNIS as an imaging tool in identifying positive surgical margins of breast cancers in an orthotopic murine model. Moreover, our results suggest that GLV-1h153 is a promising therapeutic agent in achieving local control for positive surgical margins in resected breast tumors.}, language = {en} } @article{GerovaWickeChiharaetal.2021, author = {Gerova, Milan and Wicke, Laura and Chihara, Kotaro and Schneider, Cornelius and Lavigne, Rob and Vogel, J{\"o}rg}, title = {A grad-seq view of RNA and protein complexes in Pseudomonas aeruginosa under standard and bacteriophage predation conditions}, series = {mbio}, volume = {12}, journal = {mbio}, number = {1}, doi = {10.1128/mBio.03454-20}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259054}, pages = {e03454-20}, year = {2021}, abstract = {The Gram-negative rod-shaped bacterium Pseudomonas aeruginosa is not only a major cause of nosocomial infections but also serves as a model species of bacterial RNA biology. While its transcriptome architecture and posttranscriptional regulation through the RNA-binding proteins Hfq, RsmA, and RsmN have been studied in detail, global information about stable RNA-protein complexes in this human pathogen is currently lacking. Here, we implement gradient profiling by sequencing (Grad-seq) in exponentially growing P. aeruginosa cells to comprehensively predict RNA and protein complexes, based on glycerol gradient sedimentation profiles of >73\% of all transcripts and ∼40\% of all proteins. As to benchmarking, our global profiles readily reported complexes of stable RNAs of P. aeruginosa, including 6S RNA with RNA polymerase and associated product RNAs (pRNAs). We observe specific clusters of noncoding RNAs, which correlate with Hfq and RsmA/N, and provide a first hint that P. aeruginosa expresses a ProQ-like FinO domain-containing RNA-binding protein. To understand how biological stress may perturb cellular RNA/protein complexes, we performed Grad-seq after infection by the bacteriophage ΦKZ. This model phage, which has a well-defined transcription profile during host takeover, displayed efficient translational utilization of phage mRNAs and tRNAs, as evident from their increased cosedimentation with ribosomal subunits. Additionally, Grad-seq experimentally determines previously overlooked phage-encoded noncoding RNAs. Taken together, the Pseudomonas protein and RNA complex data provided here will pave the way to a better understanding of RNA-protein interactions during viral predation of the bacterial cell. IMPORTANCE Stable complexes by cellular proteins and RNA molecules lie at the heart of gene regulation and physiology in any bacterium of interest. It is therefore crucial to globally determine these complexes in order to identify and characterize new molecular players and regulation mechanisms. Pseudomonads harbor some of the largest genomes known in bacteria, encoding ∼5,500 different proteins. Here, we provide a first glimpse on which proteins and cellular transcripts form stable complexes in the human pathogen Pseudomonas aeruginosa. We additionally performed this analysis with bacteria subjected to the important and frequently encountered biological stress of a bacteriophage infection. We identified several molecules with established roles in a variety of cellular pathways, which were affected by the phage and can now be explored for their role during phage infection. Most importantly, we observed strong colocalization of phage transcripts and host ribosomes, indicating the existence of specialized translation mechanisms during phage infection. All data are publicly available in an interactive and easy to use browser.}, language = {en} } @article{GentschevMuellerAdelfingeretal.2011, author = {Gentschev, Ivaylo and M{\"u}ller, Meike and Adelfinger, Marion and Weibel, Stephanie and Grummt, Friedrich and Zimmermann, Martina and Bitzer, Michael and Heisig, Martin and Zhang, Qian and Yu, Yong A. and Chen, Nanhai G. and Stritzker, Jochen and Lauer, Ulrich M. and Szalay, Aladar A.}, title = {Efficient Colonization and Therapy of Human Hepatocellular Carcinoma (HCC) Using the Oncolytic Vaccinia Virus Strain GLV-1h68}, series = {PLOS ONE}, volume = {6}, journal = {PLOS ONE}, number = {7}, doi = {10.1371/journal.pone.0022069}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-135319}, pages = {e22069}, year = {2011}, abstract = {Virotherapy using oncolytic vaccinia virus strains is one of the most promising new strategies for cancer therapy. In this study, we analyzed for the first time the therapeutic efficacy of the oncolytic vaccinia virus GLV-1h68 in two human hepatocellular carcinoma cell lines HuH7 and PLC/PRF/5 (PLC) in cell culture and in tumor xenograft models. By viral proliferation assays and cell survival tests, we demonstrated that GLV-1h68 efficiently colonized, replicated in, and did lyse these cancer cells in culture. Experiments with HuH7 and PLC xenografts have revealed that a single intravenous injection (i.v.) of mice with GLV-1h68 resulted in a significant reduction of primary tumor sizes compared to uninjected controls. In addition, replication of GLV-1h68 in tumor cells led to strong inflammatory and oncolytic effects resulting in intense infiltration of MHC class II-positive cells like neutrophils, macrophages, B cells and dendritic cells and in up-regulation of 13 pro-inflammatory cytokines. Furthermore, GLV-1h68 infection of PLC tumors inhibited the formation of hemorrhagic structures which occur naturally in PLC tumors. Interestingly, we found a strongly reduced vascular density in infected PLC tumors only, but not in the non-hemorrhagic HuH7 tumor model. These data demonstrate that the GLV-1h68 vaccinia virus may have an enormous potential for treatment of human hepatocellular carcinoma in man.}, language = {en} } @article{GentschevAdelfingerJosupeitetal.2012, author = {Gentschev, Ivaylo and Adelfinger, Marion and Josupeit, Rafael and Rudolph, Stephan and Ehrig, Klaas and Donat, Ulrike and Weibel, Stephanie and Chen, Nanhai G. and Yu, Yong A. and Zhang, Qian and Heisig, Martin and Thamm, Douglas and Stritzker, Jochen and MacNeill, Amy and Szalay, Aladar A.}, title = {Preclinical Evaluation of Oncolytic Vaccinia Virus for Therapy of Canine Soft Tissue Sarcoma}, series = {PLoS One}, volume = {7}, journal = {PLoS One}, number = {5}, doi = {10.1371/journal.pone.0037239}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-129998}, year = {2012}, abstract = {Virotherapy using oncolytic vaccinia virus (VACV) strains is one promising new strategy for canine cancer therapy. In this study we describe the establishment of an in vivo model of canine soft tissue sarcoma (CSTS) using the new isolated cell line STSA-1 and the analysis of the virus-mediated oncolytic and immunological effects of two different Lister VACV LIVP1.1.1 and GLV-1h68 strains against CSTS. Cell culture data demonstrated that both tested VACV strains efficiently infected and destroyed cells of the canine soft tissue sarcoma line STSA-1. In addition, in our new canine sarcoma tumor xenograft mouse model, systemic administration of LIVP1.1.1 or GLV-1h68 viruses led to significant inhibition of tumor growth compared to control mice. Furthermore, LIVP1.1.1 mediated therapy resulted in almost complete tumor regression and resulted in long-term survival of sarcoma-bearing mice. The replication of the tested VACV strains in tumor tissues led to strong oncolytic effects accompanied by an intense intratumoral infiltration of host immune cells, mainly neutrophils. These findings suggest that the direct viral oncolysis of tumor cells and the virus-dependent activation of tumor-associated host immune cells could be crucial parts of anti-tumor mechanism in STSA-1 xenografts. In summary, the data showed that both tested vaccinia virus strains and especially LIVP1.1.1 have great potential for effective treatment of CSTS.}, language = {en} } @phdthesis{Geis2005, author = {Geis, Tanja}, title = {Molekularbiologische und immunologische Untersuchungen zu klinisch relevanten immunodominanten Antigenen von Methicillin-resistenten Staphylococcus aureus-Bakterien (MRSA) f{\"u}r eine Antik{\"o}rpertherapie}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-18108}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2005}, abstract = {Zusammenfassung: Staphylococcus aureus ist einer der h{\"a}ufigsten Erreger von nosokomialen Infektionen. Die grampositiven in Haufen angeordneten kokkoiden Bakterien verursachen neben harmlosen lokal-oberfl{\"a}chlichen Hautinfektionen auch gef{\"u}rchtete lebensbedrohliche Systeminfektionen. Ein großes Problem in der Therapie von S. aureus-Infektionen stellen die zunehmenden Multiresistenzen dieses Erregers dar. Die Entwicklung neuer Antibiotika reicht hierbei auf Dauer oft nicht aus, da immer wieder neue Resistenzen der Bakterien zu erwarten sind. Es besteht daher dringender Bedarf an der Entwicklung alternativer Therapieformen im Kampf gegen multiresistente Problemkeime wie S. aureus. Eine M{\"o}glichkeit besteht in der Immuntherapie, zum Beispiel durch Gewinnung von monoklonalen Antik{\"o}rpern gegen geeignete Targetstrukturen von S. aureus. In den beiden immundominanten Antigenen IsaA und IsaB scheinen solche geeigneten Angriffsstrukturen gefunden zu sein. In dieser Arbeit wurde eine IsaB-Mutante hergestellt und nachfolgend ph{\"a}notypisch charakterisiert. Diese Arbeiten bilden die Grundlage f{\"u}r die Aufkl{\"a}rung der Funktion von IsaB. Weiterhin sollte ein humaner monoklonaler Antik{\"o}rper gegen IsaA generiert werden. Zur Testung der Antigenspezifit{\"a}t war es notwendig einen anti-IsaA-spezifischen ELISA zu entwickeln. Durch die Fusion der Hybridomzelllinie HAB mit B-Lymphozyten aus lymphatischem Gewebe septik{\"a}mischer Patienten sollten Fusionszellen gewonnen werden, die spezifische anti-IsaA-Antik{\"o}rper produzieren. Durch den Einsatz einer humanen Hybridomzelllinie sollten Kreuzspezies-Reaktionen, wie sie bei murinen therapeutischen Antik{\"o}rpern beobachtet wurden, ausgeschaltet werden. Es wurden mehrere Fusionen mit lymphatischem Material (Lymphknoten, Blut-B-Lymphozyten) durchgef{\"u}hrt. Eine spezifische Antik{\"o}rperproduktion blieb hierbei jedoch aus. Ein anti-IsaA-spezifischer ELISA konnte auf der Grundlage polyklonaler anti-IsaA-Antik{\"o}rper aus immunisierten Kaninchen erfolgreich etabliert werden. Der ELISA zeigte die h{\"o}chste Spezifit{\"a}t bei einer Antigenbeschichtung von 2 µg/ml IsaA und einer optimalen Prim{\"a}rantik{\"o}rperkonzentration von 1 : 25600. In einem weiteren Teil der Arbeit sollte die Spezifit{\"a}t des polyklonalen Antik{\"o}rpers und das Vorkommen von IsaA bei verschiedenen Staphylococcus aureus-St{\"a}mmen und anderen Bakterienspezies getestet werden. Dazu wurde mittels Western-Blot die Expression von IsaA untersucht. Ergebnis dieser Untersuchung war, dass IsaA unabh{\"a}ngig von der Herkunft, vom Infektionstyp oder Resistenzeigenschaften von allen S. aureus-Isolaten exprimiert wird. Von den anderen untersuchten Bakteriespezies zeigte der IsaA-spezifische polyklonale Antik{\"o}rper auch eine Reaktion gegen S. haemolyticus, S. hominis, S. lugdunensis, S. warnerii und S. cohnii. Ob diese Staphylokokkenarten ein IsaA-homologes Protein mit konservierten Dom{\"a}nen exprimieren, das mit dem Antiserum kreuzreagiert, m{\"u}ssen zuk{\"u}nftige Untersuchungen zeigen. Die Herstellung der IsaB-Mutante erfolgte durch Konstruktion eines Inaktivierungsplasmides (pTG1), in dem eine Erythromycinkassette zwischen ein „upstream"- und „downstream"-Fragment kloniert wurde. Als Grundlage diente der temperatursensitive „shuttle"-Vektor pBT2, der bei erh{\"o}hter Temperatur in S. aureus nicht weiter replizieren kann und dadurch homologe Fragmente auf dem Vektor mit genomischen Abschnitten rekombinieren. Die erfolgreicher Herstellung der isaB-Mutante wurde im Southern-Blot {\"u}berpr{\"u}ft. Die Mutante wurde verschiedenen vergleichenden Experimenten mit dem Wildtyp unterzogen, um Informationen {\"u}ber die m{\"o}gliche Funktion des Targetproteins IsaB im Hinblick auf Wachstum und Virulenz f{\"u}r S. aureus zu gewinnen. Die Wachstumsexperimente ergaben einen deutlichen Unterschied im Wachstumsverhalten der Mutante im Vergleich zum Wildtyp. Da die Mutante deutlich schneller wuchs als der Wildtyp scheint IsaB wichtig f{\"u}r das Wachstum von S. aureus zu sein. In vitro-Experimente zur Testung von m{\"o}glichen in vivo-Umwelteinfl{\"u}ssen bzw. Stressfaktoren auf das Wachsum der St{\"a}mme erfolgten mittels Zusetzung von Glucose und NaCl. Hier zeigte sich ein deutlicher Wachstumsvorteil des Wildtyps gegen{\"u}ber der isaB-Mutante, so dass unter extremen Umweltbedingungen IsaB S. aureus ein besseres Wachsen erm{\"o}glicht. In weiteren Experimenten wurden das H{\"a}molyse- und Proteolyseverhalten der Mutante im Vergleich zum Wildtyp untersucht. IsaB scheint Einfluss auf die Expression von H{\"a}molysinen und Proteasen zu haben, die wichtige Virulenzfaktoren von S. aureus sind. Die Untersuchungen zur Bestimmung der minimalen Hemmkonzentration (MHK) von Wildtyp und Mutante ergaben ebenfalls Unterschiede f{\"u}r verschiedene Antibiotika, so dass IsaB Einfluss auf die Antibiotikaempfindlichkeit zu nehmen scheint. Die in dieser Arbeit erzielten Ergebnisse verdeutlichen die prinzipielle Eignung von IsaB als Targetprotein f{\"u}r eine Antik{\"o}rpertherapie bei lebensbedrohlichen S. aureus-Infektionen.}, language = {de} } @article{GehrmannHertleinHopkeetal.2021, author = {Gehrmann, Robin and Hertlein, Tobias and Hopke, Elisa and Ohlsen, Knut and Lalk, Michael and Hilgeroth, Andreas}, title = {Novel small-molecule hybrid-antibacterial agents against S. aureus and MRSA strains}, series = {Molecules}, volume = {27}, journal = {Molecules}, number = {1}, issn = {1420-3049}, doi = {10.3390/molecules27010061}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-252371}, year = {2021}, abstract = {Ongoing resistance developments against antibiotics that also affect last-resort antibiotics require novel antibacterial compounds. Strategies to discover such novel structures have been dimerization or hybridization of known antibacterial agents. We found novel antibacterial agents by dimerization of indols and hybridization with carbazoles. They were obtained in a simple one-pot reaction as bisindole tetrahydrocarbazoles. Further oxidation led to bisindole carbazoles with varied substitutions of both the indole and the carbazole scaffold. Both the tetrahydrocarbazoles and the carbazoles have been evaluated in various S. aureus strains, including MRSA strains. Those 5-cyano substituted derivatives showed best activities as determined by MIC values. The tetrahydrocarbazoles partly exceed the activity of the carbazole compounds and thus the activity of the used standard antibiotics. Thus, promising lead compounds could be identified for further studies.}, language = {en} } @article{GarciaBetancurGoniMorenoHorgeretal.2017, author = {Garc{\´i}a-Betancur, Juan-Carlos and Go{\~n}i-Moreno, Angel and Horger, Thomas and Schott, Melanie and Sharan, Malvika and Eikmeier, Julian and Wohlmuth, Barbara and Zernecke, Alma and Ohlsen, Knut and Kuttler, Christina and Lopez, Daniel}, title = {Cell differentiation defines acute and chronic infection cell types in Staphylococcus aureus}, series = {eLife}, volume = {6}, journal = {eLife}, number = {e28023}, doi = {10.7554/eLife.28023}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170346}, year = {2017}, abstract = {A central question to biology is how pathogenic bacteria initiate acute or chronic infections. Here we describe a genetic program for cell-fate decision in the opportunistic human pathogen Staphylococcus aureus, which generates the phenotypic bifurcation of the cells into two genetically identical but different cell types during the course of an infection. Whereas one cell type promotes the formation of biofilms that contribute to chronic infections, the second type is planktonic and produces the toxins that contribute to acute bacteremia. We identified a bimodal switch in the agr quorum sensing system that antagonistically regulates the differentiation of these two physiologically distinct cell types. We found that extracellular signals affect the behavior of the agr bimodal switch and modify the size of the specialized subpopulations in specific colonization niches. For instance, magnesium-enriched colonization niches causes magnesium binding to S. aureusteichoic acids and increases bacterial cell wall rigidity. This signal triggers a genetic program that ultimately downregulates the agr bimodal switch. Colonization niches with different magnesium concentrations influence the bimodal system activity, which defines a distinct ratio between these subpopulations; this in turn leads to distinct infection outcomes in vitro and in an in vivo murine infection model. Cell differentiation generates physiological heterogeneity in clonal bacterial infections and helps to determine the distinct infection types.}, language = {en} } @phdthesis{Galka2007, author = {Galka, Frank}, title = {Untersuchungen zum Proteom und zur Funktion von sekretierten Proteinen und {\"a}ußeren Membranvesikeln von Legionella pneumophila}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-27075}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2007}, abstract = {Das Gram-negative Bakterium Legionella pneumophila ist der Haupterreger der humanen Legion{\"a}rskrankheit, einer schweren atypischen Pneumonie. Aufgrund mangelnder Diagnostik bleibt L. pneumophila als Krankheitsverursacher jedoch oft unerkannt. Neuesten Sch{\"a}tzungen des Kompetenznetzwerkes f{\"u}r ambulant erworbene Pneumonien (CAPNETZ) zufolge k{\"o}nnten Legionellen in Deutschland f{\"u}r j{\"a}hrlich ca. 21 000 Pneumonien verantwortlich sein, etwa doppelt so viele F{\"a}lle wie bisher angenommen. Die Pathologie der humanen Infektion zeichnet sich durch extrazellul{\"a}re Effekte aus, f{\"u}r die in den letzten Jahren vielf{\"a}ltige sekretierte Effektormolek{\"u}le (SSPs) verantwortlich gemacht wurden. Dar{\"u}ber hinaus tragen spezielle Sekretionsmaschinen wie das Dot/Icm Typ-IV-Sekretionssystem sowie ersten Hinweisen entsprechend Membranvesikel, die von der {\"a}ußeren Membran der Bakterien abgeschn{\"u}rt werden (OMVs), zur intrazellul{\"a}ren Pathogenit{\"a}t von L. pneumophila bei. In der vorliegenden Dissertation bildet die umfassende Charakterisierung des Sekretoms von L. pneumophila den Schwerpunkt. Diese ist untergliedert in (i) Untersuchungen zur OMV-Produktion im Lebenszyklus von L. pneumophila, (ii) Proteomcharakterisierung der Sekretomfraktionen SSP und OMV und (iii) funktionale Analyse der Sekretomfraktionen. F{\"u}r einen Beitrag von OMVs zur L. pneumophila-Pathogenese ist deren Produktion w{\"a}hrend extra- und intrazellul{\"a}ren Wachstums essentiell. Mit Hilfe verschiedener Mikroskopie-Techniken wird in dieser Dissertation gezeigt, dass die Abschn{\"u}rung von OMVs sowohl extrazellul{\"a}r als auch intrazellul{\"a}r in Legionella-spezifischen Phagosomen stattfindet und von einer intakten Bakterienmembran erfolgt. Des Weiteren werden OMVs nicht nur w{\"a}hrend der exponentiellen, sondern auch w{\"a}hrend der station{\"a}ren Phase produziert. Diese Beobachtung ist bedeutend, weil sich L. pneumophila w{\"a}hrend der postexponentiellen Phase in die transmissive Form mit voller Virulenz differenziert und sich der Wechsel in die virulente Form folglich auch in der Zusammensetzung der OMVs widerspiegeln k{\"o}nnte. Der zweite Teil besch{\"a}ftigt sich mit der Proteomanalyse der Sekretomfraktionen. Die Proteinidentifikation ergab 181 nicht-redundante Proteine im L. pneumophila-Sekretom, von denen 107 f{\"u}r die SSP-Fraktion und 33 f{\"u}r die OMV-Fraktion hochspezifisch sind. In beiden Fraktionen sind insgesamt 22 Typ-II-Sekretionssubstrate enthalten, die verschiedene degradierende Enzymaktivit{\"a}ten aufweisen. Außerdem wurden 38 bisher putative Typ-II-Substrate, 3 Typ-IV-Substrate und 7 Eukaryoten-{\"a}hnliche Proteine detektiert. Die Analyse der Verteilung der Proteine zeigt, dass der prozentuale Anteil der „Virulenz-/Pathogenese"-Proteine in der OMV-Fraktion mit 24\% gegen{\"u}ber 11\% in der SSP-Fraktion mehr als doppelt so hoch liegt. Acht Faktoren, u. a. das Mip-Protein, einer der Haupt-Virulenzfaktoren von L. pneumophila, sind nur auf OMVs beschr{\"a}nkt. Dies k{\"o}nnte darauf hindeuten, dass OMVs als spezifische Transportmittel f{\"u}r Virulenz-assoziierte Effektoren dienen. In der funktionalen Analyse der SSP- und OMV-Fraktionen wurden anhand verschiedener Techniken Aspekte untersucht, die w{\"a}hrend des Infektionsprozesses eine Rolle spielen. Dabei zeigt sich, dass SSPs und OMVs proteo- und lipolytische Enzymaktivit{\"a}ten besitzen, die zur Zerst{\"o}rung der Alveolaroberfl{\"a}che, zur Transmigration der Bakterien durch Lungenepithelbarriere und Basallamina und letztendlich zur Ausbreitung von L. pneumophila im Lungengewebe und zur Milz beitragen k{\"o}nnten. Jedoch konnten f{\"u}r OMVs keine naheliegenden zytotoxischen oder zytolytischen Eigenschaften nachgewiesen werden. In Alveolarepithelzellen k{\"o}nnen sie ein spezifisches Zytokinsekretionsprofil induzieren, was ihre modulierenden Effekte auf Wirtszellen best{\"a}tigt. Die gezeigte Bindung von OMVs an Alveolarepithelzellen bildet die Voraussetzung f{\"u}r eine Interaktion mit den Wirtszellen. Ob dabei eine Fusion mit der Zytoplasmamembran und ein m{\"o}glicher Transfer von Effektoren in die Wirtszelle stattfinden, bleibt zu kl{\"a}ren. Abschließend werden diskutierte Funktionen sekretierter OMVs w{\"a}hrend der L. pneumophila-Infektion in einem Modell zusammengefasst. Diese neuen Ergebnisse zum Proteom des Sekretoms und zur Funktion von L. pneumophila-OMVs tragen zum besseren Verst{\"a}ndnis der Interaktion von L. pneumophila mit seiner Umwelt und der Pathogenese bei. Gleichzeitig liefern sie eine wichtige theoretische Grundlage f{\"u}r zuk{\"u}nftige Forschungsarbeiten {\"u}ber Interaktionsprozesse und beteiligter Effektoren, deren tiefgreifendes Verst{\"a}ndnis die Vorraussetzung f{\"u}r die Entwicklung neuer Strategien in der Therapie von Legionella-Infektionen bildet.}, language = {de} } @article{FoerstnerReuscherHaberzettletal.2018, author = {F{\"o}rstner, Konrad U and Reuscher, Carina M and Haberzettl, Kerstin and Weber, Lennart and Klug, Gabriele}, title = {RNase E cleavage shapes the transcriptome of Rhodobacter sphaeroides and strongly impacts phototrophic growth}, series = {Life Science Alliance}, volume = {1}, journal = {Life Science Alliance}, number = {4}, doi = {10.26508/lsa.201800080}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-177139}, pages = {e201800080}, year = {2018}, abstract = {Bacteria adapt to changing environmental conditions by rapid changes in their transcriptome. This is achieved not only by adjusting rates of transcription but also by processing and degradation of RNAs. We applied TIER-Seq (transiently inactivating an endoribonuclease followed by RNA-Seq) for the transcriptome-wide identification of RNase E cleavage sites and of 5′ RNA ends, which are enriched when RNase E activity is reduced in Rhodobacter sphaeroides. These results reveal the importance of RNase E for the maturation and turnover of mRNAs, rRNAs, and sRNAs in this guanine-cytosine-rich α-proteobacterium, some of the latter have well-described functions in the oxidative stress response. In agreement with this, a role of RNase E in the oxidative stress response is demonstrated. A remarkably strong phenotype of a mutant with reduced RNase E activity was observed regarding the formation of photosynthetic complexes and phototrophic growth, whereas there was no effect on chemotrophic growth.