@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{PapenfortVogel2014, author = {Papenfort, Kai and Vogel, J{\"o}rg}, title = {Small RNA functions in carbon metabolism and virulence of enteric pathogens}, series = {Frontiers in Cellular and Infection Microbiology}, volume = {4}, journal = {Frontiers in Cellular and Infection Microbiology}, number = {91}, issn = {2235-2988}, doi = {10.3389/fcimb.2014.00091}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-197520}, year = {2014}, abstract = {Enteric pathogens often cycle between virulent and saprophytic lifestyles. To endure these frequent changes in nutrient availability and composition bacteria possess an arsenal of regulatory and metabolic genes allowing rapid adaptation and high flexibility. While numerous proteins have been characterized with regard to metabolic control in pathogenic bacteria, small non-coding RNAs have emerged as additional regulators of metabolism. Recent advances in sequencing technology have vastly increased the number of candidate regulatory RNAs and several of them have been found to act at the interface of bacterial metabolism and virulence factor expression. Importantly, studying these riboregulators has not only provided insight into their metabolic control functions but also revealed new mechanisms of post-transcriptional gene control. This review will focus on the recent advances in this area of host-microbe interaction and discuss how regulatory small RNAs may help coordinate metabolism and virulence of enteric pathogens.}, language = {en} } @article{MietrachSchlosserGeibel2019, author = {Mietrach, Nicole and Schlosser, Andreas and Geibel, Sebastian}, title = {An extracellular domain of the EsaA membrane component of the type VIIb secretion system: expression, purification and crystallization}, series = {Acta Crystallographica Section F}, volume = {75}, journal = {Acta Crystallographica Section F}, number = {12}, doi = {10.1107/S2053230X1901495X}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-213681}, pages = {725-730}, year = {2019}, abstract = {The membrane protein EsaA is a conserved component of the type VIIb secretion system. Limited proteolysis of purified EsaA from Staphylococcus aureus USA300 identified a stable 48 kDa fragment, which was mapped by fingerprint mass spectrometry to an uncharacterized extracellular segment of EsaA. Analysis by circular dichroism spectroscopy showed that this fragment folds into a single stable domain made of mostly α-helices with a melting point of 34.5°C. Size-exclusion chromatography combined with multi-angle light scattering indicated the formation of a dimer of the purified extracellular domain. Octahedral crystals were grown in 0.2 M ammonium citrate tribasic pH 7.0, 16\% PEG 3350 using the hanging-drop vapor-diffusion method. Diffraction data were analyzed to 4.0 {\AA} resolution, showing that the crystals belonged to the enantiomorphic tetragonal space groups P41212 or P43212, with unit-cell parameters a = 197.5, b = 197.5, c = 368.3 {\AA}, α = β = γ = 90°.}, language = {en} } @phdthesis{Lerch2018, author = {Lerch, Maike Franziska}, title = {Characterisation of a novel non-coding RNA and its involvement in polysaccharide intercellular adhesin (PIA)-mediated biofilm formation of \(Staphylococcus\) \(epidermidis\)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-155777}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2018}, abstract = {Coagulase-negative staphylococci, particularly Staphylococcus epidermidis, have been recognised as an important cause of health care-associated infections due to catheterisation, and livestock-associated infections. The colonisation of indwelling medical devices is achieved by the formation of biofilms, which are large cell-clusters surrounded by an extracellular matrix. This extracellular matrix consists mainly of PIA (polysaccharide intercellular adhesin), which is encoded by the icaADBC-operon. The importance of icaADBC in clinical strains provoking severe infections initiated numerous investigations of this operon and its regulation within the last two decades. The discovery of a long transcript being located next to icaADBC, downstream of the regulator gene icaR, led to the hypothesis of a possible involvement of this transcript in the regulation of biofilm formation (Eckart, 2006). Goal of this work was to characterise this transcript, named ncRNA IcaZ, in molecular detail and to uncover its functional role in S. epidermidis. The ~400 nt long IcaZ is specific for ica-positive S. epidermidis and is transcribed in early- and mid-exponential growth phase as primary transcript. The promotor sequence and the first nucleotides of icaZ overlap with the 3' UTR of the preceding icaR gene, whereas the terminator sequence is shared by tRNAThr-4, being located convergently to icaZ. Deletion of icaZ resulted in a macroscopic biofilm-negative phenotype with highly diminished PIA-biofilm. Biofilm composition was analysed in vitro by classical crystal violet assays and in vivo by confocal laser scanning microscopy under flow conditions to display biofilm formation in real-time. The mutant showed clear defects in initial adherence and decreased cell-cell adherence, and was therefore not able to form a proper biofilm under flow in contrast to the wildtype. Restoration of PIA upon providing icaZ complementation from plasmids revealed inconsistent results in the various mutant backgrounds. To uncover the functional role of IcaZ, transcriptomic and proteomic analysis was carried out, providing some hints on candidate targets, but the varying biofilm phenotypes of wildtype and icaZ mutants made it difficult to identify direct IcaZ mRNA targets. Pulse expression of icaZ was then used as direct