}, language = {en} } @inproceedings{FoerstnerHagedornKoltzenburgetal.2011, author = {F{\"o}rstner, Konrad and Hagedorn, Gregor and Koltzenburg, Claudia and Kubke, Fabiana and Mietchen, Daniel}, title = {Collaborative platforms for streamlining workflows in Open Science}, series = {Proceedings of the 6th Open Knowledge Conference}, booktitle = {Proceedings of the 6th Open Knowledge Conference}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-101678}, year = {2011}, abstract = {Despite the internet's dynamic and collaborative nature, scientists continue to produce grant proposals, lab notebooks, data files, conclusions etc. that stay in static formats or are not published online and therefore not always easily accessible to the interested public. Because of limited adoption of tools that seamlessly integrate all aspects of a research project (conception, data generation, data evaluation, peerreviewing and publishing of conclusions), much effort is later spent on reproducing or reformatting individual entities before they can be repurposed independently or as parts of articles. We propose that workflows - performed both individually and collaboratively - could potentially become more efficient if all steps of the research cycle were coherently represented online and the underlying data were formatted, annotated and licensed for reuse. Such a system would accelerate the process of taking projects from conception to publication stages and allow for continuous updating of the data sets and their interpretation as well as their integration into other independent projects. A major advantage of such work ows is the increased transparency, both with respect to the scientific process as to the contribution of each participant. The latter point is important from a perspective of motivation, as it enables the allocation of reputation, which creates incentives for scientists to contribute to projects. Such work ow platforms offering possibilities to fine-tune the accessibility of their content could gradually pave the path from the current static mode of research presentation into a more coherent practice of open science.}, language = {en} } @phdthesis{Fuchs2000, author = {Fuchs, Sibylle Maria}, title = {Untersuchungen zur Regulation von Shiga-Toxin 2 und zur Attenuierung von enteroh{\"a}morrhagischen Escherichia coli}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-1303}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2000}, abstract = {Enteroh{\"a}morrhagische Escherichia coli (EHEC) geh{\"o}ren zu den wichtigsten der sich in j{\"u}ngster Zeit verbreitenden Pathogene und verursachen die verschiedensten Durchfallerkrankungen von unblutiger Diarrh{\"o} bis zu h{\"a}morrhagischer Kolitis, oftmals unter Auspr{\"a}gung von lebensbedrohlichen extraintestinalen Symptomen wie dem h{\"a}molytisch-ur{\"a}mischen Syndrom. Die wichtigsten Virulenzfaktoren dieser Pathogene sind Shiga-Toxine (Stx) und Faktoren, die an der Auspr{\"a}gung der sog. "attaching and effacing"-L{\"a}sionen auf Darmepithelzellen beteiligt sind. Vor allem Kinder und {\"a}ltere Menschen sind von den Infektionen, die h{\"a}ufig in Form von Ausbr{\"u}chen auftreten, betroffen. Die {\"U}bertragung erfolgt meist {\"u}ber f{\"a}kal kontaminierte Nahrungsmittel. Da die Behandlung von EHEC-Infektionen mit manchen Antibiotika die Entwicklung der extraintestinalen Symptome noch verst{\"a}rken kann, w{\"a}re die Impfung gef{\"a}hrdeter Personen der beste Weg f{\"u}r die Bek{\"a}mpfung dieser Erreger. Eine weitere M{\"o}glichkeit der Pr{\"a}vention w{\"a}re die Eradikation dieser Organismen in ihren asymptomatischen Wirten, {\"u}ber die EHEC in die menschliche Nahrungskette gelangen k{\"o}nnen. Das Ziel der vorliegenden Arbeit war unter anderem die Etablierung der Grundlagen f{\"u}r einen Lebendvakzinstamm zur Pr{\"a}vention von EHEC-Infektionen. Zu diesem Zweck wurden unterschiedliche Strategien mit dem Ziel verfolgt, einen Stx2-produzierenden EHEC-Stamm zu attenuieren. Eine Attenuierungsstrategie f{\"u}r EHEC ist die direkte Ausschaltung von Virulenzfaktor-Strukturgenen wie den Toxingenen. Zu diesem Zweck wurde eine stx2-negative Mutante des EHEC-Stammes O157:H7 86-24 durch eine Deletion im Zentrum des stx2-Genclusters konstruiert, was zur Fusion der 154 N-terminalen Aminos{\"a}uren von StxA2 mit den 62 C-terminalen Aminos{\"a}uren von StxB2 f{\"u}hrte. Die Charakterisierung der Mutante zeigte, daß der Toxin-konvertierende Bakteriophage noch intakt war. Das Fusionsprotein hatte seine zytotoxische Aktivit{\"a}t zwar vollst{\"a}ndig verloren, konnte jedoch durch Stx2-spezifisches Schweineantiserum detektiert werden. Daraus wurde geschlossen, daß das mutierte Protein einen Teil seiner antigenen Strukturen behalten hatte und daß es potentiell f{\"u}r die Impfung gegen Stx2-spezifische Sch{\"a}digungen verwendet werden k{\"o}nnte. Eine weitere Strategie mit dem Ziel der Attenuierung von EHEC-St{\"a}mmen war die Deletion von Genen, die in die Regulation von Virulenzfaktoren involviert sind. Auf diese Weise sollte die Expression von Pathogenit{\"a}tsfaktoren verhindert werden. Als erstes wurde versucht, einen postulierten bakteriophagenkodierten toxinspezifischen Regulator zu identifizieren und zu charaktierisieren, der die F{\"a}higkeit besaß, die Expression eines stx2-spezifischen Reportergens nach der Induktion des Phagen zu steigern. Eine Transposonmutagenese des Stx2-konvertierenden Phagen 933W ergab verschiedene Phagenmutanten mit ver{\"a}nderter Expression des Reportergens nach Induktion des Phagen. Die Expressionsver{\"a}nderung korrelierte nur bedingt mit der Ver{\"a}nderung der Produktion von Toxin oder Phagenpartikeln. Das Transposon der am st{\"a}rksten in ihrer Reportergenexpression reduzierten Mutante war im ORF L0065 inseriert, der unmittelbar "upstream" von den Phagengenen int/xis lokalisiert ist. Der klonierte wildtypische ORF war nicht in der Lage, die Transposonmutante in trans zu komplementieren. Daraus wurde geschlossen, daß der Ph{\"a}notyp der Mutante durch einen polaren Effekt des Transposons auf int/xis bedingt sein k{\"o}nnte, da eine reduzierte Phagengenomexcision eine Verminderung der Phageninduktion verursachen w{\"u}rde, was sich entsprechend auf die Reportergenexpression auswirken k{\"o}nnte. Neely et al. (1998) identifizierten den Phagen-Antiterminator Q als einen m{\"o}glichen Kandidaten f{\"u}r den postulierten phagenkodierten stx2-Regulator. Eine Deletion dieses zentralen Phagenregulators k{\"o}nnte durch die St{\"o}rung der regul{\"a}ren Phagenfunktionen zur Attenuierung von EHEC beitragen. Als zweites wurde in einem Projekt von Dr. I. M{\"u}hldorfer anhand von recA-negativen Mutanten der EHEC-St{\"a}mme O157:H7 86-24 und EDL933 in verschiedenen M{\"a}usemodellen demonstriert, daß die Deletion von recA einen massiven Virulenzverlust und damit eine Attenuierung der St{\"a}mme zur Folge hatte. Die dadurch bedingte drastische Reduktion der Toxinproduktion konnte indirekt auf das Fehlen von recA zur{\"u}ckgef{\"u}hrt werden. Im Gegensatz dazu ver{\"a}nderte die Deletion von recA im UPEC-Stamm O6:K15:H31 536 die Virulenz dieses Stammes nicht. Im Rahmen dieser Arbeit erfolgte die Auswertung der Ergebnisse der Virulenztests. Die Deletion von recA ist außerdem eine wichtige Sicherheitsmaßnahme f{\"u}r eine Pr{\"a}vention der Integration von Fremd-DNA in Lebendvakzine und damit f{\"u}r die Verhinderung der Reversion dieser St{\"a}mme zur Pathogenit{\"a}t. Als drittes wurden die Auswirkungen der Deletion des Gens leuX, das f{\"u}r die seltenere Leucin-spezifische tRNA5Leu kodiert, auf die Expression von EHEC-Virulenzfaktoren anhand einer leuX-Deletionsmutante des EHEC-Stammes O157:H7 86-24 untersucht. Die Deletion dieser tRNA im UPEC-Stamm 536 f{\"u}hrt wegen der dadurch reduzierten Expression verschiedener Virulenzfaktoren zu einer Attenuierung des Stammes. Es wurde gezeigt, daß wie in UPEC auch in EHEC die Produktion von Flagellen und Enterobaktin beeintr{\"a}chtigt war. Zus{\"a}tzlich war die H{\"a}minverwertung reduziert. Außerdem verminderte die Deletion von leuX die Expression nicht-identifizierter Proteine der {\"a}ußeren und inneren Membran sowie eines mit Typ 1-Fimbrien-spezifischem Serum kreuzreaktiven Antigens. Im Gegensatz dazu wurden die Stx2-Produktion sowie die in vivo-Virulenz des Stammes in M{\"a}usen nicht beeinflußt. Die Enteroh{\"a}molyse sowie die Expression von Intimin waren verst{\"a}rkt. Die Expression der typischen EHEC-Virulenzfaktoren war demnach in der leuX-Mutante nicht reduziert. Der Einfluß von leuX auf die Expression dieser Faktoren war offensichtlich nicht auf eine Translationsreduktion durch die fehlende Bereitstellung der tRNA beschr{\"a}nkt, sondern scheint weitere Mechanismen zu involvieren. Eine wirkliche Attenuierung von EHEC kann durch die Deletion von leuX wahrscheinlich nicht erzielt werden.}, subject = {EHEC}, language = {de} } @phdthesis{Fuchs2006, author = {Fuchs, Maura-Maria}, title = {Ph{\"a}no- und genotypische Charakterisierung extraintestinal pathogener E.coli St{\"a}mme}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-22050}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2006}, abstract = {Ziel der vorliegenden Arbeit war die Untersuchung der genetischen Variabilit{\"a}t verschiedener uropathogener E. coli St{\"a}mme. Diese wurden mittels ph{\"a}notypischer Tests, Multiplex-PCR, Pulsfeldgelelektophorese und DNA-DNA Hybridisierung unter Verwendung zweier verschiedener DNA-Makroarrays durchgef{\"u}hrt. Die Stammauswahl umfasste 12 uropathogene und f{\"a}kale Isolate von Patientinnen mit chronischen Harnwegsinfektionen. Die St{\"a}mme wurden zu unterschiedlichen Zeiten der Infektion abgenommen und schon in Vorg{\"a}ngerarbeiten genauer ph{\"a}notypisch und genotypisch untersucht. Es wurde in diesem Zusammenhang u. a. vermutet, dass sich die St{\"a}mme im fortschreitenden Verlauf der Infektion m{\"o}glicherweise in ihrer genetischen Ausstattung ver{\"a}ndert hatten. Der Gegenstand dieser Arbeit war nun der Ausschluss von Kontaminationen oder Mischkulturen unter diesen 12 St{\"a}mmen und die genauere Untersuchung der Genomver{\"a}nderungen bzw. Genomplastizit{\"a}t im Verlauf der Infektion. Neben diesen St{\"a}mmen wurden 18 weitere St{\"a}mme ausgew{\"a}hlt. Neun davon waren ah{\"a}molytische Mutanten der St{\"a}mme 536, 764 und 768. Durch den Vergleich mit dem jeweiligen Wildtyp sollte gekl{\"a}rt werden, ob sich ein identischer Mechanismus hinter dem Verlust der H{\"a}molysef{\"a}higkeit (Deletion einer PAI durch site-spezifische Rekombination) finden l{\"a}sst oder jede dieser Mutanten verschiedene eher zuf{\"a}llige Deletionen aufweist. Des weiteren wurden drei uropathogene Isolate untersucht, die die F{\"a}higkeit zur Bildung von Curli verloren haben sowie zwei UPEC St{\"a}mme, bei denen unter Antibiotikaeinfluss die Bildung von L-Formen beobachtet wurde. Durch den Einsatz von DNA-Arrays sollte auf Genomebene nach m{\"o}glichen Ursachen f{\"u}r die jeweiligen Ph{\"a}notypen gesucht werden.}, language = {de} } @article{FroehlichPapenfortBergeretal.2012, author = {Fr{\"o}hlich, Kathrin S. and Papenfort, Kai and Berger, Allison A. and Vogel, J{\"o}rg}, title = {A conserved RpoS-dependent small RNA controls the synthesis of major porin OmpD}, series = {Nucleic Acids Research}, volume = {40}, journal = {Nucleic Acids Research}, number = {8}, doi = {10.1093/nar/gkr1156}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-134230}, pages = {3623-3640}, year = {2012}, abstract = {A remarkable feature of many small non-coding RNAs (sRNAs) of Escherichia coli and Salmonella is their accumulation in the stationary phase of bacterial growth. Several stress response regulators and sigma factors have been reported to direct the transcription of stationary phase-specific sRNAs, but a widely conserved sRNA gene that is controlled by the major stationary phase and stress sigma factor, Sigma(S) (RpoS), has remained elusive. We have studied in Salmonella the conserved SdsR sRNA, previously known as RyeB, one of the most abundant stationary phase-specific sRNAs in E. coli. Alignments of the sdsR promoter region and genetic analysis strongly suggest that this sRNA gene is selectively transcribed by Sigma(S). We show that SdsR down-regulates the synthesis of the major Salmonella porin OmpD by Hfq-dependent base pairing; SdsR thus represents the fourth sRNA to regulate this major outer membrane porin. Similar to the InvR, MicC and RybB sRNAs, SdsR recognizes the ompD mRNA in the coding sequence, suggesting that this mRNA may be primarily targeted downstream of the start codon. The SdsR-binding site in ompD was localized by 3'-RACE, an experimental approach that promises to be of use in predicting other sRNA-target interactions in bacteria.}, language = {en} } @article{FroehlichHanekePapenfortetal.2016, author = {Fr{\"o}hlich, Kathrin S. and Haneke, Katharina and Papenfort, Kai and Vogel, J{\"o}rg}, title = {The target spectrum of SdsR small RNA in Salmonella}, series = {Nucleic Acids Research}, volume = {44}, journal = {Nucleic Acids Research}, number = {21}, doi = {10.1093/nar/gkw632}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-175365}, pages = {10406-10422}, year = {2016}, abstract = {Model enteric bacteria such as Escherichia coli and Salmonella enterica express hundreds of small non-coding RNAs (sRNAs), targets for most of which are yet unknown. Some sRNAs are remarkably well conserved, indicating that they serve cellular functions that go beyond the necessities of a single species. One of these 'core sRNAs' of largely unknown function is the abundant ∼100-nucleotide SdsR sRNA which is transcribed by the general stress σ-factor, σ\(^{S}\) and accumulates in stationary phase. In Salmonella, SdsR was known to inhibit the synthesis of the species-specific porin, OmpD. However, sdsR genes are present in almost all enterobacterial genomes, suggesting that additional, conserved targets of this sRNA must exist. Here, we have combined SdsR pulse-expression with whole genome transcriptomics to discover 20 previously unknown candidate targets of SdsR which include mRNAs coding for physiologically important regulators such as the carbon utilization regulator, CRP, the nucleoid-associated chaperone, StpA and the antibiotic resistance transporter, TolC. Processing of SdsR by RNase E results in two cellular SdsR variants with distinct target spectra. While the overall physiological role of this orphan core sRNA remains to be fully understood, the new SdsR targets present valuable leads to determine sRNA functions in resting bacteria.}, language = {en} } @phdthesis{Froehlich2012, author = {Fr{\"o}hlich, Kathrin}, title = {Assigning functions to Hfq-dependent small RNAs in the model pathogen Salmonella Typhimurium}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85488}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Non-coding RNAs constitute a major class of regulators involved in bacterial gene expression. A group of riboregulators of heterogeneous size and shape referred to as small regulatory RNAs (sRNAs) control trans- or cis-encoded genes through direct base-pairing with their mRNAs. Although mostly inhibiting their target mRNAs, several sRNAs also induce gene expression. An important co-factor for sRNA activity is the RNA chaperone, Hfq, which is able to rearrange intramolecular secondary structures and to promote annealing of complementary RNA sequences. In addition, Hfq protects unpaired RNA from degradation by ribonucleases and thus increases sRNA stability. Co-immunoprecipitation of RNA with the Hfq protein, and further experimental as well as bioinformatical studies performed over the last decade suggested the presence of more than 150 different sRNAs in various Enterobacteria including Escherichia coli and Salmonellae. So-called core sRNAs are considered to fulfill central cellular activities as deduced from their high degree of conservation among different species. Approximately 25 core sRNAs have been implicated in gene regulation under a variety of environmental responses. However, for the majority of sRNAs, both the riboregulators' individual biological roles as well as modes of action remain to be elucidated. The current study aimed to define the cellular functions of the two highly conserved, Hfq-dependent sRNAs, SdsR and RydC, in the model pathogen Salmonella Typhimurium. SdsR had been known as one of the most abundant sRNAs during stationary growth phase in E. coli. Examination of the conservation patterns in the sdsR promoter region in combination with classic genetic analyses revealed SdsR as the first sRNA under direct transcriptional control of the alternative σ factor σS. In Salmonella, over-expression of SdsR down-regulates the synthesis of the major porin OmpD, and the interaction site in the ompD mRNA coding sequence was mapped by a 3'RACE-based approach. At the post-transcriptional level, expression of ompD is controlled by three additional sRNAs, but SdsR plays a specific role in porin regulation during the stringent response. Similarly, RydC, the second sRNA adressed in this study, was initially discovered in E. coli but appeared to be conserved in many related γ-proteobacteria. An interesting aspect of this Hfq-dependent sRNAs is its secondary structure involving a pseudo-knot configuration, while the 5' end remains single stranded. A transcriptomic approach combining RydC pulse-expression and scoring of global mRNA changes on microarrays was employed to identify the targets of this sRNA. RydC specifically activated expression of the longer of two versions of the cfa mRNA encoding for the phospholipid-modifying enzyme cyclopropane fatty acid synthase. Employing its conserved single-stranded 5' end, RydC acts as a positive regulator and masks a recognition site of the endoribonuclease, RNase E, in the cfa leader.}, subject = {Small RNA}, language = {en} } @article{FrischholzRoellinghoffMoll1994, author = {Frischholz., S. and R{\"o}llinghoff, M. and Moll, Heidrun}, title = {Cutaneous leishmaniasis: Co-ordinate expression of granzyme A and lymphokines by CD4\(^+\) T cells from susceptible mice.}, series = {Immunology}, volume = {82}, journal = {Immunology}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-30954}, pages = {255 -- 260}, year = {1994}, abstract = {We have recently demonstrated that the frequency ofT cells expressing granzyme A is significantly higher in skin lesions and spleens of susceptible BALB/c mice compared with resistant C57BL/6 mice infected with Leishmania major, a cause of human cutaneous leishmaniasis. In the present study, we have performed in vitro studies to characterize the subpopulation, the antigen responsiveness and the lymphokine production pattern of granzyme A-expressing T cells in L. major-infected mice. Using a limiting dilution system for functional analysis of selected T cells at the clonallevel, we could show that granzyme A activity in infected BALB/c mice can be assigned to L. major-reactive CD4\(^+\) T cells secreting interleukin-2 (IL-2) and IL-4. Granzyme A production was most pronounced in the early phase of infection. On the other hand, granzyme A expression could not be detected in C57BL/6-derived T cells responding to L. major. The da ta support the suggestion that granzyme A is produced by L. major-responsive CD4\(^+\) T cells facilitating lesion formation and the dissemination of infection.}, language = {en} } @phdthesis{Friedrich2009, author = {Friedrich, Torben}, title = {New statistical Methods of Genome-Scale Data Analysis in Life Science - Applications to enterobacterial Diagnostics, Meta-Analysis of Arabidopsis thaliana Gene Expression and functional Sequence Annotation}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-39858}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2009}, abstract = {Recent progresses and developments in molecular biology provide a wealth of new but insufficiently characterised data. This fund comprises amongst others biological data of genomic DNA, protein sequences, 3-dimensional protein structures as well as profiles of gene expression. In the present work, this information is used to develop new methods for the characterisation and classification of organisms and whole groups of organisms as well as to enhance the automated gain and transfer of information. The first two presented approaches (chapters 4 und 5) focus on the medically and scientifically important enterobacteria. Its impact in medicine and molecular biology is founded in versatile mechanisms of infection, their fundamental function as a commensal inhabitant of the intestinal tract and their use as model organisms as they are easy to cultivate. Despite many studies on single pathogroups with clinical distinguishable pathologies, the genotypic factors that contribute to their diversity are still partially unknown. The comprehensive genome comparison described in Chapter 4 was conducted with numerous enterobacterial strains, which cover nearly the whole range of clinically relevant diversity. The genome comparison constitutes the basis of a characterisation of the enterobacterial gene pool, of a reconstruction of evolutionary processes and of comprehensive analysis of specific protein families in enterobacterial subgroups. Correspondence analysis, which is applied for the first time in this context, yields qualitative statements to bacterial subgroups and the respective, exclusively present protein families. Specific protein families were identified for the three major subgroups of enterobacteria namely the genera Yersinia and Salmonella as well as to the group of Shigella and E. coli by applying statistical tests. In conclusion, the genome comparison-based methods provide new starting points to infer specific genotypic traits of bacterial groups from the transfer of functional annotation. Due to the high medical importance of enterobacterial isolates their classification according to pathogenicity has been in focus of many studies. The microarray technology offers a fast, reproducible and standardisable means of bacterial typing and has been proved in bacterial diagnostics, risk assessment and surveillance. The design of the diagnostic microarray of enterobacteria described in chapter 5 is based on the availability of numerous enterobacterial genome sequences. A novel probe selection strategy based on the highly efficient algorithm of string search, which considers both coding and non-coding regions of genomic DNA, enhances pathogroup detection. This principle reduces the risk of incorrect typing due to restrictions to virulence-associated capture probes. Additional capture probes extend the spectrum of applications of the microarray to simultaneous diagnostic or surveillance of antimicrobial resistance. Comprehensive test hybridisations largely confirm the reliability of the selected capture probes and its ability to robustly classify enterobacterial strains according to pathogenicity. Moreover, the tests constitute the basis of the training of a regression model for the classification of pathogroups and hybridised amounts of DNA. The regression model features a continuous learning capacity leading to an enhancement of the prediction accuracy in the process of its application. A fraction of the capture probes represents intergenic DNA and hence confirms the relevance of the underlying strategy. Interestingly, a large part of the capture probes represents poorly annotated genes suggesting the existence of yet unconsidered factors with importance to the formation of respective virulence phenotypes. Another major field of microarray applications is gene expression analysis. The size of gene expression databases rapidly increased in recent years. Although they provide a wealth of expression data, it remains challenging to integrate results from different studies. In chapter 6 the methodology of an unsupervised meta-analysis of genome-wide A. thaliana gene expression data sets is presented, which yields novel insights in function and regulation of genes. The application of kernel-based principal component analysis in combination with hierarchical clustering identified three major groups of contrasts each sharing overlapping expression profiles. Genes associated with two groups are known to play important roles in Indol-3 acetic acid (IAA) mediated plant growth and development as well as in pathogen defence. Yet uncharacterised serine-threonine kinases could be assigned to novel functions in pathogen defence by meta-analysis. In general, hidden interrelation between genes regulated under different conditions could be unravelled by the described approach. HMMs are applied to the functional characterisation of proteins or the detection of genes in genome sequences. Although HMMs are technically mature and widely applied in computational biology, I demonstrate the methodical optimisation with respect to the modelling accuracy on biological data with various distributions of sequence lengths. The subunits of these models, the states, are associated with a certain holding time being the link to length distributions of represented sequences. An adaptation of simple HMM topologies to bell-shaped length distributions described in chapter 7 was achieved by serial chain-linking of single states, while residing in the class of conventional HMMs. The impact of an optimisation of HMM topologies was underlined by performance evaluations with differently adjusted HMM topologies. In summary, a general methodology was introduced to improve the modelling behaviour of HMMs by topological optimisation with maximum likelihood and a fast and easily implementable moment estimator. Chapter 8 describes the application of HMMs to the prediction of interaction sites in protein domains. As previously demonstrated, these sites are not trivial to predict because of varying degree in conservation of their location and type within the domain family. The prediction of interaction sites in protein domains is achieved by a newly defined HMM topology, which incorporates both sequence and structure information. Posterior decoding is applied to the prediction of interaction sites providing additional information of the probability of an interaction for all sequence positions. The implementation of interaction profile HMMs (ipHMMs) is based on the well established profile HMMs and inherits its known efficiency and sensitivity. The large-scale prediction of interaction sites by ipHMMs explained protein dysfunctions caused by mutations that are associated to inheritable diseases like different types of cancer or muscular dystrophy. As already demonstrated by profile HMMs, the ipHMMs are suitable for large-scale applications. Overall, the HMM-based method enhances the prediction quality of interaction sites and improves the understanding of the molecular background of inheritable diseases. With respect to current and future requirements I provide large-scale solutions for the characterisation of biological data in this work. All described methods feature a highly portable character, which allows for the transfer to related topics or organisms, respectively. Special emphasis was put on the knowledge transfer facilitated by a steadily increasing wealth of biological information. The applied and developed statistical methods largely provide learning capacities and hence benefit from the gain of knowledge resulting in increased prediction accuracies and reliability.}, subject = {Genomik}, language = {en} } @article{FrankMarcudeOliveiraAlmeidaPetersenetal.2015, author = {Frank, Benjamin and Marcu, Ana and de Oliveira Almeida Petersen, Antonio Luis and Weber, Heike and Stigloher, Christian and Mottram, Jeremy C. and Scholz, Claus J{\"u}rgen and Schurigt, Uta}, title = {Autophagic digestion of Leishmania major by host macrophages is associated with differential expression of BNIP3, CTSE, and the miRNAs miR-101c, miR-129, and miR-210}, series = {Parasites \& Vectors}, volume = {8}, journal = {Parasites \& Vectors}, number = {404}, doi = {10.1186/s13071-015-0974-3}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-124997}, year = {2015}, abstract = {Background Autophagy participates in innate immunity by eliminating intracellular pathogens. Consequently, numerous microorganisms have developed strategies to impair the autophagic machinery in phagocytes. In the current study, interactions between Leishmania major (L. m.) and the autophagic machinery of bone marrow-derived macrophages (BMDM) were analyzed. Methods BMDM were generated from BALB/c mice, and the cells were infected with L. m. promastigotes. Transmission electron microscopy (TEM) and electron tomography were used to investigate the ultrastructure of BMDM and the intracellular parasites. Affymetrix® chip analyses were conducted to identify autophagy-related messenger RNAs (mRNAs) and microRNAs (miRNAs). The protein expression levels of autophagy related 5 (ATG5), BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), cathepsin E (CTSE), mechanistic target of rapamycin (MTOR), microtubule-associated proteins 1A/1B light chain 3B (LC3B), and ubiquitin (UB) were investigated through western blot analyses. BMDM were transfected with specific small interfering RNAs (siRNAs) against autophagy-related genes and with mimics or inhibitors of autophagy-associated miRNAs. The infection rates of BMDM were determined by light microscopy after a parasite-specific staining. Results The experiments demonstrated autophagy induction in BMDM after in vitro infection with L. m.. The results suggested a putative MTOR phosphorylation-dependent counteracting mechanism in the early infection phase and indicated that intracellular amastigotes were cleared by autophagy in BMDM in the late infection phase. Transcriptomic analyses and specific downregulation of protein expression with siRNAs suggested there is an association between the infection-specific over expression of BNIP3, as well as CTSE, and the autophagic activity of BMDM. Transfection with mimics of mmu-miR-101c and mmu-miR-129-5p, as well as with an inhibitor of mmu-miR-210-5p, demonstrated direct effects of the respective miRNAs on parasite clearance in L. m.-infected BMDM. Furthermore, Affymetrix® chip analyses revealed a complex autophagy-related RNA network consisting of differentially expressed mRNAs and miRNAs in BMDM, which indicates high glycolytic and inflammatory activity in the host macrophages. Conclusions Autophagy in L. m.-infected host macrophages is a highly regulated cellular process at both the RNA level and the protein level. Autophagy has the potential to clear parasites from the host. The results obtained from experiments with murine host macrophages could be translated in the future to develop innovative and therapeutic antileishmanial strategies for human patients.}, language = {en} } @phdthesis{Frank2015, author = {Frank, Benjamin}, title = {Untersuchungen zur Autophagieinduktion in Leishmania major-infizierten Knochenmarksmakrophagen}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-137277}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2015}, abstract = {Die von der WHO zu den 17 wichtigsten NTDs gez{\"a}hlte Leishmaniose wird durch intrazellul{\"a}re Parasiten der Gattung Leishmania hervorgerufen. Der Lebenszyklus der Parasiten besteht aus zwei Phasen. Die l{\"a}nglichen und beweglichen Promastigoten kennzeichnen die Phase in der Sandm{\"u}cke - der Vektor der Leishmaniose. Hingegen ist die Phase im S{\"a}ugerwirt durch runde unbewegliche Amastigoten charakterisiert. Aufgrund des Mangels an potenten antileishmanialen Therapien wurde in der vorliegenden Arbeit die Interaktion zwischen L. m. Parasiten und der Hauptwirtszelle, der Makrophage, v. a. in Hinblick auf autophage Prozesse in den infizierten Makrophagen n{\"a}her untersucht, um demgem{\"a}ß neue Erkenntnisse zu gewinnen, welche bei der Herstellung zuk{\"u}nftiger anti-leishmanialer Medikamente helfen k{\"o}nnten. Bei der Autophagie handelt es sich um einen katabolen Prozess, wodurch Zellen bei Nahrungsmangel oder zellul{\"a}rem Stress ihre Hom{\"o}ostase erhalten k{\"o}nnen. Durch diesen Prozess k{\"o}nnen {\"u}berfl{\"u}ssige oder besch{\"a}digte Organellen recycelt werden, um die Funktionen der Zelle aufrechtzuerhalten. Daneben {\"u}bernimmt Autophagie auch eine essenzielle Rolle bei der Abwehr von ins Zytosol eindringenden Pathogenen. Mittels des neu etablierten totalen Autophagiescore konnte festgestellt werden, dass Autophagie in L. m.-infizierten BMDM induziert wird. Die intrazellul{\"a}ren Amastigoten werden durch Autophagie in den BMDM verdaut. Die erh{\"o}hte autophage Aktivit{\"a}t konnte zudem durch Western-Blot-Analysen der autophagierelevanten Proteine ATG5, LC3B und UB best{\"a}tigt werden. Die molekulargenetischen Untersuchungen von L. m.-infizier-ten BMDM mithilfe von Affymetrix Microarrays f{\"u}hrten zu einem Netzwerk aus autophagierelevanten und infektionsspezifischen Genen, welches als LISA bezeichnet worden ist. Hier hat sich ebenfalls eine starke Verkn{\"u}pfung von autophagierelevanten Genen und den Genen der Glykolyse, einem zweiten katabolen Prozess, gezeigt. Zudem konnten zwei weitere autophagierelevante und infektionsspezifische Gene außerhalb von LISA identifiziert werden, n{\"a}mlich Bnip3 und Ctse, welche im Anschluss genauer untersucht worden sind. Bei beiden Genen konnte auf Proteinebene gezeigt werden, dass sie in L. m.-infizierten BMDM signifikant erh{\"o}ht sind. Durch siRNA-Analysen konnte {\"u}berdies beobachtet werden, dass beide f{\"u}r die erfolgreiche Elimination der Amastigoten essenziell sind. Somit konnte mit den Proteinen BNIP3 und CTSE zwei potenzielle neue Ansatzpunkte f{\"u}r m{\"o}gliche zuk{\"u}nftige antileishmaniale Therapien gefunden werden. Auch die in LISA enthaltenen Gene stellen prinzipiell vielversprechende Ziele f{\"u}r k{\"u}nftige Medikamente gegen Leishmaniose dar. Durch all diese Untersuchungen kommt man dem Ziel einer neuen, gezielten und nebenwirkungs{\"a}rmeren Behandlung der Leishmaniose einen Schritt n{\"a}her.}, subject = {Autophagie}, language = {de} } @phdthesis{Frank2010, author = {Frank, Astrid Christina}, title = {Untersuchungen zur Verbreitung von Pathogenit{\"a}tsinseln unter pathogenen Escherichia coli}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-54111}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2010}, abstract = {Die Ergebnisse dieser Arbeit zeigen erstmals die weite Verbreitung des IS100 innerhalb der Spezies E. coli und große {\"A}hnlichkeiten bez{\"u}glich der chromosomalen Lokalisationen einzelner Kopien in einem heterogenen Kollektiv von E. coli-St{\"a}mmen.}, subject = {Escherichia coli}, language = {de} } @phdthesis{Fluegel1999, author = {Fl{\"u}gel, Manfred}, title = {Molekularbiologische Studien zur Pathogenit{\"a}t und {\"O}kologie von Legionella pneumophila}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-1178}, school = {Universit{\"a}t W{\"u}rzburg}, year = {1999}, abstract = {Legionella pneumophila, der Erreger der Legion{\"a}rskrankheit, ist ein Umweltkeim mit Verbreitung in aquatischen Habitaten. Die Keime sind in der Lage, sich intrazellul{\"a}r in eukaryontischen Zellen, wie Makrophagen, Monozyten und Protozoen zu vermehren. Die erfolgreiche Besiedlung {\"o}kologischer Nischen, aber auch das Virulenzpotential des Keimes k{\"o}nnen dabei von Umweltfaktoren abh{\"a}ngen. Die Erforschung {\"o}kologischer Zusammenh{\"a}nge kann daher f{\"u}r das Verst{\"a}ndnis der bakteriellen Virulenz von großer Bedeutung sein. Ausgangspunkt dieser Arbeit ist die in der sp{\"a}ten station{\"a}ren Phase des Bakterienwachstums von L. pneumophila zu beobachtende intensive Pigmentierung des Kulturmediums. F{\"u}r diesen Ph{\"a}notyp ist das Legiolysin (Lly) verantwortlich. Es ist eines der wenigen bekannten Genprodukte von L. pneumophila, die einen Einfluß auf das {\"U}berleben des Keimes in der Umwelt haben. Legiolysin kann daher als Fitnessfaktor bezeichnet werden. Um die {\"o}kologischen Zusammenh{\"a}nge der Pigmentgenerierung n{\"a}her zu untersuchen, wurde das lly-positive Plasmid pEWL 1 subkloniert und sequenziert. Neben lly konnten in diesem Genabschnitt durch Sequenzvergleiche der abgeleiteten Aminos{\"a}uresequenzen sechs weitere Leseraster detektiert werden. Die drei unmittelbar benachbarten Gene von lly kodieren dabei f{\"u}r Proteine mit Funktionalit{\"a}t in Bezug zur lly-Determinante, w{\"a}hrend die drei upstream von lly liegenden Leseraster Homologien zu Proteinen aufweisen, die an Transportprozessen beteiligt sind. Die L{\"a}nge des RNA-Transkripts von lly konnte im Northern-Blot mit ungef{\"a}hr 1,8 kb bestimmt werden und l{\"a}ßt damit auf die gemeinsame Transkription des lly-Gens mit dem unmittelbar upstream liegenden Leseraster schließen. Durch Sequenzanalysen konnte aufgezeigt werden, daß das f{\"u}r diesen Ph{\"a}notyp verantwortliche Legiolysin-Gen f{\"u}r eine p-Hydroxyphenylpyruvat-Dioxygenase (HPPD) kodiert. Dieses Enzym katalysiert die Umsetzung von p-Hydroxyphenylpyruvat zu Homogentisat (HGA). In Zusammenarbeit mit Prof. P. Proksch (Pharmazeutische Biologie, W{\"u}rzburg) konnte im Vergleich zu lly-negativen L. pneumophila- und rekombinanten E. coli-St{\"a}mmen in den Kultur{\"u}berst{\"a}nden von lly-positiven St{\"a}mmen auf Basis einer Hochdruck-Fl{\"u}ssigkeits-Chromatographie (HPLC) HGA nachgewiesen werden. Es konnte somit gezeigt werden, daß das Legiolysin-Gen f{\"u}r ein Protein mit HPPD- Aktivit{\"a}t kodiert, und in die Degradation der aromatischen Aminos{\"a}uren Phenylalanin und Tyrosin involviert ist. Des weiteren wurden chromosomale Integrationsmutationen der aus L. pneumophila stammenden Gene lly und mip ("macrophage infectivity potentiator") in E. coli K-12 St{\"a}mmen erstellt. Diese wurden nachfolgend in {\"o}kologisch ausgerichteten Langzeitstudien eingesetzt. Die chromosomale Integration von lly erfolgte als ortsspezifische Rekombination in die l att - site von E. coli WM 2269. Die Integration von mip erfolgte in die fim-Region des E. coli K-12-Stammes AAEC 160. Der zweite Teil der vorliegenden Arbeit befaßt sich mit {\"o}kologischen Studien zur Persistenz von L. pneumophila in der Umwelt. Durch die Bestrahlung mit Licht {\"u}ber einen Verlauf von sieben Tagen konnte gezeigt werden, daß die Exprimierung von lly zu einer Persistenz unter Lichtstreß f{\"u}hrt. Dieser Lichtschutz k{\"o}nnte in der Umwelt, aber auch bei der Sanierung von Wasserleitungssystemen mittels UV-Licht Relevanz aufweisen. Die Assoziation von L. pneumophila JR32 und JR32-1 (lly-negativ) mit dem Cyanobakterium Fischerella wurde in Mikrokosmen {\"u}ber einen Verlauf von sieben Tagen beobachtet. Dabei konnte gezeigt werden, daß Legionella in Assoziation mit Fischerella bzw. in dessen {\"U}berstand zu persistieren vermag, w{\"a}hrend dies den Bakterien in frischem Fischerella-Medium nicht m{\"o}glich war. Wie die Coinkubation der Fischerellen mit L. pneumophila JR32-1 zeigte, spielt die Expression von lly dabei keine Rolle. Ein Wachstum der Bakterienkulturen konnte weder im Fischerella-{\"U}berstand, noch in Fischerella-Medium beobachtet werden. Durch Rasterelektronenmikroskopie konnte der adh{\"a}sive Charakter der Assoziation von L. pneumophila zu Fischerella dokumentiert werden. Durch Persistenzstudien von L. pneumophila und E. coli in Boden wurde die {\"U}berlebensf{\"a}higkeit der Mikroorganismen in suboptimaler Umgebung getestet. F{\"u}r Legionella ist eine rapide Abnahme der Zellzahl schon nach kurzer Zeit zu detektieren. Nach sechs Tagen konnten keine Zellen mehr kultiviert werden. Die Defizienz der Pathogenit{\"a}ts- und Umweltfaktoren Mip, Fla (Flagellin) und Lly hatte dabei keinen Einfluß auf die Persistenz der Bakterien im Boden. Zudem sind die zuvor generierten rekombinanten E. coli-Klone AAEC 160-1 und WM 2269-1 mit genomischer mip- bzw. lly-Integration eingesetzt worden. Innerhalb von vier Wochen konnte f{\"u}r diese St{\"a}mme eine kontinuierliche Reduktion der Zellzahlen beobachtet werden. Somit erwies sich keiner der Organismen als erfolgreicher Besiedler der Bodenprobe. Die Studien zum Verlust der Kultivierbarkeit von L. pneumophila und E. coli erfolgten in autoklaviertem Leitungswasser bzw. PBS und zwei unterschiedlich behandelten Varianten von Mainwasser. Neben sterilfiltriertem Mainwasser fand die Inokulierung der Organismen auch in einem Ansatz mit autoklaviertem Mainwasser statt. Es konnte gezeigt werden, daß L. pneumophila in Leitungswasser und Mainwasser außerordentlich gut zu persistieren vermochte. Zudem konnte dokumentiert werden, daß auch E. coli DH5a in ein lebensf{\"a}higes, aber nicht mehr kultivierbares Ruhestadium (viable but nonculturable, VBNC) eintreten kann. Parallel zur Ermittlung der CFU-Werte wurden w{\"a}hrend des Verlaufs der Experimente die Lebendzellzahlen durch Fluoreszenzf{\"a}rbungen bestimmt. Der {\"U}bergang von L. pneumophila in das VBNC-Ruhestadium wurde zudem durch in situ - Hybridisierungen mit fluoreszenzmarkierten 16 S rRNA-Oligonukleotidsonden dokumentiert. Im Naturhaushalt spielt dieser {\"U}bergang zu VBNC-Stadien bei Umweltbakterien zur {\"U}berwindung ung{\"u}nstiger Phasen eine große Rolle. Schließlich erfolgten Experimente zur Reaktivierung der VBNC-Ruhestadien. Diese Reaktivierung ist abh{\"a}ngig von speziesspezifischen Triggern und kann bei L. pneumophila durch Coinkubation mit Acanthamoeba castellanii erfolgen.}, subject = {Legionella pneumophila}, language = {de} } @article{FischerBangLudwigetal.1992, author = {Fischer, G. and Bang, H. and Ludwig, B. and Mann, K. H. and Hacker, J{\"o}rg}, title = {Mip protein of Legionella pneumophila exhibits peptidyl-prolyl cis-trans-isomerase (PPIase) activity}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59778}, year = {1992}, abstract = {Legfonells pneumoph/la is an intracellular paraslte which ts able to survtve and multipJy in human monocytes and alveolar macrophages. The Mtp (macrophage lnfectiv1ty potentlator) protein has been shown to be an essential virulente factor. A search of translated nuclelt .acld data ba.ses has shown that the Mip proteJn from strain Wadsworth possesses reglons homologaus to those found in the FK.506-bindfng proteins (FKBPs) of several different eukaryotlc organisms. FKBPs are abte to bind to the fmmunosuppressant macrollde FK506 and possess peptidyf .. prolyl cisltrans Isomerase (PPiase) activlty. The gene coding for the Mlp proteln was cloned from the ehromo. some of L. pneumophila straln Philadelph·a I and sequenced. II was synthesl\%ed in Escherichla coll ·K- 12 and alter purlfication it exhibited PPiase activity catalyslng the slow clsltrans lsomerization of prolyl peptlde bonds. ln ollgopeptides. Mip ls inhibi~ted by FK506 and fully reslstant to cyclosporln A, as was also found for the recently characterlzed FKBP-type PPiases of eukaryotes. However, the N-terminal extenslon of Mip and/or the substltutrons of the vari· ab1e amlno acrds ln the C-termlnal FKBP core Iead to variatlons,. when compared with eukaryotlc FKBPs, Jn substrate specfflclty wlth the Oligopeptide substrates of' type Suc-Aia-Xaa-Pro-Phe·4·nitroanUide. Never· theless, the Legionella Mip factor represents a bacte· rial gene product whtch shares some characteristics normally found in eukaryotic proteins. ln view of the activity of PPiases in protein-folding reactlonsf such prokaryotic FKBP analogues may represent a new class of bacterial. pathogenicity factors.}, subject = {Infektionsbiologie}, language = {en} } @article{FirdessaGoodAmstaldenetal.2015, author = {Firdessa, Rebuma and Good, Liam and Amstalden, Maria Cecilia and Chindera, Kantaraja and Kamaruzzaman, Nor Fadhilah and Schultheis, Martina and R{\"o}ger, Bianca and Hecht, Nina and Oelschlaeger, Tobias A. and Meinel, Lorenz and L{\"u}hmann, Tessa and Moll, Heidrun}, title = {Pathogen- and host-directed antileishmanial effects mediated by polyhexanide (PHMB)}, series = {PLoS Neglected Tropical Diseases}, volume = {9}, journal = {PLoS Neglected Tropical Diseases}, number = {10}, doi = {10.1371/journal.pntd.0004041}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-148162}, pages = {e0004041}, year = {2015}, abstract = {Background Cutaneous leishmaniasis (CL) is a neglected tropical disease caused by protozoan parasites of the genus Leishmania. CL causes enormous suffering in many countries worldwide. There is no licensed vaccine against CL, and the chemotherapy options show limited efficacy and high toxicity. Localization of the parasites inside host cells is a barrier to most standard chemo- and immune-based interventions. Hence, novel drugs, which are safe, effective and readily accessible to third-world countries and/or drug delivery technologies for effective CL treatments are desperately needed. Methodology/Principal Findings Here we evaluated the antileishmanial properties and delivery potential of polyhexamethylene biguanide (PHMB; polyhexanide), a widely used antimicrobial and wound antiseptic, in the Leishmania model. PHMB showed an inherent antileishmanial activity at submicromolar concentrations. Our data revealed that PHMB kills Leishmania major (L. major) via a dual mechanism involving disruption of membrane integrity and selective chromosome condensation and damage. PHMB's DNA binding and host cell entry properties were further exploited to improve the delivery and immunomodulatory activities of unmethylated cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODN). PHMB spontaneously bound CpG ODN, forming stable nanopolyplexes that enhanced uptake of CpG ODN, potentiated antimicrobial killing and reduced host cell toxicity of PHMB. Conclusions Given its low cost and long history of safe topical use, PHMB holds promise as a drug for CL therapy and delivery vehicle for nucleic acid immunomodulators.}, language = {en} } @phdthesis{Fieseler2005, author = {Fieseler, Lars}, title = {Entdeckung des neuen Candidatus Phylums Poribacteria}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-13283}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2005}, abstract = {Marine Schw{\"a}mme (Porifera) sind sessile Invertebraten, deren Biomasse bis zu 60\% von assoziierten Mikroorganismen gebildet werden kann. Dieses mikrobielle Konsortium ist phylogenetisch komplex, die monophyletischen Abstammungslinien sind hochgradig wirtsspezifisch und bisher konnte kein Vertreter dieser Mikroflora kultiviert werden. In seiner Zusammensetzung unterscheidet sich dieses Konsortium sowohl von der Mikroflora mariner Sedimente, als auch vom marinen Bakterioplankton. Durch 16S rRNA Sequenzanalysen und Fluoreszenz in situ Hybridisierung (FISH) konnte w{\"a}hrend dieser Arbeit das neue Candidatus Phylum Poribacteria kultivierungsunabh{\"a}ngig identifiziert werden. Poribacteria bilden definitionsgem{\"a}ß ein unabh{\"a}ngiges Candidatus Phylum, da sie weniger als 75\% Sequenzhomologie innerhalb der 16S rRNA zu anderen prokaryontischen Phyla zeigen. Sie sind verwandt mit Planctomycetes. Der Name „Poribacteria" wurde gew{\"a}hlt, da diese Organismen spezifisch mit marinen Porifera assoziiert zu sein scheinen. Bisher konnten Poribacteria in Porifera der Ordnungen Verongida, Haplosclerida und Lithistida nachgewiesen werden, w{\"a}hrend sie in den Ordnungen Poecilosclerida, Agelasida, Halichondrida und Hadromerida nicht nachweisbar waren. Im marinen Sediment und im Bakterioplankton wurden Poribacteria ebenfalls nicht detektiert. Durch FISH Analysen wurde deutlich, dass Poribacteria in A. aerophoba (Verongida) eine abundante Fraktion der assoziierten Mikroflora bilden. Da Vertreter des mikrobiellen Konsortiums mariner Schw{\"a}mme bisher nicht kultiviert werden konnten, wurde das „Metagenom" dieser Mikroorganismen durch die ex situ Isolierung hoch molekularer DNA direkt kloniert. Eine Charakterisierung von Metagenomen erlaubt unabh{\"a}ngig von der Kultivierbarkeit der entsprechenden Organismen direkte Einblicke in deren Genotyp und liefert so eine erste Verbindung zwischen phylogenetischer Diversit{\"a}t und physiologischen Eigenschaften. F{\"u}r die Erstellung der Metagenombank wurde mikrobielle Biomasse aus A. aerophoba vom Mesohyl getrennt und lysiert und die gereinigte DNA in Fosmid Vektoren in E. coli kloniert. Die resultierende Metagenombank APAE02 umfasst ca. 1,1 Gb hoch molekularer prokaryontischer genomischer DNA. Eine Bestimmung der in dieser Metagenombank archivierten mikrobiellen Diversit{\"a}t lieferte zus{\"a}tzlich zu bekannten 16S rRNA kodierenden Loci aus Cyanobacteria, Chloroflexi, Acidobacteria und Gammaproteobacteria einen 16S rRNA kodierenden poribakteriellen Fosmidklon. Die Annotation der flankierenden genomischen Regionen des 16S rRNA Gens f{\"u}hrte zur Detektion eines unterbrochenen rrn Operons, eines wahrscheinlich neuen Transporters, einer neuen Molybd{\"a}n enthaltenen Oxidoreduktase und orthologer „open reading frames" (ORFs) aus Rhodopirellula baltica (Planctomycetes) in Poribacteria. Die Charakterisierung dieses 38,7 kb DNA Fragmentes stellt die Basis f{\"u}r weitere genomische Untersuchungen an Poribacteria dar. Metagenombanken repr{\"a}sentieren eine reichhaltige Quelle zum Nachweis neuer Enzyme oder Biosyntheseoperons. Somit konnten in der Metagenombank APAE02 neuartige Typ I Polyketidsynthasen (PKS) nachgewiesen werden. Phylogenetische Analysen der Ketosynthasedom{\"a}ne zeigten, dass diese Systeme nicht herk{\"o}mmlichen Typ I cis-AT bzw. trans-AT (Acyltransferase) PKS Systemen zugeordnet werden k{\"o}nnen. Die kodierenden Bereiche der PKS Systeme sind mit nur ca. 10 kb relativ klein. Im Gegensatz zu der Organisation sich wiederholender multipler Module herk{\"o}mmlicher PKS Typ I Systeme bestehen sie nur aus einem einzigen Modul und k{\"o}nnten vermutlich bei der Synthese von Fetts{\"a}uren beteiligt sein. Die Struktur und Funktion der Produkte ist bisher unbekannt. Generell ist durch in silico Analysen eine Abbildung des „funktionellen Repertoires" unkultivierter Mikroorganismen m{\"o}glich. Es w{\"a}re denkbar, dass durch weitere Studien fundierte Einblicke in den Genpool der Poribacteria und anderer Organismen des mikrobiellen Konsortiums aus Poriferen er{\"o}ffnet werden, um metabolische Eigenschaften zu rekonstruieren und die Mechanismen zur Interaktion mit dem Wirt verstehen zu k{\"o}nnen.}, subject = {Schw{\"a}mme}, language = {de} } @article{FellerThomKochetal.2013, author = {Feller, Tatjana and Thom, Pascal and Koch, Natalie and Spiegel, Holger and Addai-Mensah, Otchere and Fischer, Rainer and Reimann, Andreas and Pradel, Gabriele and Fendel, Rolf and Schillberg, Stefan and Scheuermayer, Matthias and Schinkel, Helga}, title = {Plant-Based Production of Recombinant Plasmodium Surface Protein Pf38 and Evaluation of its Potential as a Vaccine Candidate}, series = {PLOS ONE}, volume = {8}, journal = {PLOS ONE}, number = {11}, issn = {1932-6203}, doi = {10.1371/journal.pone.0079920}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-128221}, pages = {e79920}, year = {2013}, abstract = {Pf38 is a surface protein of the malarial parasite Plasmodium falciparum. In this study, we produced and purified recombinant Pf38 and a fusion protein composed of red fluorescent protein and Pf38 (RFP-Pf38) using a transient expression system in the plant Nicotiana benthamiana. To our knowledge, this is the first description of the production of recombinant Pf38. To verify the quality of the recombinant Pf38, plasma from semi-immune African donors was used to confirm specific binding to Pf38. ELISA measurements revealed that immune responses to Pf38 in this African subset were comparable to reactivities to AMA-1 and \(MSP1_{19}\). Pf38 and RFP-Pf38 were successfully used to immunise mice, although titres from these mice were low (on average 1:11.000 and 1:39.000, respectively). In immune fluorescence assays, the purified IgG fraction from the sera of immunised mice recognised Pf38 on the surface of schizonts, gametocytes, macrogametes and zygotes, but not sporozoites. Growth inhibition assays using \(\alpha Pf38\) antibodies demonstrated strong inhibition \((\geq 60 \\% ) \) of the growth of blood-stage P. falciparum. The development of zygotes was also effectively inhibited by \(\alpha Pf38\) antibodies, as determined by the zygote development assay. Collectively, these results suggest that Pf38 is an interesting candidate for the development of a malaria vaccine.}, language = {en} } @article{FanEbnerReichertetal.2019, author = {Fan, Sook-Ha and Ebner, Patrick and Reichert, Sebstian and Hertlein, Tobias and Zabel, Susanne and Lankapalli, Aditya Kumar and Nieselt, Kay and Ohlsen, Knut and G{\"o}tz, Friedrich}, title = {MpsAB is important for Staphylococcus aureus virulence and growth at atmospheric CO2 levels}, series = {Nature Communications}, volume = {10}, journal = {Nature Communications}, doi = {10.1038/s41467-019-11547-5}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-227624}, year = {2019}, abstract = {The mechanisms behind carbon dioxide (CO2) dependency in non-autotrophic bacterial isolates are unclear. Here we show that the Staphylococcus aureus mpsAB operon, known to play a role in membrane potential generation, is crucial for growth at atmospheric CO2 levels. The genes mpsAB can complement an Escherichia coli carbonic anhydrase (CA) mutant, and CA from E. coli can complement the S. aureus delta-mpsABC mutant. In comparison with the wild type, S. aureus mps mutants produce less hemolytic toxin and are less virulent in animal models of infection. Homologs of mpsA and mpsB are widespread among bacteria and are often found adjacent to each other on the genome. We propose that MpsAB represents a dissolved inorganic carbon transporter, or bicarbonate concentrating system, possibly acting as a sodium bicarbonate cotransporter.}, language = {en} } @article{FanLiChaoetal.2015, author = {Fan, Ben and Li, Lei and Chao, Yanjie and F{\"o}rstner, Konrad and Vogel, J{\"o}rg and Borriss, Rainer and Wu, Xiao-Qin}, title = {dRNA-Seq Reveals Genomewide TSSs and Noncoding RNAs of Plant Beneficial Rhizobacterium Bacillus amyloliquefaciens FZB42}, series = {PLoS One}, volume = {10}, journal = {PLoS One}, number = {11}, doi = {10.1371/journal.pone.0142002}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-138369}, pages = {e0142002}, year = {2015}, abstract = {Bacillus amyloliquefaciens subsp. plantarum FZB42 is a representative of Gram-positive plant-growth-promoting rhizobacteria (PGPR) that inhabit plant root environments. In order to better understand the molecular mechanisms of bacteria-plant symbiosis, we have systematically analyzed the primary transcriptome of strain FZB42 grown under rhizospheremimicking conditions using differential RNA sequencing (dRNA-seq). Our analysis revealed 4,877 transcription start sites for protein-coding genes, identified genes differentially expressed under different growth conditions, and corrected many previously mis-annotated genes. We also identified a large number of riboswitches and cis-encoded antisense RNAs, as well as trans-encoded small noncoding RNAs that may play important roles in the gene regulation of Bacillus. Overall, our analyses provided a landscape of Bacillus primary transcriptome and improved the knowledge of rhizobacteria-host interactions.}, language = {en} } @phdthesis{FajardoMoser2006, author = {Fajardo-Moser, Marcela}, title = {Untersuchung der Legionella-Infektion in der genetisch manipulierbaren Am{\"o}be Dictyostelium discoideum}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-21355}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2006}, abstract = {Die haploide Am{\"o}be Dictyostelium discoideum hat sich als geeinet erwiesen f{\"u}r die Untersuchung der zellul{\"a}ren Aspekte der Legionella Infektion. Nach der Aufnahme befindet sich L. pneumophila innerhalb eines unreifen Phagosoms das weder anges{\"a}uert wird noch mit Lysosomen fusioniert. In dieser Studie wurden die Wirtzellfaktoren untersucht, die Legionella eine erfolgreiche Kolonizierung des Wirt erm{\"o}glichen. Phagozytoseversuche mit spezifischen zellul{\"a}ren Inhibitoren und die Analyse der Aufnahme in definierten Wirtzell-Mutanten haben gezeigt, daß das zytoplasmatische Kalziumniveau, Zytoskelettproteine und die Kalzium-bindenden Proteine des ERs, Calreticulin und Calnexin, spezifisch die Aufnahme und das intrazellul{\"a}re Wachstum von L. pneumophila beeinflussen. Mikroskopisches Untersuchungen mit GFP-markierten Calnexin und Calreticulin haben gezeigt, dass beide Proteine spezifisch in den "phagocytic cups" der L. pneumophila-infizierten Wirtszellen akkumulieren. Beide Proteine umh{\"u}llten die replikative Vakuole von L. pneumophila w{\"a}hrend der gesamten Replikation des Bakteriums. Die kumulativen Effekte intrazellul{\"a}ren Kalziumniveaus, die r{\"a}umliche Verteilung von Calnexin und Calreticulin und die Defekte Aufnahme und intrazellul{\"a}re Vermehrung von L. pneumophila im Calnexin- und Calreticulin-minus der Zellen deuten darauf hin, daß diese Faktoren ein Teil des Regulationssystems sind, der zu der Bildung der spezifische Vakuole von L. pneumophila f{\"u}hrt.}, subject = {Dictyostelium discoideum}, language = {de} } @article{EulalioFroehlichManoetal.2011, author = {Eulalio, Ana and Fr{\"o}hlich, Kathrin S. and Mano, Miguel and Giacca, Mauro and Vogel, J{\"o}rg}, title = {A Candidate Approach Implicates the Secreted Salmonella Effector Protein SpvB in P-Body Disassembly}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-68928}, year = {2011}, abstract = {P-bodies are dynamic aggregates of RNA and proteins involved in several post-transcriptional regulation processes. Pbodies have been shown to play important roles in regulating viral infection, whereas their interplay with bacterial pathogens, specifically intracellular bacteria that extensively manipulate host cell pathways, remains unknown. Here, we report that Salmonella infection induces P-body disassembly in a cell type-specific manner, and independently of previously characterized pathways such as inhibition of host cell RNA synthesis or microRNA-mediated gene silencing. We show that the Salmonella-induced P-body disassembly depends on the activation of the SPI-2 encoded type 3 secretion system, and that the secreted effector protein SpvB plays a major role in this process. P-body disruption is also induced by the related pathogen, Shigella flexneri, arguing that this might be a new mechanism by which intracellular bacterial pathogens subvert host cell function.}, subject = {Salmonella}, language = {en} } @article{EspinaPaganLopezetal.2015, author = {Espina, Laura and Pag{\´a}n, Rafael and L{\´o}pez, Daniel and Garc{\´i}a-Gonzalo, Diego}, title = {Individual Constituents from Essential Oils Inhibit Biofilm Mass Production by Multi-Drug Resistant Staphylococcus aureus}, series = {Molecules}, volume = {20}, journal = {Molecules}, doi = {10.3390/molecules200611357}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-151845}, pages = {11357 -- 11372}, year = {2015}, abstract = {Biofilm formation by Staphylococcus aureus represents a problem in both the medical field and the food industry, because the biofilm structure provides protection to embedded cells and it strongly attaches to surfaces. This circumstance is leading to many research programs seeking new alternatives to control biofilm formation by this pathogen. In this study we show that a potent inhibition of biofilm mass production can be achieved in community-associated methicillin-resistant S. aureus (CA-MRSA) and methicillin-sensitive strains using plant compounds, such as individual constituents (ICs) of essential oils (carvacrol, citral, and (+)-limonene). The Crystal Violet staining technique was used to evaluate biofilm mass formation during 40 h of incubation. Carvacrol is the most effective IC, abrogating biofilm formation in all strains tested, while CA-MRSA was the most sensitive phenotype to any of the ICs tested. Inhibition of planktonic cells by ICs during initial growth stages could partially explain the inhibition of biofilm formation. Overall, our results show the potential of EOs to prevent biofilm formation, especially in strains that exhibit resistance to other antimicrobials. As these compounds are food additives generally recognized as safe, their anti-biofilm properties may lead to important new applications, such as sanitizers, in the food industry or in clinical settings.}, language = {en} } @article{EskenGorisGadkarietal.2020, author = {Esken, Jens and Goris, Tobias and Gadkari, Jennifer and Bischler, Thorsten and F{\"o}rstner, Konrad U. and Sharma, Cynthia M. and Diekert, Gabriele and Schubert, Torsten}, title = {Tetrachloroethene respiration in Sulfurospirillum species is regulated by a two-component system as unraveled by comparative genomics, transcriptomics, and regulator binding studies}, series = {MicrobiologyOpen}, volume = {9}, journal = {MicrobiologyOpen}, number = {12}, doi = {10.1002/mbo3.1138}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-225754}, year = {2020}, abstract = {Energy conservation via organohalide respiration (OHR) in dehalogenating Sulfurospirillum species is an inducible process. However, the gene products involved in tetrachloroethene (PCE) sensing and signal transduction have not been unambiguously identified. Here, genome sequencing of Sulfurospirillum strains defective in PCE respiration and comparative genomics, which included the PCE-respiring representatives of the genus, uncovered the genetic inactivation of a two-component system (TCS) in the OHR gene region of the natural mutants. The assumption that the TCS gene products serve as a PCE sensor that initiates gene transcription was supported by the constitutive low-level expression of the TCS operon in fumarate-adapted cells of Sulfurospirillum multivorans. Via RNA sequencing, eight transcriptional units were identified in the OHR gene region, which includes the TCS operon, the PCE reductive dehalogenase operon, the gene cluster for norcobamide biosynthesis, and putative accessory genes with unknown functions. The OmpR-family response regulator (RR) encoded in the TCS operon was functionally characterized by promoter-binding assays. The RR bound a cis-regulatory element that contained a consensus sequence of a direct repeat (CTATW) separated by 17 bp. Its location either overlapping the -35 box or 50 bp further upstream indicated different regulatory mechanisms. Sequence variations in the regulator binding sites identified in the OHR gene region were in accordance with differences in the transcript levels of the respective gene clusters forming the PCE regulon. The results indicate the presence of a fine-tuned regulatory network controlling PCE metabolism in dehalogenating Sulfurospirillum species, a group of metabolically versatile organohalide-respiring bacteria.}, language = {en} } @article{EneLohseVladuetal.2016, author = {Ene, Iuliana V. and Lohse, Matthew B. and Vladu, Adrian V. and Morschh{\"a}user, Joachim and Johnson, Alexander D. and Bennett, Richard J.}, title = {Phenotypic Profiling Reveals that Candida albicans Opaque Cells Represent a Metabolically Specialized Cell State Compared to Default White Cells}, series = {mBio}, volume = {7}, journal = {mBio}, number = {6}, doi = {10.1128/mBio.01269-16}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-165818}, pages = {e01269-16}, year = {2016}, abstract = {The white-opaque switch is a bistable, epigenetic transition affecting multiple traits in Candida albicans including mating, immunogenicity, and niche specificity. To compare how the two cell states respond to external cues, we examined the fitness, phenotypic switching, and filamentation properties of white cells and opaque cells under 1,440 different conditions at 25°C and 37°C. We demonstrate that white and opaque cells display striking differences in their integration of metabolic and thermal cues, so that the two states exhibit optimal fitness under distinct conditions. White cells were fitter than opaque cells under a wide range of environmental conditions, including growth at various pHs and in the presence of chemical stresses or antifungal drugs. This difference was exacerbated at 37°C, consistent with white cells being the default state of C. albicans in the mammalian host. In contrast, opaque cells showed greater fitness than white cells under select nutritional conditions, including growth on diverse peptides at 25°C. We further demonstrate that filamentation is significantly rewired between the two states, with white and opaque cells undergoing filamentous growth in response to distinct external cues. Genetic analysis was used to identify signaling pathways impacting the white-opaque transition both in vitro and in a murine model of commensal colonization, and three sugar sensing pathways are revealed as regulators of the switch. Together, these findings establish that white and opaque cells are programmed for differential integration of metabolic and thermal cues and that opaque cells represent a more metabolically specialized cell state than the default white state.}, language = {en} } @article{EmmrichMollSimon1985, author = {Emmrich, F. and Moll, Heidrun and Simon, Markus M.}, title = {Recombinant human interleukin 2 acts as a B cell growth and differentiation promoting factor}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-34132}, year = {1985}, abstract = {Human B cells appropriately activated by a B cell mitogen are rendered susceptible to human Interleukin 2 (IL-2) as demonstrated with recombinant human IL-2 (rec. h IL-2). They show increased proliferation and drastically enhanced immunoglobulin secretion. Susceptibility to IL-2 is accompanied with the expression of the IL-2 receptor (Tac antigen) on B cells. The data suggest that IL-2 is one of the lymphokines directly involved in the activation of B lymphocytes.}, language = {en} } @article{ElMoualiGerovacMineikaitėetal.2021, author = {El Mouali, Youssef and Gerovac, Milan and Mineikaitė, Raminta and Vogel, J{\"o}rg}, title = {In vivo targets of Salmonella FinO include a FinP-like small RNA controlling copy number of a cohabitating plasmid}, series = {Nucleic Acids Research}, volume = {49}, journal = {Nucleic Acids Research}, number = {9}, doi = {10.1093/nar/gkab281}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-261072}, pages = {5319-5335}, year = {2021}, abstract = {FinO-domain proteins represent an emerging family of RNA-binding proteins (RBPs) with diverse roles in bacterial post-transcriptional control and physiology. They exhibit an intriguing targeting spectrum, ranging from an assumed single RNA pair (FinP/traJ) for the plasmid-encoded FinO protein, to transcriptome-wide activity as documented for chromosomally encoded ProQ proteins. Thus, the shared FinO domain might bear an unusual plasticity enabling it to act either selectively or promiscuously on the same cellular RNA pool. One caveat to this model is that the full suite of in vivo targets of the assumedly highly selective FinO protein is unknown. Here, we have extensively profiled cellular transcripts associated with the virulence plasmid-encoded FinO in Salmonella enterica. While our analysis confirms the FinP sRNA of plasmid pSLT as the primary FinO target, we identify a second major ligand: the RepX sRNA of the unrelated antibiotic resistance plasmid pRSF1010. FinP and RepX are strikingly similar in length and structure, but not in primary sequence, and so may provide clues to understanding the high selectivity of FinO-RNA interactions. Moreover, we observe that the FinO RBP encoded on the Salmonella virulence plasmid controls the replication of a cohabitating antibiotic resistance plasmid, suggesting cross-regulation of plasmids on the RNA level.}, language = {en} } @article{EhrigKilincChenetal.2013, author = {Ehrig, Klaas and Kilinc, Mehmet O. and Chen, Nanhai G. and Stritzker, Jochen and Buckel, Lisa and Zhang, Qian and Szalay, Aladar A.}, title = {Growth inhibition of different human colorectal cancer xenografts after a single intravenous injection of oncolytic vaccinia virus GLV-1h68}, series = {Journal of Translational Medicine}, volume = {11}, journal = {Journal of Translational Medicine}, number = {79}, doi = {10.1186/1479-5876-11-79}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-129619}, year = {2013}, abstract = {Background: Despite availability of efficient treatment regimens for early stage colorectal cancer, treatment regimens for late stage colorectal cancer are generally not effective and thus need improvement. Oncolytic virotherapy using replication-competent vaccinia virus (VACV) strains is a promising new strategy for therapy of a variety of human cancers. Methods: Oncolytic efficacy of replication-competent vaccinia virus GLV-1h68 was analyzed in both, cell cultures and subcutaneous xenograft tumor models. Results: In this study we demonstrated for the first time that the replication-competent recombinant VACV GLV-1h68 efficiently infected, replicated in, and subsequently lysed various human colorectal cancer lines (Colo 205, HCT-15, HCT-116, HT-29, and SW-620) derived from patients at all four stages of disease. Additionally, in tumor xenograft models in athymic nude mice, a single injection of intravenously administered GLV-1h68 significantly inhibited tumor growth of two different human colorectal cell line tumors (Duke's type A-stage HCT-116 and Duke's type C-stage SW-620), significantly improving survival compared to untreated mice. Expression of the viral marker gene ruc-gfp allowed for real-time analysis of the virus infection in cell cultures and in mice. GLV-1h68 treatment was well-tolerated in all animals and viral replication was confined to the tumor. GLV-1h68 treatment elicited a significant up-regulation of murine immune-related antigens like IFN-γ, IP-10, MCP-1, MCP-3, MCP-5, RANTES and TNF-γ and a greater infiltration of macrophages and NK cells in tumors as compared to untreated controls. Conclusion: The anti-tumor activity observed against colorectal cancer cells in these studies was a result of direct viral oncolysis by GLV-1h68 and inflammation-mediated innate immune responses. The therapeutic effects occurred in tumors regardless of the stage of disease from which the cells were derived. Thus, the recombinant vaccinia virus GLV-1h68 has the potential to treat colorectal cancers independently of the stage of progression.}, language = {en} } @phdthesis{Eckart2006, author = {Eckart, Martin}, title = {Analyse einer chromosomalen Deletion und Entdeckung einer neuartigen nicht-translatierten RNA in Staphylococcus epidermidis}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-21448}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2006}, abstract = {Staphylococcus epidermidis ist ein wichtiger Bestandteil der gesunden Hautflora des Menschen, gleichzeitig aber auch der h{\"a}ufigste Erreger nosokomialer Infektionen bei immunsupprimierten Patienten. Die Forschungsarbeiten haben sich in den vergangenen Jahren besonders auf Faktoren und Mechanismen konzentriert, welche zur Etablierung der Spezies als Pathogen beigetragen haben. Eine typische Eigenschaft klinischer Isolate ist die F{\"a}higkeit, auf k{\"u}nstlichen Oberfl{\"a}chen Biofilme zu bilden. Gegenstand der vorliegenden Arbeit war die Untersuchung der IS-vermittelten Genomflexibilit{\"a}t und der Vergleich der Genomstruktur nosokomialer und kommensaler Isolate von S. epidermidis. Dazu wurde eine 260 kb große spontane Deletion im Chromosom des Biofilm-bildenden Stammes S. epidermidis 307 sequenziert und annotiert, von der bekannt war, dass sie durch eine homologe Rekombination zweier IS256-Kopien ausgel{\"o}st wurde. Auf dem deletierten Fragment fanden sich neben dem ica-Operon zahlreiche potentielle Virulenz-assoziierte Gene. Das {\"u}berraschende Ergebnis dieser Analyse war jedoch die Identifizierung eines neuartigen SCC-Elementes, das den rechten Rand der Deletion begrenzt. Dies ist die erste Beschreibung eines SSC-Elementes mit einer CcrC-Rekombinase, dem das Methicillin-Resistenzgen mecA fehlt. In der N{\"a}he des Rekombinasegens fand sich S.-haemolyticus-spezifische DNA. Weitere Untersuchungen zeigten, dass das SCC-Element durch die IS256/Tn4001 -vermittelte Deletion in der Mutante verk{\"u}rzt wurde. Genomvergleiche ica-positiver und ica-negativer St{\"a}mme von S. epidermidis mittels Microarrays, sowie in-silico-Analysen zweier vollst{\"a}ndiger S.-epidermidis-Genome ergaben, dass sich kommensale und pathogene St{\"a}mme in nur wenigen Faktoren unterscheiden. Diese befinden sich jedoch fast alle in einer Region um den oriC, in dessen N{\"a}he auch die Insertionsstelle f{\"u}r die SCCmec-Inseln lokalisiert ist. Das deletierte DNA-Fragment von S. epidermidis 307 konnte ebenfalls dieser Region zugeordnet werden, die offenbar eine Zone hoher genomischer Flexibilit{\"a}t darstellt, in der fremde DNA integriert werden kann. Im Rahmen dieser Arbeit konnte außerdem gezeigt werden, dass das ica-Operon diesen variablen Genomabschnitt begrenzt. Die exakte Grenze zwischen ica-positiven und ica-negativen St{\"a}mmen bildet eine Thr-tRNA, die in einer 1,4 kb großen intergenischen Region stromabw{\"a}rts des icaR-Regulatorgens lokalisiert ist. In unmittelbarer Nachbarschaft konnte ein Transkript nachgewiesen werden, welches nur in ica-positiven S. epidermidis vorkommt. Gest{\"u}tzt auf bioinformatische Analysen wurden zun{\"a}chst die Polarit{\"a}t und L{\"a}nge des Transkriptes, sowie der korrespondierende Promotor experimentell bestimmt. Demnach handelt es sich um eine 487 nt lange RNA, die entgegengesetzt zur Thr-tRNA orientiert ist und mit dieser teilweise {\"u}berlappt. Da die Nukelotid-Sequenz weder eine Ribosomen- Bindungsstelle noch einen gr{\"o}ßeren Leserahmen aufweist, ist es sehr wahrscheinlich, dass es sich um eine nicht-translatierte RNA handelt. Qualitative RT-PCR-Experimente zeigten, dass die IGRica-RNA kostitutiv exprimiert wird. Spontane IS256 -Insertionsmutanten in der Promotorregion der IGRica-RNA wiesen ph{\"a}notypisch eine deutlich verminderte Biofilmbildung auf. Daher ist zu vermuten, dass die IGRica-RNA in die Regulation dieses wichtigen Virulenzfaktors involviert ist. In der vorliegenden Arbeit konnte weiterhin gezeigt werden, dass S. epidermidis das in vielen Spezies konservierte Hfq-Protein exprimiert, welches ein Bindungspartner und Chaperon f{\"u}r zahlreiche regulatorische RNAs darstellt. gel-shift-Experimente mit rekombinantem Hfq-Protein aus S. epidermidis ergaben jedoch keine nachweisbare Wechselwirkung der IGRica-RNA mit diesem Faktor in vitro. Insgesamt hat die Studie gezeigt, dass S. epidermidis eine Spezies mit einer außerordentlich hohen Genomflexibilit{\"a}t ist, die sich haupts{\"a}chlich in einer bestimmten Genomregion abspielt und f{\"u}r die IS-Elemente von besonderer Bedeutung sind. Die Identifizierung von DNA aus anderen Staphylokokken-Arten in dieser Region zeigt, dass S. epidermidis zu horizontalem Gentransfer {\"u}ber Speziesgrenzen hinweg in der Lage ist. Dies, sowie die Daten zur neuen SCC-Kassette, unterstreichen die Bedeutung von S. epidermidis als Reservoir f{\"u}r die Entwicklung neuartiger Resistenz- und Virulenzdeterminanten, die gerade im Krankenhausmilieu auf Spezies mit h{\"o}herem Virulenzpotential {\"u}bertragen werden k{\"o}nnen und so einen wichtigen Faktor f{\"u}r die anhaltende Problematik nosokomialer Infektionen mit S. aureus darstellen. Die Entdeckung einer RNA mit m{\"o}glicherweise regulatorischer Funktion ist der erste Faktor dieser Art, der - abgesehen von einigen phylogenetisch hoch konservierten RNAs - bisher in S. epidermidis nachgewiesen wurde. Die funktionale Analyse der IGRica-RNA und die Suche nach weiteren regulatorischen RNAs in Staphylokokken er{\"o}ffnen ein Forschungsfeld, das zum besseren Verst{\"a}ndnis der Lebensweise dieser bedeutenden Erreger beitragen kann.}, subject = {Staphylococcus epidermis}, language = {de} } @article{DaeullaryImdahlDietrichetal.2023, author = {D{\"a}ullary, Thomas and Imdahl, Fabian and Dietrich, Oliver and Hepp, Laura and Krammer, Tobias and Fey, Christina and Neuhaus, Winfried and Metzger, Marco and Vogel, J{\"o}rg and Westermann, Alexander J. and Saliba, Antoine-Emmanuel and Zdzieblo, Daniela}, title = {A primary cell-based in vitro model of the human small intestine reveals host olfactomedin 4 induction in response to Salmonella Typhimurium infection}, series = {Gut Microbes}, volume = {15}, journal = {Gut Microbes}, number = {1}, doi = {10.1080/19490976.2023.2186109}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-350451}, year = {2023}, abstract = {Infection research largely relies on classical cell culture or mouse models. Despite having delivered invaluable insights into host-pathogen interactions, both have limitations in translating mechanistic principles to human pathologies. Alternatives can be derived from modern Tissue Engineering approaches, allowing the reconstruction of functional tissue models in vitro. Here, we combined a biological extracellular matrix with primary tissue-derived enteroids to establish an in vitro model of the human small intestinal epithelium exhibiting in vivo-like characteristics. Using the foodborne pathogen Salmonella enterica serovar Typhimurium, we demonstrated the applicability of our model to enteric infection research in the human context. Infection assays coupled to spatio-temporal readouts recapitulated the established key steps of epithelial infection by this pathogen in our model. Besides, we detected the upregulation of olfactomedin 4 in infected cells, a hitherto unrecognized aspect of the host response to Salmonella infection. Together, this primary human small intestinal tissue model fills the gap between simplistic cell culture and animal models of infection, and shall prove valuable in uncovering human-specific features of host-pathogen interplay.}, language = {en} } @phdthesis{Dunkel2013, author = {Dunkel, Nico}, title = {Regulation of virulence-associated traits of the human fungal pathogen Candida albicans by nitrogen availability}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-83076}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Nitrogen-regulated pathogenesis describes the expression of virulence attributes as direct response to the quantity and quality of an available nitrogen source. As consequence of nitrogen availability, the opportunistic human fungal pathogen Candida albicans changes its morphology and secretes aspartic proteases [SAPs], both well characterized virulence attributes. C. albicans, contrarily to its normally non-pathogenic relative Saccharomyces cerevisiae, is able to utilize proteins, which are considered as abundant and important nitrogen source within the human host. To assimilate complex proteinaceous matter, extracellular proteolysis is followed by uptake of the degradation products through dedicated peptide transporters (di-/tripeptide transporters [PTRs] and oligopeptide transporters [OPTs]). The expression of both traits is transcriptionally controlled by Stp1 - the global regulator of protein utilization - in C. albicans. The aim of the present study was to elucidate the regulation of virulence attributes of the pathogenic fungus C. albicans by nitrogen availability in more detail. Within a genome wide binding profile of Stp1, during growth with proteins, more than 600 Stp1 target genes were identified, thereby confirming its role in the usage of proteins, but also other nitrogenous compounds as nitrogen source. Moreover, the revealed targets suggest an involvement of Stp1 in the general adaption to nutrient availability as well as in the environmental stress response. With the focus on protein utilization and nitrogen-regulated pathogenesis, the regulation of the major secreted aspartic protease Sap2 - additionally one of the prime examples of allelic heterogeneity in C. albicans - was investigated in detail. Thereby, the heterogezygous SAP2 promoter helped to identify an unintended genomic alteration as the true cause of a growth defect of a C. albicans mutant. Additionally, the promoter region, which was responsible for the differential activation of the SAP2 alleles, was delimited. Furthermore, general Sap2 induction was demonstrated to be mediated by distinct cis-acting elements that are required for a high or a low activity of SAP2 expression. For the utilization of proteins as nitrogen source it is also crucial to take up the peptides that are produced by extracellular proteolysis. Therefore, the function and importance of specific peptide transporters was investigated in C. albicans mutants, unable to use peptides as nitrogen source (opt1Δ/Δ opt2Δ/Δ opt3Δ/Δ opt4Δ/Δ opt5Δ/Δ ptr2Δ/Δ ptr22Δ/Δ septuple null mutants). The overexpression of individual transporters in these mutants revealed differential substrate specificities and expanded the specificity of the OPTs to dipeptides, a completely new facet of these transporters. The peptide-uptake deficient mutants were further used to elucidate, whether indeed proteins and peptides are an important in vivo nitrogen source for C. albicans. It was found that during competitive colonization of the mouse intestine these mutants exhibited wild-type fitness, indicating that neither proteins nor peptides are primary nitrogen sources required to efficiently support growth of C. albicans in the mouse gut. Adequate availability of the preferred nitrogen source ammonium represses the utilization of proteins and other alternative nitrogen sources, but also the expression of virulence attributes, like Sap secretion and nitrogen-starvation induced filamentation. In order to discriminate, whether ammonium availability is externally sensed or determined inside the cell by C. albicans, the response to exterior ammonium concentrations of ammonium-uptake deficient mutants (mep1Δ/Δ mep2Δ/Δ null mutants) was investigated. This study showed that presence of an otherwise suppressing ammonium concentration did not inhibit Sap2 proteases secretion and arginine-induced filamentation in these mutants. Conclusively, ammonium availability is primarily determined inside the cell in order to control the expression of virulence traits. In sum, the present work contributes to the current understanding of how C. albicans regulates expression of virulence-associated traits in response to the presence of available nitrogen sources - especially proteins and peptides - in order to adapt its lifestyle within a human host.}, subject = {Candida albicans}, language = {en} } @article{DugarSvenssonBischleretal.2016, author = {Dugar, Gaurav and Svensson, Sarah L. and Bischler, Thorsten and Waldchen, Sina and Reinhardt, Richard and Sauer, Markus and Sharma, Cynthia M.}, title = {The CsrA-FliW network controls polar localization of the dual-function flagellin mRNA in Campylobacter jejuni}, series = {Nature Communications}, volume = {7}, journal = {Nature Communications}, doi = {10.1038/ncomms11667}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-173201}, year = {2016}, abstract = {The widespread CsrA/RsmA protein regulators repress translation by binding GGA motifs in bacterial mRNAs. CsrA activity is primarily controlled through sequestration by multiple small regulatory RNAs. Here we investigate CsrA activity control in the absence of antagonizing small RNAs by examining the CsrA regulon in the human pathogen Campylobacter jejuni. We use genome-wide co-immunoprecipitation combined with RNA sequencing to show that CsrA primarily binds flagellar mRNAs and identify the major flagellin mRNA (flaA) as the main CsrA target. The flaA mRNA is translationally repressed by CsrA, but it can also titrate CsrA activity. Together with the main C. jejuni CsrA antagonist, the FliW protein, flaA mRNA controls CsrA-mediated post-transcriptional regulation of other flagellar genes. RNA-FISH reveals that flaA mRNA is expressed and localized at the poles of elongating cells. Polar flaA mRNA localization is translation dependent and is post-transcriptionally regulated by the CsrA-FliW network. Overall, our results suggest a role for CsrA-FliW in spatiotemporal control of flagella assembly and localization of a dual-function mRNA.}, language = {en} } @phdthesis{Dude2009, author = {Dude, Marie-Adrienne}, title = {Die Expression der Multiadh{\"a}sionsdom{\"a}nenproteine PfCCp5 und PfFNPA in Plasmodium falciparum und Cysteinprotease-Inhibitoren als potentielle Wirkstoffe gegen Malaria}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-45863}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2009}, abstract = {Der Erreger der Malaria tropica, Plasmodium falciparum, ist f{\"u}r eine j{\"a}hrliche Todesrate von {\"u}ber einer Million Menschen verantwortlich. Rasch zunehmende Erregerresistenzen gegen g{\"a}ngige Antimalariamedikamente und das Fehlen eines Impfstoffes machen die Suche nach neuen therapeutischen Ans{\"a}tzen und Medikamenten unerl{\"a}sslich. Sexualstadienspezifische Oberfl{\"a}chenproteine des Parasiten sind attraktive Zielstrukturen f{\"u}r die Entwicklung von TBV, welche eine Entwicklung von P. falciparum in der M{\"u}cke unterbrechen. Die Suche nach multiplen tier- oder bakterien{\"a}hnlichen, extrazellul{\"a}ren Adh{\"a}sionsdom{\"a}nen im Genom von P. falciparum f{\"u}hrte zur Identifizierung einer Familie von sechs Proteinen mit hochkonservierten Adh{\"a}sionsmodulen, die vermutlich an Parasit-Parasit- oder Parasit-Wirtsinteraktionen beteiligt sind, was sie zu potentiellen Kandidaten f{\"u}r Komponenten von TBV macht. Aufgrund ihrer gemeinsamen LCCL-Dom{\"a}ne wurden diese Proteine PfCCp1 bis PfCCp5 sowie PfFNPA benannt. PfFNPA besitzt keine LCCL-Dom{\"a}ne, es ist jedoch {\"a}hnlich aufgebaut wie PfCCp5 und wurde daher mit in die PfCCp-Familie integriert. Die in der parasitophoren Vakuole reifer Gametozyten lokalisierenden PfCCp1- bis PfCCp3-Proteine werden w{\"a}hrend der Gametogenese teilweise freigesetzt und umgeben matrix{\"a}hnlich entstehende Exflagellationszentren. In PfCCp2- und PfCCp3-defizienten Parasiten ist die Wanderung der Sporozoiten aus den Mitteldarmoozysten in die Speicheldr{\"u}sen der M{\"u}cke blockiert. Sexualstadien-spezifische Expression und eine wichtige Funktion bei der Entwicklung des Erregers in der M{\"u}cke sind die Hauptkriterien f{\"u}r potentielle TBV-Kandidaten. Diese viel versprechenden Daten waren Anlass, in der vorliegenden Arbeit, die bisher nur hypothetischen PfCCp5- und PfFNPA-Proteine genauer zu untersuchen. Expressionsstudien von PfCCp5 und PfFNPA mittels RT-PCR, Western-Blot-, Immunfluoreszenz- und Transmissionselektronenmikroskopischen-Analysen zeigten, dass sie sowohl plasmamembranassoziiert in der parasitophoren Vakuole als auch intrazellul{\"a}r in reifen Gametozyten exprimiert werden. Beide Proteine sind in Gameto-zyten ab dem Stadium II detektierbar und weisen in unreifen Gametozyten ein punktiertes Expressionsmuster auf. In reifen Gametozyten konzentriert sich ihre Expression dagegen v. a. auf die Zellpole. Ferner werden PfCCp5 und PfFNPA auf der Oberfl{\"a}che von Makrogameten, jedoch nicht in Mikrogameten und Ookineten exprimiert. Zus{\"a}tzlich wird PfCCp5 in einem Teil reifer Schizonten eines gametozyten-bildenden Parasiten-Stammes exprimiert. Durch Integration eines Komplementations-Konstukts in die 3-untranslatierte Region von PfCCp5 bzw. PfFNPA konnte gezeigt werden, dass beide Gene genetisch manipulierbar sind. Mit PfCCp5- bzw. PfFNPA-KO-Konstrukten transfizierte WT-Parasiten wachsen nach erfolgter positiver Selektion jedoch nicht mehr. Diese Daten lassen vermuten, dass PfCCp5 und PfFNPA eine essentielle Funktion in den Blutstadien bzw. bei Gametozytenbildung haben. Zur weiteren Analyse von PfFNPA wurde ein verk{\"u}rztes Protein durch Integration eines weiteren PfFNPA-KO-Konstrukts in den Locus von WT-Parasiten generiert. Erste Analysen des PfFNPA-KO-Ph{\"a}notyps deuten darauf hin, dass durch die Ausschaltung der 3'-Region des Gens das Protein nicht mehr korrekt exprimiert wird, obwohl keine morphologischen Ver{\"a}nderungen der Blutstadien des Parasiten feststellbar sind. Außerdem werden PfCCp5 und PfFNPA ko-abh{\"a}ngig in PfCCp1-, PfCCp2- und PfCCp3-KO-Gametozyten exprimiert. Ko-Immunpr{\"a}zipitationsstudien zeigten, dass beide Proteine mit den anderen PfCCp-Mitgliedern interagieren. Affinit{\"a}tschromato-graphiestudien deckten dann direkte Interaktionen einzelner PfCCp-Dom{\"a}nen auf. Hierbei sind v. a. die LCCL-, die SR- und die NEC- Dom{\"a}ne an Proteininteraktionen beteiligt, was die Hypothese einer Komplexbildung der PfCCp-Familie w{\"a}hrend der Gametogenese des Erregers st{\"u}tzt. Transmissionsblockierungsstudien sollen nun die Eignung ausgew{\"a}hlter PfCCp-Proteine als TBV-Komponenten n{\"a}her beleuchten. Zunehmende Resistenzen gegen gebr{\"a}uchliche Malariamedikamente veranlassen zur Suche nach neuen Angriffspunkten zur Behandlung der Erkrankung. Die maßgeblich an der H{\"a}moglobinhydrolyse beteiligten plasmodialen Cysteinproteasen Falcipain-2 und Falcipain-3 sind m{\"o}gliche Ziele f{\"u}r die Entwicklung neuer Antimalariawirkstoffe. In der vorliegenden Arbeit wurden peptidomimetische 1,4-Benzodiazepin- und nicht-peptidische Etacryns{\"a}urederivate in vitro auf ihre antiplasmodiale Wirkung an P. falciparum-Blutstadien getestet. Ein erstes Screening hatte gezeigt, dass die eine Vinylsulfonkopfgruppe tragenden 1,4 Benzodiazepinderivate rekombinant exprimiertes Falcipain-2 irreversibel hemmen. In vitro konnte dann auch eine antiplasmodiale Aktivit{\"a}t f{\"u}r diese Verbindungen festgestellt werden. Dockingstudien und HPLC-Assays mit den Etacryns{\"a}urederivaten deckten eine Hemmung der Cysteinprotease Papain und der SARS-Mpro-Hauptprotease der Coronaviren auf. Weiterhin konnte in einem Screening an rekombinant exprimiertem Falcipain-2 und Falcipain-3 eine inhibitorische Wirkung f{\"u}r einen Teil dieser Etacryns{\"a}urederivate festgestellt werden. Der In-vitro-Test an P. falciparum-Blutstadien deckte dann eine schwache antiplasmodiale Aktivit{\"a}t von fluorsubstituierten Etacryns{\"a}urederivaten und von Derivaten mit einer modifizierten Etacryns{\"a}urepartialstruktur auf. Der viel versprechendste Inhibitor dieser Studie wurde nun zur Identifizierung potentieller Bindungspartner mittels Affinit{\"a}tsbindungsstudien biotyniliert. Zusammenfassend besitzen beide getesteten Wirkstoffklassen eine inhibierende Aktivit{\"a}t gegen{\"u}ber Cysteinproteasen womit sie die Grundlage f{\"u}r die Entwicklung neuer, effektiverer plasmodialer Cysteinproteaseinhibitoren bieten.}, subject = {Plasmodium falciparum}, language = {de} } @phdthesis{Donat2009, author = {Donat, Stefanie}, title = {Molekulare und funktionelle Charakterisierung der Serin/Threonin-Proteinkinase PknB und -Phosphatase Stp in Staphylococcus aureus}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-41893}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2009}, abstract = {Um {\"A}nderungen in seiner Umwelt wahrnehmen zu k{\"o}nnen, ben{\"o}tigt S. aureus unterschiedliche Signaltransduktionssysteme. In dieser Arbeit wurde erstmals die Eukaryoten-{\"a}hnliche Serin/Threonin-Proteinkinase (STPK) PknB umfassend charakterisiert. Die posttranslationale Proteinmodifikation mittels Phosphorylierung spielt sowohl in Eukaryoten als auch in Prokaryoten eine wichtige Rolle. Man glaubte lange, dass die Phosphorylierung von Serin-, Threonin- und Tyrosinresten ein nur auf Eukaryoten beschr{\"a}nkter Regulationsmechanismus ist. Dagegen wurde die Phosphorylierung an Histidin- und Aspartatresten durch die Zweikomponenten-Systeme allein den Prokaryoten zugeordnet. Die Genomanalysen der letzten Jahre identifizierten jedoch STPKs und Serin/Threonin-Proteinphosphatasen (STPP) in nahezu allen prokaryotischen Genomen. Auch S. aureus codiert f{\"u}r eine STPK, die eine hohe Homologie zu den beschriebenen STPKs aufweist. In dieser Arbeit wurden mittels Microarray-Analyse einer \&\#916;pknB-Mutante im Stamm 8325 erste Hinweise zur Funktion von PknB als Regulator der Zellwandsynthese sowie zentraler Stoffwechselwege gewonnen. Es wurden mittels Phosphopreoteom-Analysen in vivo-Substrate identifiziert und weiterhin die Kinase biochemisch charakterisiert.}, subject = {Staphylococcus aureus}, language = {de} } @article{DischingerHeckelBischleretal.2021, author = {Dischinger, Ulrich and Heckel, Tobias and Bischler, Thorsten and Hasinger, Julia and K{\"o}nigsrainer, Malina and Schmitt-B{\"o}hrer, Angelika and Otto, Christoph and Fassnacht, Martin and Seyfried, Florian and Hankir, Mohammed Khair}, title = {Roux-en-Y gastric bypass and caloric restriction but not gut hormone-based treatments profoundly impact the hypothalamic transcriptome in obese rats}, series = {Nutrients}, volume = {14}, journal = {Nutrients}, number = {1}, issn = {2072-6643}, doi = {10.3390/nu14010116}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-252392}, year = {2021}, abstract = {Background: The hypothalamus is an important brain region for the regulation of energy balance. Roux-en-Y gastric bypass (RYGB) surgery and gut hormone-based treatments are known to reduce body weight, but their effects on hypothalamic gene expression and signaling pathways are poorly studied. Methods: Diet-induced obese male Wistar rats were randomized into the following groups: RYGB, sham operation, sham + body weight-matched (BWM) to the RYGB group, osmotic minipump delivering PYY3-36 (0.1 mg/kg/day), liraglutide s.c. (0.4 mg/kg/day), PYY3-36 + liraglutide, and saline. All groups (except BWM) were kept on a free choice of high- and low-fat diets. Four weeks after interventions, hypothalami were collected for RNA sequencing. Results: While rats in the RYGB, BWM, and PYY3-36 + liraglutide groups had comparable reductions in body weight, only RYGB and BWM treatment had a major impact on hypothalamic gene expression. In these groups, hypothalamic leptin receptor expression as well as the JAK-STAT, PI3K-Akt, and AMPK signaling pathways were upregulated. No significant changes could be detected in PYY3-36 + liraglutide-, liraglutide-, and PYY-treated groups. Conclusions: Despite causing similar body weight changes compared to RYGB and BWM, PYY3-36 + liraglutide treatment does not impact hypothalamic gene expression. Whether this striking difference is favorable or unfavorable to metabolic health in the long term requires further investigation.}, language = {en} } @article{DingemansMonsieursYuetal.2016, author = {Dingemans, Josef and Monsieurs, Pieter and Yu, Sung-Huan and Crabb{\´e}, Aur{\´e}lie and F{\"o}rstner, Konrad U. and Malfroot, Anne and Cornelis, Pierre and Van Houdt, Rob}, title = {Effect of Shear Stress on Pseudomonas aeruginosa Isolated from the Cystic Fibrosis Lung}, series = {mBio}, volume = {7}, journal = {mBio}, number = {4}, doi = {10.1128/mBio.00813-16}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-165821}, pages = {e00813-16}, year = {2016}, abstract = {Chronic colonization of the lungs by Pseudomonas aeruginosa is one of the major causes of morbidity and mortality in cystic fibrosis (CF) patients. To gain insights into the characteristic biofilm phenotype of P. aeruginosa in the CF lungs, mimicking the CF lung environment is critical. We previously showed that growth of the non-CF-adapted P. aeruginosa PAO1 strain in a rotating wall vessel, a device that simulates the low fluid shear (LS) conditions present in the CF lung, leads to the formation of in-suspension, self-aggregating biofilms. In the present study, we determined the phenotypic and transcriptomic changes associated with the growth of a highly adapted, transmissible P. aeruginosa CF strain in artificial sputum medium under LS conditions. Robust self-aggregating biofilms were observed only under LS conditions. Growth under LS conditions resulted in the upregulation of genes involved in stress response, alginate biosynthesis, denitrification, glycine betaine biosynthesis, glycerol metabolism, and cell shape maintenance, while genes involved in phenazine biosynthesis, type VI secretion, and multidrug efflux were downregulated. In addition, a number of small RNAs appeared to be involved in the response to shear stress. Finally, quorum sensing was found to be slightly but significantly affected by shear stress, resulting in higher production of autoinducer molecules during growth under high fluid shear (HS) conditions. In summary, our study revealed a way to modulate the behavior of a highly adapted P. aeruginosa CF strain by means of introducing shear stress, driving it from a biofilm lifestyle to a more planktonic lifestyle.}, language = {en} } @article{DimastrogiovanniFroehlichBandyraetal.2014, author = {Dimastrogiovanni, Daniela and Fr{\"o}hlich, Kathrin S. and Bandyra, Katarzyna J. and Bruce, Heather A. and Hohensee, Susann and Vogel, J{\"o}rg and Luisi, Ben F.}, title = {Recognition of the small regulatory RNA RydC by the bacterial Hfq protein}, series = {eLife}, volume = {3}, journal = {eLife}, number = {e05375}, issn = {2050-084X}, doi = {10.7554/eLife.05375}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-114191}, year = {2014}, abstract = {Bacterial small RNAs (sRNAs) are key elements of regulatory networks that modulate gene expression. The sRNA RydC of Salmonella sp. and Escherichia coli is an example of this class of riboregulators. Like many other sRNAs, RydC bears a 'seed' region that recognises specific transcripts through base-pairing, and its activities are facilitated by the RNA chaperone Hfq. The crystal structure of RydC in complex with E. coli Hfq at 3.48 angstrom resolution illuminates how the protein interacts with and presents the sRNA for target recognition. Consolidating the protein-RNA complex is a host of distributed interactions mediated by the natively unstructured termini of Hfq. Based on the structure and other data, we propose a model for a dynamic effector complex comprising Hfq, small RNA, and the cognate mRNA target.}, language = {en} } @phdthesis{Dietrich2000, author = {Dietrich, Claudia}, title = {Molekularbiologische Studien zur Bedeutung der Flagelle f{\"u}r die Virulenz von Legionella pneumophila}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-1081}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2000}, abstract = {Legionella pneumophila, der Erreger der Legion{\"a}rskrankheit, ist ein fakultativ intrazellul{\"a}res, ubiquit{\"a}r vorkommendes Umweltbakterium. Die Rolle, die Flagelle und Motilit{\"a}t der Legionellen bei der Infektion von Protozoen oder humanen Zellen spielen k{\"o}nnen, ist bisher noch nicht gekl{\"a}rt. Um etwas {\"u}ber noch unbekannte Flagellengene und deren Organisation in Legionella zu erfahren, wurde mit Hilfe einer Cosmid-Genbank des Stammes L. pneumophila Philadelphia I die flaA-Region n{\"a}her charakterisiert. Im 5´-Bereich von flaA konnten auf dem Gegenstrang zwei Stoffwechselgene (accD und folC) identifiziert werden, im 3´-Bereich schliessen sich die Flagellengene flaG, fliD und fliS, sowie zwei offene Leseraster mit Homologien zu den erst k{\"u}rzlich bei Legionella beschriebenen Genen enhA und milA an. Zur Untersuchung des Einflusses der Flagelle auf den Infektionsverlauf wurde die flaA-negative Mutante KH3, bei der das flaA-Gen durch Insertion einer Kanamycin-Kassette unterbrochen worden war, wieder komplementiert. Dies gelang durch Reintegration des intakten flaA-Gens mit Hilfe des „Suicide"-Vektors pMSS704 in das Chromosom von KH3, wodurch Stamm CD10 entstand. Durch Westernblot-Analyse konnte gezeigt werden, dass der Stamm wieder in der Lage war, Flagellin zu exprimieren. Elektronenmikroskopische Aufnahmen best{\"a}tigten außerdem das Vorhandensein intakter Flagellen. Das Verhalten von flagellierten und nicht flagellierten Legionellen bei der Infektion von Wirtszellen wurde hinsichtlich Auffinden, Adh{\"a}renz, Invasion, intrazellul{\"a}rer Vermehrung und Lyse der Zellen untersucht. Als Wirtszellen wurden sowohl Protozoen (Acanthamoeba castellanii), als auch humane Zellen (HL-60 Zellen und frisch isolierte Blutmonozyten) verwendet. Dabei wurde deutlich, dass die Flagelle f{\"u}r das Erreichen der Wirtszellen eine wichtige Funktion hat. Wurde der Motilit{\"a}tsdefekt der flaA-Mutanten durch Zentrifugation auf die Zielzellen aufgehoben, so konnten mit den gew{\"a}hlten Versuchsbedingungen bez{\"u}glich des Adh{\"a}renzverm{\"o}gens der St{\"a}mme keine Unterschiede detektiert werden. Es wurde jedoch eine signifikante Reduktion der Invasionseffizienz f{\"u}r die nicht flagellierten Legionellen beobachtet. Diese war bei den humanen Zellen besonders ausgepr{\"a}gt. Hinsichtlich der intrazellul{\"a}ren Vermehrung konnte keine Attenuierung der Mutante festgestellt werden. Allerdings f{\"u}hrte vermutlich die Reduktion der Invasivit{\"a}t zu einer geringeren Ausbreitungsgeschwindigkeit im HL-60 Modell, die bei niedriger Infektionsdosis mit einer verlangsamten Wachstumsrate der Bakterien einherging. Durch Sequenzierung des Genbank-Cosmids 12/44, auf welchem die Gene fliA und motA lokalisiert waren, konnten im „upstream"-Bereich von fliA zwei putative Flagellenregulatorgene identifiziert werden (motR und flhF). Im 3´-Bereich von motA schließt sich, um 26 bp {\"u}berlappend, das Gen motB an, welches f{\"u}r den Motor der Flagelle eine Rolle spielt, gefolgt von einem Leseraster unbekannter Funktion und einem ORF mit Homologien zu prfB. Durch Insertion einer Kanamycin-Kassette in das motA-Gen von L. pneumophila Corby konnte in dieser Arbeit eine motA-negative Mutante hergestellt werden. Westernblot-Analyse und elektronenmikroskopische Untersuchungen best{\"a}tigten, dass es weiterhin zur Expression und zur Polymerisation des Flagellins kommt. Lichtmikroskopisch war jedoch zu beobachten, dass die hergestellte Mutante im Gegensatz zum Wildtyp durch den fehlerhaften Flagellenmotor nicht mehr in der Lage ist, gerichtete Strecken zu schwimmen. Untersuchungen mit den Wirtszellen A. castellanii und humanen HL-60 Zellen belegten, wie schon bei der flaA-Mutante, eine Beteiligung der Motilit{\"a}t an Vorg{\"a}ngen wie Auffinden der Zielzelle und deren Invasion, wohingegen die Adh{\"a}renz und die intrazellul{\"a}re Vermehrung nicht beeintr{\"a}chtigt waren. Eine Southernblot-Analyse des erst k{\"u}rzlich beschriebenen Transkriptionsregulators FlaR ergab, dass es sich hierbei vermutlich um einen L. pneumophila-spezifischen Regulationsfaktor handelt, welcher in Kombination mit dem „upstream" auf dem Gegenstrang liegenden ORF234 vorkommt. Fusionen der Promotorbereiche mit dem Reportergen gfp zeigten, dass beide Gene auch in Legionella aktiv sind und temperaturabh{\"a}ngig reguliert werden.}, subject = {Legionella pneumophila}, language = {de} } @article{DembekBarquistBoinettetal.2015, author = {Dembek, Marcin and Barquist, Lars and Boinett, Christine J. and Cain, Amy K. and Mayho, Matthew and Lawley, Trevor D. and Fairweather, Neil F. and Fagan, Robert P.}, title = {High-throughput analysis of gene essentiality and sporulation in Clostridium difficile}, series = {mBio}, volume = {6}, journal = {mBio}, number = {2}, doi = {10.1128/mBio.02383-14}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-143745}, pages = {e02383-14}, year = {2015}, abstract = {Clostridium difficile is the most common cause of antibiotic-associated intestinal infections and a significant cause of morbidity and mortality. Infection with C. difficile requires disruption of the intestinal microbiota, most commonly by antibiotic usage. Therapeutic intervention largely relies on a small number of broad-spectrum antibiotics, which further exacerbate intestinal dysbiosis and leave the patient acutely sensitive to reinfection. Development of novel targeted therapeutic interventions will require a detailed knowledge of essential cellular processes, which represent attractive targets, and species-specific processes, such as bacterial sporulation. Our knowledge of the genetic basis of C. difficile infection has been hampered by a lack of genetic tools, although recent developments have made some headway in addressing this limitation. Here we describe the development of a method for rapidly generating large numbers of transposon mutants in clinically important strains of C. difficile. We validated our transposon mutagenesis approach in a model strain of C. difficile and then generated a comprehensive transposon library in the highly virulent epidemic strain R20291 (027/BI/NAP1) containing more than 70,000 unique mutants. Using transposon-directed insertion site sequencing (TraDIS), we have identified a core set of 404 essential genes, required for growth in vitro. We then applied this technique to the process of sporulation, an absolute requirement for C. difficile transmission and pathogenesis, identifying 798 genes that are likely to impact spore production. The data generated in this study will form a valuable resource for the community and inform future research on this important human pathogen.}, language = {en} } @article{CzimmererDanielHorvathetal.2018, author = {Czimmerer, Zsolt and Daniel, Bence and Horvath, Attila and R{\"u}ckerl, Dominik and Nagy, Gergely and Kiss, Mate and Peloquin, Matthew and Budai, Marietta M. and Cuaranta-Monroy, Ixchelt and Simandi, Zoltan and Steiner, Laszlo and Nagy Jr., Bela and Poliska, Szilard and Banko, Csaba and Bacso, Zsolt and Schulman, Ira G. and Sauer, Sascha and Deleuze, Jean-Francois and Allen, Judith E. and Benko, Szilvia and Nagy, Laszlo}, title = {The Transcription Factor STAT6 Mediates Direct Repression of Inflammatory Enhancers and Limits Activation of Alternatively Polarized Macrophages}, series = {Immunity}, volume = {48}, journal = {Immunity}, doi = {10.1016/j.immuni.2017.12.010}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-223380}, pages = {75-90}, year = {2018}, abstract = {The molecular basis of signal-dependent transcriptional activation has been extensively studied in macrophage polarization, but our understanding remains limited regarding the molecular determinants of repression. Here we show that IL-4-activated STAT6 transcription factor is required for the direct transcriptional repression of a large number of genes during in vitro and in vivo alternative macrophage polarization. Repression results in decreased lineage-determining transcription factor, p300, and RNA polymerase II binding followed by reduced enhancer RNA expression, H3K27 acetylation, and chromatin accessibility. The repressor function of STAT6 is HDAC3 dependent on a subset of IL-4-repressed genes. In addition, STAT6-repressed enhancers show extensive overlap with the NF-κB p65 cistrome and exhibit decreased responsiveness to lipopolysaccharide after IL-4 stimulus on a subset of genes. As a consequence, macrophages exhibit diminished inflammasome activation, decreased IL-1β production, and pyroptosis. Thus, the IL-4-STAT6 signaling pathway establishes an alternative polarization-specific epigenenomic signature resulting in dampened macrophage responsiveness to inflammatory stimuli.}, language = {en} } @article{CullLimaPradoGodinhoFernandesRodriguesetal.2014, author = {Cull, Benjamin and Lima Prado Godinho, Joseane and Fernandes Rodrigues, Juliany Cola and Frank, Benjamin and Schurigt, Uta and Williams, Roderick AM and Coombs, Graham H and Mottram, Jeremy C}, title = {Glycosome turnover in Leishmania major is mediated by autophagy}, series = {Autophagy}, volume = {10}, journal = {Autophagy}, number = {12}, doi = {10.4161/auto.36438}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-150277}, pages = {2143-2157}, year = {2014}, abstract = {Autophagy is a central process behind the cellular remodeling that occurs during differentiation of Leishmania, yet the cargo of the protozoan parasite's autophagosome is unknown. We have identified glycosomes, peroxisome-like organelles that uniquely compartmentalize glycolytic and other metabolic enzymes in Leishmania and other kinetoplastid parasitic protozoa, as autophagosome cargo. It has been proposed that the number of glycosomes and their content change during the Leishmania life cycle as a key adaptation to the different environments encountered. Quantification of RFP-SQL-labeled glycosomes showed that promastigotes of L. major possess ~20 glycosomes per cell, whereas amastigotes contain ~10. Glycosome numbers were significantly greater in promastigotes and amastigotes of autophagy-defective L. major Δatg5 mutants, implicating autophagy in glycosome homeostasis and providing a partial explanation for the previously observed growth and virulence defects of these mutants. Use of GFP-ATG8 to label autophagosomes showed glycosomes to be cargo in ~15\% of them; glycosome-containing autophagosomes were trafficked to the lysosome for degradation. The number of autophagosomes increased 10-fold during differentiation, yet the percentage of glycosome-containing autophagosomes remained constant. This indicates that increased turnover of glycosomes was due to an overall increase in autophagy, rather than an upregulation of autophagosomes containing this cargo. Mitophagy of the single mitochondrion was not observed in L. major during normal growth or differentiation; however, mitochondrial remnants resulting from stress-induced fragmentation colocalized with autophagosomes and lysosomes, indicating that autophagy is used to recycle these damaged organelles. These data show that autophagy in Leishmania has a central role not only in maintaining cellular homeostasis and recycling damaged organelles but crucially in the adaptation to environmental change through the turnover of glycosomes.}, language = {en} } @article{CorreiaSantosBischlerWestermannetal.2021, author = {Correia Santos, Sara and Bischler, Thorsten and Westermann, Alexander J. and Vogel, J{\"o}rg}, title = {MAPS integrates regulation of actin-targeting effector SteC into the virulence control network of Salmonella small RNA PinT}, series = {Cell Reports}, volume = {34}, journal = {Cell Reports}, number = {5}, doi = {10.1016/j.celrep.2021.108722}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259134}, pages = {108722}, year = {2021}, abstract = {A full understanding of the contribution of small RNAs (sRNAs) to bacterial virulence demands knowledge of their target suites under infection-relevant conditions. Here, we take an integrative approach to capturing targets of the Hfq-associated sRNA PinT, a known post-transcriptional timer of the two major virulence programs of Salmonella enterica. Using MS2 affinity purification and RNA sequencing (MAPS), we identify PinT ligands in bacteria under in vitro conditions mimicking specific stages of the infection cycle and in bacteria growing inside macrophages. This reveals PinT-mediated translational inhibition of the secreted effector kinase SteC, which had gone unnoticed in previous target searches. Using genetic, biochemical, and microscopic assays, we provide evidence for PinT-mediated repression of steC mRNA, eventually delaying actin rearrangements in infected host cells. Our findings support the role of PinT as a central post-transcriptional regulator in Salmonella virulence and illustrate the need for complementary methods to reveal the full target suites of sRNAs.}, language = {en} } @phdthesis{Cho2001, author = {Cho, Seung-Hak}, title = {Epidemiologische und molekulare Untersuchungen zur Biofilmbildung in Staphylococcus epidermidis und Staphylococcus aureus}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-1181296}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2001}, abstract = {Staphylococcus aureus und Staphylococcus epidermidis geh{\"o}ren zu den h{\"a}ufigsten Erregern nosokomialer Infektionen bei immunsupprimierten Patienten. Gleichzeitig bilden diese Bakterien einen wesentlichen Teil der gesunden Hautflora des Menschen. Bisher ist wenig dar{\"u}ber bekannt, ob es Unterschiede in der genetischen Ausstattung zwischen klinischen und kommensalen Isolaten gibt und welche Faktoren zur Etablierung von Staphylokokken im Hospitalmilieu beitragen. Die Ergebnisse der vorliegenden Arbeit zeigen, daß die F{\"a}higkeit zur Biofilmbildung offensichtlich ein wesentliches Merkmal pathogener Staphylokokken ist. Die Expression dieses Virulenzfaktors ist dabei hochvariabel und h{\"a}ngt von der genetischen Ausstattung der St{\"a}mme mit dem f{\"u}r die Biofilmbildung verantwortlichen ica-Operon, bestimmten Umweltfaktoren und dem Einfluß von Insertionssequenzen ab. In einer epidemiologische Untersuchung wurde gezeigt, daß in S. epidermidis das ica-Operon h{\"a}ufiger in klinischen als in kommensalen St{\"a}mmen vorkommt. Der {\"u}berwiegende Teil dieser ica-positiven St{\"a}mme bildete ph{\"a}notypisch einen Biofilm aus. Im Unterschied dazu enthielten alle untersuchten S. aureus-St{\"a}mme, unabh{\"a}ngig von ihrer Herkunft, das vollst{\"a}ndige ica-Gencluster, wobei jedoch keiner dieser St{\"a}mme unter Laborbedingungen einen Biofilm bildete. Durch subinhibitorischen Konzentrationen bestimmter Antibiotika bzw. durch Osmostress ließ sich die Biofilmbildung in 30 Prozent der S. aureus-St{\"a}mme induzieren. Ebenso konnte in ica-positiven S. epidermidis-St{\"a}mmen die Biofilmbildung dirch diese Umweltfaktoren stimuliert werden. Die Studie ergab auch, daß es einen Zusammenhang zwischen der Biofilmbildung, der Antibiotikaresistenz und dem Vorkommen der Insertionssequenz IS256 gibt. So war IS256 signifikant h{\"a}ufig in klinischen S. epidermidis und S. aureus-St{\"a}mmen nachweisbar, w{\"a}hrend es keinen Unterschied im Auftreten von IS257 zwischen klinischen und saprophyt{\"a}ren Isolaten gab. Die IS256-positiven S. epidermidis-St{\"a}mme wiesen {\"u}berdurchschnittlich oft das ica-Operon auf und waren gegen mindestens zwei Antibiotika gleichzeitig resistent. Weiterhin konnte gezeigt werden, daß IS256 an der Phasenvariation der Biofilmbildung in vivo beteiligt ist. Bei einem klinischen S. epidermidis-Stamm, der von einem Patienten mit einer Katheter-assoziierten Harnwegsinfektion isoliert wurde, wurde die Insertion des Elementes im icaC-Gen nachgewiesen, was in einem Biofilm-negativen Ph{\"a}notyp resultierte. Subkultivierung der Insertionsmutante f{\"u}hrte nach wenigen Passagen zur Ausbildung eines Biofilms. Die Nukleotidsequenzierung ergab die vollst{\"a}ndige Exzision von IS256 aus dem icaC-Gen einschließlich der duplizierten Zielsequenz von sieben Basenpaaren. Diese Daten stimmen vollst{\"a}ndig mit den zuvor in einer in-vitro-Studie erhaltenenen Ergebnissen {\"u}berein und sie zeigen, daß IS256 die Expression des ica-Operons offensichtlich auch in vivo w{\"a}hrend einer Infektion beeinflußt. Bei S. aureus konnte in dieser Arbeit ebenfalls eine Phasenvariation der Biofilmexpression nachgewiesen werden. Durch Mehrfachpassagen wurden aus ehemals Biofilm-negativen Einzelkolonien mehrere Biofilmproduzenten gewonnen, die auch wieder zum Biofilm-negativen Ph{\"a}notyp revertieren konnten. Die DNA-Analyse mittels Pulsfeldgelelektrophorese zeigte, daß es in den varianten St{\"a}mmen zu gr{\"o}ßeren DNA-Rearrangements gekommen war, die neben der variablen Biofilmbildung auch mit Unterschieden in der Expression des alternativen Transkriptionsfaktors SigmaB einhergingen. Die Nukleotidsequenzierung des sigB-Systems ergab in den Varianten mehrere Punktmutationen in den SigB-Regulatorgenen rsbU und rsbW. Dies legt nahe, daß der SigB-Genlokus einer starken genetischen Variabilit{\"a}t unterliegt, die wiederum pleiotrope Effekte auf die Genexpression in S. aureus aus{\"u}bt. Durch Northern-Blot-Analysen konnte allerdings gezeigt werden, daß die Biofilmbildung in den S. aureus-Varianten nicht mit der ver{\"a}nderten SigB-Expression in Zusammenhang steht.}, subject = {Staphylococcus aureus}, language = {de} } @article{ChenYuZhangetal.2011, author = {Chen, Nanhai G. and Yu, Yong A. and Zhang, Qian and Szalay, Aladar A.}, title = {Replication efficiency of oncolytic vaccinia virus in cell cultures prognosticates the virulence and antitumor efficacy in mice}, series = {Journal of Translational Medicine}, volume = {9}, journal = {Journal of Translational Medicine}, number = {164}, doi = {10.1186/1479-5876-9-164}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142268}, pages = {1-11}, year = {2011}, abstract = {Background: We have shown that insertion of the three vaccinia virus (VACV) promoter-driven foreign gene expression cassettes encoding Renilla luciferase-Aequorea GFP fusion protein, beta-galactosidase, and beta-glucuronidase into the F14.5L, J2R, and A56R loci of the VACV LIVP genome, respectively, results in a highly attenuated mutant strain GLV 1h68. This strain shows tumor specific replication and is capable of eradicating tumors with little or no virulence in mice. This study aimed to distinguish the contribution of added VACV promoter-driven transcriptional units as inserts from the effects of insertional inactivation of three viral genes, and to determine the correlation between replication efficiency of oncolytic vaccinia virus in cell cultures and the virulence and antitumor efficacy in mice Methods: A series of recombinant VACV strains was generated by replacing one, two, or all three of the expression cassettes in GLV 1h68 with short non coding DNA sequences. The replication efficiency and tumor cell killing capacity of these newly generated VACV strains were compared with those of the parent virus GLV-1h68 in cell cultures. The virus replication efficiency in tumors and antitumor efficacy as well as the virulence were evaluated in nu/nu (nude) mice bearing human breast tumor xenografts. Results: we found that virus replication efficiency increased with removal of each of the expression cassettes. The increase in virus replication efficiency was proportionate to the strength of removed VACV promoters linked to foreign genes. The replication efficiency of the new VACV strains paralleled their cytotoxicity in cell cultures. The increased replication efficiency in tumor xenografts resulted in enhanced antitumor efficacy in nude mice. Similarly, the enhanced virus replication efficiency was indicative of increased virulence in nude mice. Conclusions: These data demonstrated that insertion of VACV promoter-driven transcriptional units into the viral genome for the purpose of insertional mutagenesis did modulate the efficiency of virus replication together with antitumor efficacy as well as virulence. Replication efficiency of oncolytic VACV in cell cultures can predict the virulence and therapeutic efficacy in nude mice. These findings may be essential for rational design of safe and potent VACV strains for vaccination and virotherapy of cancer in humans and animals.}, language = {en} } @article{ChakrabortyKathariouHackeretal.1987, author = {Chakraborty, Trinad and Kathariou, Sophia and Hacker, J{\"o}rg and Hof, Herbert and Huhle, Burkhard and Wagner, Wilma and Kuhn, Michael and Goebel, Werner}, title = {Molecular analysis of bacterial cytolysins}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-40328}, year = {1987}, abstract = {Results of molecular and pathogenic studies of three different bacterial hemolysins (cytolysins) are presented. These exoproteins derive from the two gram-negative bacteria Escherichia coli and Aeromonas hydrophila and from the gram-positive pathogen Listeria monocytogenes. The hemolysin of E. coli is determined by an 8-kilobase (kb) region that includes four clustered genes (hlyC, hlyA, hlyB, and hlyD). This hemolysin determinant is part either of large transmissible plasmids or of the chromosome. The genes located chromosomally are found predominantly in E. coli strains that can cause pyelonephritis and/or other extraintestinal infections. A detailed analysis of the chromosomal hly determinants of one nephropathogenic E. coli strain revealed the existence of specific, large chromosomal insertions 75 kb and lOO kb in size that carry the hly genes but that also influence the expression of other virulence properties, i.e., adhesion and serum resistance. The direct involvement of E. coli hemolysin in virulence could be demonstrated in several model systems. The genetic determinants for hemolysin (cytolysin) formation in , A. hydrophila (aerolysin) and L. monocytogenes (listeriolysin) are less complex. Both cytolysins seem to be encoded by single genes, although two loci (aerB and aerC) that affect the expression and activity of aerolysin have been identified distal and proximal to the structural gene for aerolysin (aerA). Cytolysin-negative mutants of both bacteria were obtained by site-specific deletion and/or transposon mutagenesis. These mutants show a drastic reduction in the virulence of the respective bacteria.}, language = {en} } @article{CalderonPenaSuarezetal.2019, author = {Calderon, Dayana and Pe{\~n}a, Luis and Suarez, Ang{\´e}lica and Villamil, Carolina and Ramirez-Rojas, Adan and Anzola, Juan M. and Garc{\´i}a-Betancur, Juan C. and Cepeda, Martha L. and Uribe, Daniel and Del Portillo, Patricia and Mongui, Alvaro}, title = {Recovery and functional validation of hidden soil enzymes in metagenomic libraries}, series = {MicrobiologyOpen}, volume = {8}, journal = {MicrobiologyOpen}, doi = {10.1002/mbo3.572}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-222016}, year = {2019}, abstract = {The vast microbial diversity on the planet represents an invaluable source for identifying novel activities with potential industrial and therapeutic application. In this regard, metagenomics has emerged as a group of strategies that have significantly facilitated the analysis of DNA from multiple environments and has expanded the limits of known microbial diversity. However, the functional characterization of enzymes, metabolites, and products encoded by diverse microbial genomes is limited by the inefficient heterologous expression of foreign genes. We have implemented a pipeline that combines NGS and Sanger sequencing as a way to identify fosmids within metagenomic libraries. This strategy facilitated the identification of putative proteins, subcloning of targeted genes and preliminary characterization of selected proteins. Overall, the in silico approach followed by the experimental validation allowed us to efficiently recover the activity of previously hidden enzymes derived from agricultural soil samples. Therefore, the methodology workflow described herein can be applied to recover activities encoded by environmental DNA from multiple sources.}, language = {en} } @article{BoehmTorsinTintetal.2017, author = {B{\"o}hm, Lena and Torsin, Sanda and Tint, Su Hlaing and Eckstein, Marie Therese and Ludwig, Tobias and P{\´e}rez, J. Christian}, title = {The yeast form of the fungus Candida albicans promotes persistence in the gut of gnotobiotic mice}, series = {PLoS Pathogens}, volume = {13}, journal = {PLoS Pathogens}, number = {10}, doi = {10.1371/journal.ppat.1006699}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-159120}, pages = {e1006699}, year = {2017}, abstract = {Many microorganisms that cause systemic, life-threatening infections in humans reside as harmless commensals in our digestive tract. Yet little is known about the biology of these microbes in the gut. Here, we visualize the interface between the human commensal and pathogenic fungus Candida albicans and the intestine of mice, a surrogate host. Because the indigenous mouse microbiota restricts C. albicans settlement, we compared the patterns of colonization in the gut of germ free and antibiotic-treated conventionally raised mice. In contrast to the heterogeneous morphologies found in the latter, we establish that in germ free animals the fungus almost uniformly adopts the yeast cell form, a proxy of its commensal state. By screening a collection of C. albicans transcription regulator deletion mutants in gnotobiotic mice, we identify several genes previously unknown to contribute to in vivo fitness. We investigate three of these regulators—ZCF8, ZFU2 and TRY4—and show that indeed they favor the yeast form over other morphologies. Consistent with this finding, we demonstrate that genetically inducing non-yeast cell morphologies is detrimental to the fitness of C. albicans in the gut. Furthermore, the identified regulators promote adherence of the fungus to a surface covered with mucin and to mucus-producing intestinal epithelial cells. In agreement with this result, histology sections indicate that C. albicans dwells in the murine gut in close proximity to the mucus layer. Thus, our findings reveal a set of regulators that endows C. albicans with the ability to endure in the intestine through multiple mechanisms.}, language = {en} } @article{BurySoundararajanBhartietal.2018, author = {Bury, Susanne and Soundararajan, Manonmani and Bharti, Richa and von B{\"u}nau, Rudolf and F{\"o}rstner, Konrad U. and Oelschlaeger, Tobias A.}, title = {The probiotic escherichia coli strain Nissle 1917 combats lambdoid bacteriophages stx and lambda}, series = {Frontiers in Microbiology}, volume = {9}, journal = {Frontiers in Microbiology}, doi = {10.3389/fmicb.2018.00929}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-221960}, year = {2018}, abstract = {Shiga toxin (Stx) producing E. coli (STEC) such as Enterohemorrhagic E. coli (EHEC) are the major cause of foodborne illness in humans. In vitro studies showed the probiotic Escherichia coil strain Nissle 1917 (EcN) to efficiently inhibit the production of Stx. Life threatening EHEC strains as for example the serotype 0104:H4, responsible for the great outbreak in 2011 in Germany, evolutionary developed from certain E. coll strains which got infected by stx2-encoding lambdoid phages turning the E. coil into lysogenic and subsequently Stx producing strains. Since antibiotics induce stx genes and Stx production, EHEC infected persons are not recommended to be treated with antibiotics. Therefore, EcN might be an alternative medication. However, because even commensal E. coli strains might be converted into Stx-producers after becoming host to a stx encoding prophage, we tested EcN for stx-phage genome integration. Our experiments revealed the resistance of EcN toward not only stx-phages but also against lambda-phages. This resistance was not based on the lack of or by mutated phage receptors. Rather it involved the expression of a phage repressor (pr) gene of a defective prophage in EcN which was able to partially protect E. coli K-12 strain MG1655 against stx and lambda phage infection. Furthermore, we observed EcN to inactivate phages and thereby to protect E. coli K-12 strains against infection by stx- as well as lambda-phages. Inactivation of lambda-phages was due to binding of lambda-phages to LamB of EcN whereas inactivation of stx-phages was caused by a thermostable protein of EcN. These properties together with its ability to inhibit Stx production make EcN a good candidate for the prevention of illness caused by EHEC and probably for the treatment of already infected people.}, language = {en} } @article{BruchhagenJarickMewisetal.2018, author = {Bruchhagen, Christin and Jarick, Marcel and Mewis, Carolin and Hertlein, Tobias and Niemann, Silke and Ohlsen, Knut and Peters, Georg and Planz, Oliver and Ludwig, Stephan and Ehrhardt, Christina}, title = {Metabolic conversion of CI-1040 turns a cellular MEK-inhibitor into an antibacterial compound}, series = {Scientific Reports}, volume = {8}, journal = {Scientific Reports}, doi = {10.1038/s41598-018-27445-7}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-221648}, year = {2018}, abstract = {Influenza virus (IV) infections cause severe respiratory illnesses that can be complicated by bacterial super-infections. Previously, we identified the cellular Raf-MEK-ERK cascade as a promising antiviral target. Inhibitors of MEK, such as CI-1040, showed potent antiviral activity. However, it remained unclear if this inhibitor and its active form, ATR-002, might sensitize host cells to either IV or secondary bacterial infections. To address these questions, we studied the anti-pathogen activity of ATR-002 in comparison to CI-1040, particularly, its impact on Staphylococcus aureus (S. aureus), which is a major cause of IV super-infections. We analysed IV and S. aureus titres in vitro during super-infection in the presence and absence of the drugs and characterized the direct impact of ATR-002 on bacterial growth and phenotypic changes. Importantly, neither CI-1040 nor ATR-002 treatment led to increased bacterial titres during super-infection, indicating that the drug does not sensitize cells for bacterial infection. In contrast, we rather observed reduced bacterial titres in presence of ATR-002. Surprisingly, ATR-002 also led to reduced bacterial growth in suspension cultures, reduced stress- and antibiotic tolerance without resistance induction. Our data identified for the first time that a particular MEK-inhibitor metabolite exhibits direct antibacterial activity, which is likely due to interference with the bacterial PknB kinase/Stp phosphatase signalling system.}, language = {en} } @phdthesis{Bourdet2011, author = {Bourdet, Patric}, title = {Entwicklung einer auf Antik{\"o}rpern basierten Therapie von chirurgischen Infektionen verursacht durch methicillinresistente und -sensible Staphylococcus aureus (MRSA und MSSA)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-56199}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2011}, abstract = {Staphylococcus aureus ist einer der h{\"a}ufigsten Erreger von nosokomialen Infektionen. Diese grampositiven Bakterien verursachen neben harmlosen oberfl{\"a}chlichen Hautinfektionen auch lebensbedrohliche Systeminfektionen. Ein großes Problem in der Therapie von S. aureus-Infektionen stellen die zunehmenden Multiresistenzen dar. Die Entwicklung neuer Antibiotika wird zuk{\"u}nftig wahrscheinlich nicht ausreichen, da immer wieder neue Resistenzen der Bakterien zu erwarten sind. Es besteht daher dringender Bedarf an der Entwicklung alternativer Therapieformen im Kampf gegen multiresistente Problemkeime wie S. aureus. Eine M{\"o}glichkeit besteht in der Immuntherapie, zum Beispiel durch Gewinnung von monoklonalen Antik{\"o}rpern gegen geeignete Targetstrukturen von S. aureus. Ziel dieser Arbeit war es, zun{\"a}chst zwei Proteine IsaA und IsaB herzustellen, um diese Proteine f{\"u}r Immunisierungsstudien zu nutzen. Zun{\"a}chst wurde das gereinigte IsaA-Protein verwendet, um ein Kaninchen zu immunisieren. Mit den daraus gewonnenen Antik{\"o}rpern wurden dann erste Tierversuche begonnen, um die Bedingungen f{\"u}r den therapeutischen Einatz von gegen IsaA-gerichteten Antik{\"o}rpern zu ermitteln und die Wirksamkeit einer Antik{\"o}rper-Behandlung zu evaluieren. F{\"u}r die Herstellung der gew{\"u}nschten Proteine wurden die Gensequenzen zun{\"a}chst aus verschiedenen S. aureus-St{\"a}mmen mittels PCR amplifiziert und in den kommerziellen Expressionsvektor pQE30 kloniert. Die amplifizierte Gensequenz stammt aus den klinischen St{\"a}mmen 418 (IsaA) bzw. 134 (IsaB). Nach der Klonierung wurden geeignete Expressions- und Reinigungsstrategien entwickelt. Dabei wurden folgende Bedingungen als optimal f{\"u}r Wachstum und {\"U}berexpression herausgearbeitet: IsaA: Induktion der {\"U}berexpression mit 100 µM IPTG, 3 h Wachstum bei 37°C. IsaB: Induktion der {\"U}berexpression mit 100 µM IPTG, 4 h Wachstum bei 37°C. Es stellte sich auch heraus, dass IsaA zun{\"a}chst in nur unzureichender Quantit{\"a}t vorhanden bzw. exprimiert worden war. Die Vermutung, dass IsaA {\"u}berwiegend im Pellet in sogenannten Einschlussk{\"o}rpern (inclusion bodies) eingeschlossen war, erkl{\"a}rte dieses Ph{\"a}nomen. Das Protein konnte erfolgreich aus dem Pellet isoliert werden. Die Produktion und Aufreinigung beider Proteine IsaA und IsaB unter optimierten Bedingungen ergab, dass beide Proteine nun in ausreichender Menge und Konzentration f{\"u}r die folgende Immunisierung und die weiteren Arbeiten vorlagen. Aus Kaninchen, die mit IsaA immunisiert wurden, konnten polyklonale Antik{\"o}rper gewonnen werden, die die Grundlage f{\"u}r einen ersten Tierversuch mit 24 Ratten bildeten. Hierbei zeigte sich, dass die Tiere, die mit 1.000.000.000 Bakterien infiziert worden waren deutlich st{\"a}rkere Infektionszeichen aufwiesen als diejenigen, die mit 100.000.000 Bakterien infiziert worden waren. Weiterhin wurde deutlich, dass die Tiere, die Serum (mit Antik{\"o}rper gegen IsaA) erhalten hatten, gegen{\"u}ber den Vergleichstieren mit Placebo einen deutlichen Vorteil hinsichtlich Infektionszeichen und Immunantwort hatten. Somit belegen die tierexperimentiellen Ergebnisse in dieser Arbeit erstmalig den therapeutischen Nutzen von Antik{\"o}rpern gegen IsaA. IsaA ist demnach ein geeignetes Target f{\"u}r eine Immuntherapie gegen S. aureus.}, subject = {MRSA}, language = {de} } @phdthesis{BoltUlschmid2004, author = {Bolt-Ulschmid, Julia Katharina}, title = {Charakterisierung von Adenylatkinasen aus Plasmodium falciparum und Thioredoxinreduktase-assoziierten Proteinen aus Dipteren}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-10752}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2004}, abstract = {In S{\"a}ugetieren existieren im wesentlichen zwei Abwehrsysteme gegen oxidativen Streß, in welchen die Glutathionreduktase (GR) und Thioredoxinreduktase (TrxR) Schl{\"u}sselenzyme sind. Ein einzelnes Gen der Taufliege, genannt dmtrxr-1, kodiert sowohl f{\"u}r die durch alternatives Splicing entstehende cytoplasmatische und mitochondriale Form der DmTrxR-1. Zum Teil innerhalb des dmtrxr-1-Gens findet sich auf dem Komplement{\"a}rstrang ein weiteres Gen, welches sniffer genannt wurde. In Kooperation wurde nachgewiesen, daß dieses Gen essentiell zur Verhinderung alterungsbedingter Neurodegeneration ist. Durch biochemische Charakterisierung konnte das rekombinant hergestellte Produkt dieses Gens in der vorliegenden Arbeit als Carbonylreduktase, ein zu den Kurzketten-Dehydrogenasen (short-chain dehydrogenases) geh{\"o}rendes Enzym, identifiziert werden. Sniffer weist das f{\"u}r Carbonylreduktasen typische Substratspektrum mit Phenanthrenequinone als bestem Substrat auf und wird von Flavonoiden wie Quercetin und Rutin sowie Hydroxymercuribenzoat gehemmt. In verschiedenen Ans{\"a}tzen konnten Kristalle des rekombinanten Proteins gewonnen werden, die inzwischen in Kooperation vermessen wurden und so zu einer Kristallstruktur mit einer Aufl{\"o}sung von 1,7 Angstr{\"o}m f{\"u}hrten. Durch diese Arbeiten konnte zum ersten Mal eine Verbindung zwischen einem charakterisierten Gen (snifffer), oxidativem Streß und neurodegenerativen Effekten auf molekularer Ebene nachgewiesen werden. Parasiten haben w{\"a}hrend ihres Lebenszyklus einen hohen Bedarf an Energie und sind abh{\"a}ngig von einer starken Syntheseleistung. Zur Bew{\"a}ltigung dieses Stresses ben{\"o}tigen sie hohe Aktivit{\"a}ten an Adenylatkinase (AK; ATP + AMP \&\#61683; 2 ADP) und GTP-AMP-Phosphotransferase (GAK; GTP + AMP \&\#61683; GDP + ADP). Beide Enzyme wurden in Blutstadien des Malariaparasiten Plasmodium falciparum identifiziert und die entsprechenden Gene der PfAK und PfGAK auf den Chromosomen 10 und 4 respektive lokalisiert. Klonierung und heterologe Expression in E. coli ergab enzymatisch aktive Proteine mit einer Gr{\"o}ße von 28,9 (PfAK), bzw. 28,0 kDa (PfGAK). Das rekombinante Protein der PfAK entspricht in seinen biochemischen Charakteristika denen der authentischen PfAK. Dies gilt auch f{\"u}r eine m{\"o}gliche Assoziation mit einem stabilisierenden Protein mit einem Molekulargewicht von ca. 70 kDa und der hohen Substratspezifit{\"a}t f{\"u}r das Monophosphat-Nukleotid AMP. Die Spezifit{\"a}t f{\"u}r das Triphosphat-Substrat ist weniger stringent. Das beste Triphosphat-Substrat ist ATP mit einem Vmax-Wert von 75 U/mg und einem kcat von 2800 min-1. Die Sequenz der PfAK enth{\"a}lt eine amphiphatische Helix, welche als notwendig f{\"u}r die Translokation zytosolischer Adenylatkinasen in den Intermembranraum der Mitochondrien beschrieben wurde. Die PfGAK bevorzugt GTP und AMP als Substrat (100 U/mg; kcat = 2800 min-1 bei 25°C) und zeigt als Besonderheit keine messbare Aktivit{\"a}t mit ATP. Im Gegensatz zu ihrem Ortholog im Menschen (AK3) enth{\"a}lt die Sequenz der PfGAK ein Zinkfinger-Motiv und bindet Eisenionen. Erste Immunfluoreszenz-Analysen lokalisieren die PfGAK in den Mitochondrien. PfAK und PfGAK werden von den Dinukleosid-Pentaphosphat-Verbindungen AP5A beziehungsweise GP5A gehemmt. Die Ki-Werte liegen mit ca. 0.2 µM ungef{\"a}hr 250-fach niedriger als die KM-Werte der entsprechenden Nukleotidsubstrate. Zur L{\"o}sung der vor allem im Rahmen einer rationalen Medikamentenentwicklung notwendigen Kristallstruktur des Zielmolek{\"u}ls konnten bereits Kristalle der PfGAK erhalten werden.}, subject = {Taufliege}, language = {de} } @article{BogdanMollSolbachetal.1990, author = {Bogdan, Christian and Moll, Heidrun and Solbach, Werner and R{\"o}llinghoff, Martin}, title = {Tumor necrosis factor-\(\alpha\) in combination with interferon-\(\gamma\), but not with interleukin 4 activates murine macrophages for elimination of Leishmania major amastigotes}, volume = {20}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-31614}, pages = {1131 -- 1135}, year = {1990}, abstract = {We have previously shown that during an infection with Leishmania major, susceptible BALB/c mice, as opposed to mice of a resistant strain (C57BLl6), are primed by lipopolysaccharide for the production of high levels of tumor necrosis factor-\(\alpha\) (TNF-\(\alpha\)) which is known to be a potent maerophage M\(\Phi\) stimulator in other parasitic diseases. In the present study we investigated whether TNF-\(\alpha\) activates M\(\Phi\) for killing of L. major parasites. In the absence of interferon-y (IFN-\(\gamma\)) or lipopolysaccharide, TNF-\(\alpha\) (0.025-25000 U/ml) failed to activate peritoneal exudate M\(\Phi\) from BALB/c mice for killling of L. major amastigotes. In the presence of suboptimal doses of IFN-\(\gamma\) (5 or 10 Vlml), however, TNF-\(\alpha\) mediated a rapid elimination of intracellular parasites, which was highly significant compared to IFN-\(\gamma\) alone. Tbe combination of TNF with interleukin 4, in contrast, was inactive in this respect and allowed survival of intracellular parasites. From these data we conelude that the presence of IFN-\(\gamma\) is crucial for TNF-\(\alpha\)-mediated killing of L. major parasites by M\(\Phi\). Disease progression in susceptible mice therefore seems to be a consequence of a deficiency of IFN-\(\gamma\) and a predominance of interleukin 4 rather than the result of an excess amount of TNF-\(\alpha\).}, subject = {Infektionsbiologie}, language = {en} } @article{BoesSpiegelVoepeletal.2015, author = {Boes, Alexander and Spiegel, Holger and Voepel, Nadja and Edgue, Gueven and Beiss, Veronique and Kapelski, Stephanie and Fendel, Rolf and Scheuermayer, Matthias and Pradel, Gabriele and Bolscher, Judith M. and Behet, Marije C. and Dechering, Koen J. and Hermsen, Cornelus C. and Sauerwein, Robert W. and Schillberg, Stefan and Reimann, Andreas and Fischer, Rainer}, title = {Analysis of a multi-component multi-stage malaria vaccine candidate—tackling the cocktail challenge}, series = {PLoS ONE}, volume = {10}, journal = {PLoS ONE}, number = {7}, doi = {10.1371/journal.pone.0131456}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-173092}, pages = {e0131456}, year = {2015}, abstract = {Combining key antigens from the different stages of the P. falciparum life cycle in the context of a multi-stage-specific cocktail offers a promising approach towards the development of a malaria vaccine ideally capable of preventing initial infection, the clinical manifestation as well as the transmission of the disease. To investigate the potential of such an approach we combined proteins and domains (11 in total) from the pre-erythrocytic, blood and sexual stages of P. falciparum into a cocktail of four different components recombinantly produced in plants. After immunization of rabbits we determined the domain-specific antibody titers as well as component-specific antibody concentrations and correlated them with stage specific in vitro efficacy. Using purified rabbit immune IgG we observed strong inhibition in functional in vitro assays addressing the pre-erythrocytic (up to 80\%), blood (up to 90\%) and sexual parasite stages (100\%). Based on the component-specific antibody concentrations we calculated the IC50 values for the pre-erythrocytic stage (17-25 μg/ml), the blood stage (40-60 μg/ml) and the sexual stage (1.75 μg/ml). While the results underline the feasibility of a multi-stage vaccine cocktail, the analysis of component-specific efficacy indicates significant differences in IC50 requirements for stage-specific antibody concentrations providing valuable insights into this complex scenario and will thereby improve future approaches towards malaria vaccine cocktail development regarding the selection of suitable antigens and the ratios of components, to fine tune overall and stage-specific efficacy.}, language = {en} } @article{BlaettnerDasPaprotkaetal.2016, author = {Bl{\"a}ttner, Sebastian and Das, Sudip and Paprotka, Kerstin and Eilers, Ursula and Krischke, Markus and Kretschmer, Dorothee and Remmele, Christian W. and Dittrich, Marcus and M{\"u}ller, Tobias and Schuelein-Voelk, Christina and Hertlein, Tobias and Mueller, Martin J. and Huettel, Bruno and Reinhardt, Richard and Ohlsen, Knut and Rudel, Thomas and Fraunholz, Martin J.}, title = {Staphylococcus aureus Exploits a Non-ribosomal Cyclic Dipeptide to Modulate Survival within Epithelial Cells and Phagocytes}, series = {PLoS Pathogens}, volume = {12}, journal = {PLoS Pathogens}, number = {9}, doi = {10.1371/journal.ppat.1005857}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-180380}, year = {2016}, abstract = {Community-acquired (CA) Staphylococcus aureus cause various diseases even in healthy individuals. Enhanced virulence of CA-strains is partly attributed to increased production of toxins such as phenol-soluble modulins (PSM). The pathogen is internalized efficiently by mammalian host cells and intracellular S. aureus has recently been shown to contribute to disease. Upon internalization, cytotoxic S. aureus strains can disrupt phagosomal membranes and kill host cells in a PSM-dependent manner. However, PSM are not sufficient for these processes. Here we screened for factors required for intracellular S. aureus virulence. We infected escape reporter host cells with strains from an established transposon mutant library and detected phagosomal escape rates using automated microscopy. We thereby, among other factors, identified a non-ribosomal peptide synthetase (NRPS) to be required for efficient phagosomal escape and intracellular survival of S. aureus as well as induction of host cell death. By genetic complementation as well as supplementation with the synthetic NRPS product, the cyclic dipeptide phevalin, wild-type phenotypes were restored. We further demonstrate that the NRPS is contributing to virulence in a mouse pneumonia model. Together, our data illustrate a hitherto unrecognized function of the S. aureus NRPS and its dipeptide product during S. aureus infection.}, language = {en} } @article{BlumOttCrossetal.1991, author = {Blum, G. and Ott, M. and Cross, A. and Hacker, J{\"o}rg}, title = {Virulence determinants of Escherichia coli O6 extraintestinal isolates analysed by Southern hybridizations and DNA long range mapping techniques}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59717}, year = {1991}, abstract = {A total of 16 Escherichia coli 06 strains isolated from cases of extraintestinal infections were analysed for the genetic presence and phenotypic expression of fimbrial adhesins ( P, S/FIC, type I), aerobactin and hemolysin. ln addition restriction fragment length polymorphisms (RFLPs) of Xbal-cleaved genomic DNA of seven selected strains, separated by orthogonal field alternation gel electrophoresis {OFAGE) were determined and virulence-associated DNA probes were used for Southern hybridization studies of the Xbal-cleaved genomic DNAs. The virulence characteristics and hybridization patterns obtained differed between the various isolates. ln three isolates hemolysin genes and P fimbrial determinants were located on the same Xbal fragments. Furthermore, multiple copies of FIC determinants (foc) could be detected in two strains. Our data show that the new technique of pulse field electrophoresis tagether with Southern hybridization represents a powerful tool for the genetic analysis of pathogenic bacteria.}, subject = {Infektionsbiologie}, language = {en} } @article{BlankFuchsRappersbergeretal.1993, author = {Blank, Christine and Fuchs, Harald and Rappersberger, Klemens and R{\"o}llinghoff, Martin and Moll, Heidrun}, title = {Parasitism of epidermal Langerhans cells in experimental cutaneous leishmaniasis with Leishmania major}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-45850}, year = {1993}, abstract = {Murine epidermal Langerhans cells (LC) have been demonstrated to stimulate a vigorous T cell response to Leishmania major, a cause of human cutaneous leishmaniasis. It was therefore of interest to analyze whether LC can take up viable parasites. Epidermal cells were obtained from mouse ear skin for incubation with L. major and subsequent detection of intracellular parasites by cytochemistry. Freshly isolated LC, but not cultured LC, phagocytosed L. major and the uptake was inhibited by antibodies to the complement receptor type 3. Electron microscopic studies revealed the presence of viable amastigotes within Le. Moreover, with double-Iabeling techniques, L. major-containing LC could also be detected in infected skin. The results demonstrate that LC can internalize L. major. Since the number of organisms per infected LC remained consistently low, the prime task of LC may not be the promotion of parasite spreading but the presentation of L. major antigen to T cells and, thus, the regulation of the cellular immunity during cutaneous leishmaniasis.}, language = {en} } @phdthesis{Biswas2005, author = {Biswas, Kajal}, title = {Analysis of Nitrogen starvation induced filamentous growth and characterization of putative essential genes in the human fungal pathogen, Candida albicans}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-11554}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2005}, abstract = {1. Zusammenfassung Candida albicans ist ein opportunistisch pathogener Hefepilz, der sowohl oberfl{\"a}chliche Infektionen der Schleimhaut als auch lebensbedrohliche systemische Infektionen hervorrufen kann. Obwohl die F{\"a}higkeit von C.albicans Infektionen auszul{\"o}sen weitgehend vom Immunstatus des Wirts abh{\"a}ngt, besitzt der Pilz doch auch spezifische Eigenschaften, die eine Kolonisierung, Disseminierung und Anpassung an unterschiedliche Wirtsnischen erm{\"o}glichen und ihn vom harmlosen Kommensalen zum gef{\"a}hrlichen Krankheitsserreger werden lassen. Unter bestimmten Umweltbedingungen geht C.albicans vom Wachstum als sprossende Hefe zum invasiven, filament{\"o}sen Wachstum {\"u}ber, das eine wichtige Rolle in der Pathogenit{\"a}t des Pilzes spielt. Stickstoffmangel ist eines der Signale, die das filament{\"o}se Wachstum in C.albicans induzieren, und die Kontrolle der Morphogenese durch die Verf{\"u}gbarkeit von Stickstoff wurde in dieser Arbeit detailliert untersucht. Ammonium ist f{\"u}r Hefepilze eine bevorzugte Stickstoffquelle, die {\"u}ber spezifische Transporter in die Zelle aufgenommen wird. In der vorliegenden Arbeit konnte gezeigt werden, dass C.albicans zwei Ammoniumpermeasen besitzt, deren Expression durch Stickstoffmangel induziert wird. W{\"a}hrend die Deletion von CaMEP1 oder CaMEP2 keinen Einfluss auf das Wachstum bei limitierenden Ammoniumkonzentrationen hatte, konnten \&\#61508;mep1 \&\#61508;mep2 Doppelmutanten bei Ammoniumkonzentrationen unter 5 mM nicht mehr wachsen. Im Gegensatz zu \&\#61508;mep1 Mutanten bildeten \&\#61508;mep2 Mutanten unter Stickstoffmangel keine Hyphen mehr und wuchsen ausschließlich in der Hefeform. CaMep2p hat also nicht nur eine Funktion als Ammoniumtransporter, sondern spielt auch eine Rolle bei der Induktion des filament{\"o}sen Wachstums. Weitere Experimente zeigten, dass CaMep2p ein weniger effizienter Ammoniumtransporter als CaMep1p ist, daf{\"u}r aber st{\"a}rker exprimiert wird, und dass dieser Unterschied wichtig f{\"u}r die Signalfunktion von CaMep2p ist. Durch Deletionsanalysen konnte bewiesen werden, dass die C-terminale, cytoplasmatische Dom{\"a}ne von CaMep2p essentiell f{\"u}r die Induktion des Hyphenwachstums ist, f{\"u}r den Ammoniumtransport jedoch nicht ben{\"o}tigt wird, und diese beiden Funktionen von CaMep2p daher voneinander getrennt werden k{\"o}nnen. In C.albicans gibt es mindestens zwei Signalwege die das filament{\"o}se Wachstum steuern, eine MAP-Kinase-Kaskade und einen cAMP-abh{\"a}ngigen Signalweg, die in den Transkriptionsfaktoren Cph1p bzw. Efg1p enden. Bei Inaktivierung des einen oder des anderen Signalwegs induziert Stickstoffmangel kein filament{\"o}ses Wachstum mehr. Ein hyperaktives CaMEP2 Allel konnte den filament{\"o}sen Wachstumsdefekt sowohl von \&\#61508;cph1 als auch \&\#61508;efg1 Mutanten aufheben, nicht jedoch den einer \&\#61508;cph1 \&\#61508;efg1 Doppelmutante oder einer Mutante, der das G-Protein Ras1p fehlte, das beide Signalwege aktiviert. Umgekehrt wurde der filament{\"o}se Wachstumsdefekt von \&\#61472;\&\#61508;mep2 Mutanten durch ein dominant-aktives RAS1 Allel bzw. durch die Zugabe von cAMP aufgehoben. Diese Ergebnisse deuten darauf hin, dass CaMep2p bei Stickstoffmangel sowohl den MAP-Kinase- als auch den cAMP-abh{\"a}ngigen Signalweg aktiviert, um filament{\"o}ses Wachstum zu induzieren. In gen{\"u}gend hohen Konzentrationen reprimierte Ammonium das filament{\"o}se Wachstum selbst wenn die Signalwege artifiziell aktiviert waren. Die bevorzugte Stickstoffquelle Ammonium ist deshalb ein Inhibitor der Morphogenese, der durch denselben Transporter in die Zelle aufgenommen wird, der bei Stickstoffmangel das filament{\"o}se Wachstum von C.albicans induziert. Obwohl ein genaues Verst{\"a}ndnis der Virulenzmechanismen von C.albicans auch neue Ans{\"a}tze zur Bek{\"a}mpfung von Infektionen durch diesen Pilz liefern kann, ist doch die Identifizierung und Charakterisierung von essentiellen Genen als potentielle Ziele f{\"u}r die Entwicklung neuer Antimykotika eine Strategie, die von der pharmazeutischen Industrie favorisiert wird. Aus diesem Grund wurden in Zusammenarbeit mit einem Industriepartner drei Gene von C.albicans ausgew{\"a}hlt, die in anderen Pilzen als essentiell beschrieben wurden, und im Rahmen dieser Arbeit funktionell charakterisiert. RAP1 codiert f{\"u}r das Repressor/Aktivator Protein 1, ein Transkriptionsfaktor und Telomerbindeprotein, das in der B{\"a}ckerhefe Saccharomyces cerevisiae essentiell ist. Die Deletion des RAP1 Gens in C.albicans beeintr{\"a}chtigte jedoch nicht die Lebensf{\"a}higkeit der Mutanten, so dass RAP1 kein vielversprechendes Ziel darstellt. CBF1 (centromere binding factor 1) ist in S.cerevisiae wichtig f{\"u}r die korrekte Chromosomenverteilung w{\"a}hrend der Mitose und außerdem auch f{\"u}r die transkriptionelle Aktivierung der Methioninbiosynthesegene; in den verwandten Hefen Kluyveromyces lactis und Candida glabrata ist CBF1 sogar essentiell. C.albicans \&\#61508;cbf1 Mutanten wiesen jedoch keinen erh{\"o}hten Chromosomenverlust auf, so dass CBF1 hier offensichtlich keine Rolle bei der Chromosomensegregation spielt. Allerdings waren die Mutanten auxotroph f{\"u}r schwefelhaltige Aminos{\"a}uren und generell stark im Wachstum beeintr{\"a}chtigt, was zeigte, dass Cbf1p f{\"u}r das normale Wachstum von C.albicans wichtig ist. YIL19 ist in S.cerevisiae ein essentielles Gen und hat eine Funktion bei der Reifung der 18S rRNA. YIL19 stellte sich auch in C.albicans als essentiell heraus. Konditionale Mutanten, in denen YIL19 durch induzierbare, FLP-vermittelte Rekombination aus dem Genom deletiert wurde, waren nicht lebensf{\"a}hig und akkumulierten rRNA Vorstufen. Durch diese Untersuchungen konnte gezeigt werden, dass YIL19 essentiell f{\"u}r diesen wichtigen zellul{\"a}ren Prozess und f{\"u}r die Lebensf{\"a}higkeit von C.albicans ist und sich m{\"o}glicherweise als Ziel f{\"u}r die Entwicklung antifungaler Substanzen eignet.}, subject = {Candida albicans}, language = {en} } @article{BischlerKopfVoss2014, author = {Bischler, Thorsten and Kopf, Matthias and Voss, Bjoern}, title = {Transcript mapping based on dRNA-seq data}, series = {BMC Bioinformatics}, volume = {15}, journal = {BMC Bioinformatics}, number = {122}, issn = {1471-2105}, doi = {10.1186/1471-2105-15-122}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-116663}, year = {2014}, abstract = {Background: RNA-seq and its variant differential RNA-seq (dRNA-seq) are today routine methods for transcriptome analysis in bacteria. While expression profiling and transcriptional start site prediction are standard tasks today, the problem of identifying transcriptional units in a genome-wide fashion is still not solved for prokaryotic systems. Results: We present RNASEG, an algorithm for the prediction of transcriptional units based on dRNA-seq data. A key feature of the algorithm is that, based on the data, it distinguishes between transcribed and un-transcribed genomic segments. Furthermore, the program provides many different predictions in a single run, which can be used to infer the significance of transcriptional units in a consensus procedure. We show the performance of our method based on a well-studied dRNA-seq data set for Helicobacter pylori. Conclusions: With our algorithm it is possible to identify operons and 5'- and 3'-UTRs in an automated fashion. This alleviates the need for labour intensive manual inspection and enables large-scale studies in the area of comparative transcriptomics.}, language = {en} } @article{BielaszewskaSchillerLammersetal.2014, author = {Bielaszewska, Martina and Schiller, Roswitha and Lammers, Lydia and Bauwens, Andreas and Fruth, Angelika and Middendorf, Barbara and Schmidt, M. Alexander and Tarr, Phillip I. and Dobrindt, Ulrich and Karch, Helge and Mellmann, Alexander}, title = {Heteropathogenic virulence and phylogeny reveal phased pathogenic metamorphosis in Escherichia coli O2:H6}, series = {EMBO Molecular Medicine}, volume = {6}, journal = {EMBO Molecular Medicine}, number = {3}, issn = {1757-4684}, doi = {10.1002/emmm.201303133}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-117254}, pages = {347-357}, year = {2014}, abstract = {Extraintestinal pathogenic and intestinal pathogenic (diarrheagenic) Escherichia coli differ phylogenetically and by virulence profiles. Classic theory teaches simple linear descent in this species, where non-pathogens acquire virulence traits and emerge as pathogens. However, diarrheagenic Shiga toxin-producing E.coli (STEC) O2:H6 not only possess and express virulence factors associated with diarrheagenic and uropathogenic E.coli but also cause diarrhea and urinary tract infections. These organisms are phylogenetically positioned between members of an intestinal pathogenic group (STEC) and extraintestinal pathogenic E.coli. STEC O2:H6 is, therefore, a 'heteropathogen,' and the first such hybrid virulent E.coli identified. The phylogeny of these E.coli and the repertoire of virulence traits they possess compel consideration of an alternate view of pathogen emergence, whereby one pathogroup of E.coli undergoes phased metamorphosis into another. By understanding the evolutionary mechanisms of bacterial pathogens, rational strategies for counteracting their detrimental effects on humans can be developed.}, language = {en} } @article{BergmillerPenaMillerBoehmetal.2011, author = {Bergmiller, Tobias and Pena-Miller, Rafael and Boehm, Alexander and Ackermann, Martin}, title = {Single-cell time-lapse analysis of depletion of the universally conserved essential protein YgjD}, series = {BMC Microbiology}, volume = {11}, journal = {BMC Microbiology}, number = {118}, doi = {10.1186/1471-2180-11-118}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142324}, pages = {1-12}, year = {2011}, abstract = {Background: The essential Escherichia coli gene ygjD belongs to a universally conserved group of genes whose function has been the focus of a number of recent studies. Here, we put ygjD under control of an inducible promoter, and used time-lapse microscopy and single cell analysis to investigate the phenotypic consequences of the depletion of YgjD protein from growing cells. Results: We show that loss of YgjD leads to a marked decrease in cell size and termination of cell division. The transition towards smaller size occurs in a controlled manner: cell elongation and cell division remain coupled, but cell size at division decreases. We also find evidence that depletion of YgjD leads to the synthesis of the intracellular signaling molecule (p) ppGpp, inducing a cellular reaction resembling the stringent response. Concomitant deletion of the relA and spoT genes - leading to a strain that is uncapable of synthesizing (p) ppGpp abrogates the decrease in cell size, but does not prevent termination of cell division upon YgjD depletion. Conclusions: Depletion of YgjD protein from growing cells leads to a decrease in cell size that is contingent on (p) ppGpp, and to a termination of cell division. The combination of single-cell time-lapse microscopy and statistical analysis can give detailed insights into the phenotypic consequences of the loss of essential genes, and can thus serve as a new tool to study the function of essential genes.}, language = {en} } @phdthesis{Bergmann2011, author = {Bergmann, Anna}, title = {Untersuchungen zur Verwertung proteinhaltiger Substrate als m{\"o}gliche Virulenzdeterminante des humanpathogenen Schimmelpilzes Aspergillus fumigatus}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-67333}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2011}, abstract = {Die asexuellen Sporen von Aspergillus fumigatus sind ubiquit{\"a}r verbreitete Luftkeime. Als Saprophyt ist dieser opportunistisch humanpathogene Pilz darauf spezialisiert, polymere Substanzen aus dem umgebenden Milieu zu zersetzen, um daraus die von ihm ben{\"o}tigten N{\"a}hrstoffe zu generieren und aufzunehmen. Die F{\"a}higkeit, verschiedene Stickstoff- und Kohlenstoffquellen zu verwerten, tr{\"a}gt dabei zu seiner Virulenz bei und hierbei scheint die extrazellul{\"a}re Proteolyse eine wichtige Rolle zu spielen. Sekretierte Proteasen, die das umgebende Gewebe w{\"a}hrend einer Infektion mit A. fumigatus erschließen, k{\"o}nnten somit zu dessen Pathogenit{\"a}t beitragen. Dementsprechend sollte im Rahmen dieser Arbeit die Bedeutung einer Regulation der extrazellul{\"a}ren proteolytischen Aktivit{\"a}t von A. fumigatus f{\"u}r dessen Virulenz untersucht werden. Dies geschah durch Untersuchungen eines konservierten Transkriptionsfaktors, PrtT. Dabei stellte sich heraus, dass PrtT die Expression der drei Hauptproteasen von A. fumigatus, Alp, Mep und Pep stark beeinflusst, in einem murinen Tiermodell der pulmonaren Aspergillose scheint dieser Regulator jedoch keine Rolle f{\"u}r die Pathogenit{\"a}t von A. fumigatus zu spielen. Um einen weiteren Aspekt des pilzlichen Aminos{\"a}urestoffwechsels zu beleuchten, wurde die Biosynthese der aromatischen Aminos{\"a}uren als m{\"o}gliche Virulenzdeterminate untersucht. F{\"u}r den Menschen sind diese Aminos{\"a}uren essentiell, weshalb dieser Syntheseweg ein m{\"o}gliches Ziel f{\"u}r antimykotische Substanzen darstellen k{\"o}nnte. Es konnten mehrere f{\"u}r A. fumigatus essentielle Komponenten des Shikimatweges identifiziert werden, des Weiteren wurden Deletionsmutanten in den Genen aroC und trpA, die f{\"u}r die Chorismatmutase bzw. Anthranilatsynthase der Biosynthese von Phenylalanin und Tyrosin bzw. Tryptophan kodieren, erzeugt und ph{\"a}notypisch charakterisiert. Deren Untersuchung in einem alternativen Tiermodell der Aspergillose zeigte eine deutlich attenuierte Virulenz. Diese Ergebnisse verdeutlichen, wie wichtig die Biosynthese der aromatischen Aminos{\"a}uren f{\"u}r das Wachstum von A. fumigatus ist, und dass ein Eingriff in diesen Syntheseweg eine lohnende Strategie zur Entwicklung neuer Antimykotika sein k{\"o}nnte. Die hier pr{\"a}sentierten Ergebnisse unterstreichen die f{\"u}r den Schimmelpilz A. fumigatus typische Redundanz bez{\"u}glich extrazellul{\"a}rer proteolytischer Enzyme und dass diese nur bedingt hinsichtlich ihres Virulenzbeitrags untersucht werden k{\"o}nnen. Im Gegensatz hierzu lassen sich bestimmte Stoffwechselwege, die oftmals durch einzigartige Genprodukte katalysiert werden, unter Umst{\"a}nden besser als unspezifische aber vielversprechende Virulenzdeterminanten identifizieren.}, subject = {Aspergillus fumigatus}, language = {de} }