fishing method and computational target predictions were executed with candidate mRNAs from aforesaid approaches. The combined data of these analyses suggested an involvement of icaR in IcaZ-mediated biofilm control. Therefore, RNA binding assays were established for IcaZ and icaR mRNA. A positive gel shift was maintained with icaR 3' UTR and with 5'/3' icaR mRNA fusion product, whereas no gel shift was obtained with icaA mRNA. From these assays, it was assumed that IcaZ regulates icaR mRNA expression in S. epidermidis. S. aureus instead lacks ncRNA IcaZ and its icaR mRNA was shown to undergo autoregulation under so far unknown circumstances by intra- or intermolecular binding of 5' UTR and 3' UTR (Ruiz de los Mozos et al., 2013). Here, the Shine-Dalgarno sequence is blocked through 5'/3' UTR base pairing and RNase III, an endoribonuclease, degrades icaR mRNA, leading to translational blockade. In this work, icaR mRNA autoregulation was therefore analysed experimentally in S. epidermidis and results showed that this specific autoregulation does not take place in this organism. An involvement of RNase III in the degradation process could not be verified here. GFP-reporter plasmids were generated to visualise the interaction, but have to be improved for further investigations. In conclusion, IcaZ was found to interact with icaR mRNA, thereby conceivably interfering with translation initiation of repressor IcaR, and thus to promote PIA synthesis and biofilm formation. In addition, the environmental factor ethanol was found to induce icaZ expression, while only weak or no effects were obtained with NaCl and glucose. Ethanol, actually is an ingredient of disinfectants in hospital settings and known as efficient effector for biofilm induction. As biofilm formation on medical devices is a critical factor hampering treatment of S. epidermidis infections in clinical care, the results of this thesis do not only contribute to better understanding of the complex network of biofilm regulation in staphylococci, but may also have practical relevance in the future.}, subject = {Biofilm}, 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{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{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} } @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} } @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{SchneiderDobrindtMiddendorfetal.2011, author = {Schneider, Gy{\"o}rgy and Dobrindt, Ulrich and Middendorf, Barbara and Hochhut, Bianca and Szij{\´a}rt{\´o}, Valeria and Em{\´o}dy, Levente and Hacker, J{\"o}rg}, title = {Mobilisation and remobilisation of a large archetypal pathogenicity island of uropathogenic \(Escherichia\) \(coli\) \(in\) \(vitro\) support the role of conjugation for horizontal transfer of genomic islands}, series = {BMC Microbiology}, volume = {11}, journal = {BMC Microbiology}, doi = {10.1186/1471-2180-11-210}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-140975}, pages = {210}, year = {2011}, abstract = {Background: A substantial amount of data has been accumulated supporting the important role of genomic islands (GEIs) - including pathogenicity islands (PAIs) - in bacterial genome plasticity and the evolution of bacterial pathogens. Their instability and the high level sequence similarity of different (partial) islands suggest an exchange of PAIs between strains of the same or even different bacterial species by horizontal gene transfer (HGT). Transfer events of archetypal large genomic islands of enterobacteria which often lack genes required for mobilisation or transfer have been rarely investigated so far. Results: To study mobilisation of such large genomic regions in prototypic uropathogenic E. coli (UPEC) strain 536, PAI II(536) was supplemented with the mob(RP4) region, an origin of replication (oriV(R6K)), an origin of transfer (oriT(RP4)) and a chloramphenicol resistance selection marker. In the presence of helper plasmid RP4, conjugative transfer of the 107-kb PAI II(536) construct occured from strain 536 into an E. coli K-12 recipient. In transconjugants, PAI II(536) existed either as a cytoplasmic circular intermediate (CI) or integrated site-specifically into the recipient's chromosome at the leuX tRNA gene. This locus is the chromosomal integration site of PAI II(536) in UPEC strain 536. From the E. coli K-12 recipient, the chromosomal PAI II(536) construct as well as the CIs could be successfully remobilised and inserted into leuX in a PAI II(536) deletion mutant of E. coli 536. Conclusions: Our results corroborate that mobilisation and conjugal transfer may contribute to evolution of bacterial pathogens through horizontal transfer of large chromosomal regions such as PAIs. Stabilisation of these mobile genetic elements in the bacterial chromosome result from selective loss of mobilisation and transfer functions of genomic islands.}, language = {en} } @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{HillStritzkerScadengetal.2011, author = {Hill, Philip J. and Stritzker, Jochen and Scadeng, Miriam and Geissinger, Ulrike and Haddad, Daniel and Basse-L{\"u}sebrink, Thomas C. and Gbureck, Uwe and Jakob, Peter and Szalay, Aladar A.}, title = {Magnetic Resonance Imaging of Tumors Colonized with Bacterial Ferritin-Expressing \(Escherichia\) \(coli\)}, series = {PLoS ONE}, volume = {6}, journal = {PLoS ONE}, number = {10}, doi = {10.1371/journal.pone.0025409}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-140920}, pages = {e25409}, year = {2011}, abstract = {Background: Recent studies have shown that human ferritin can be used as a reporter of gene expression for magnetic resonance imaging (MRI). Bacteria also encode three classes of ferritin-type molecules with iron accumulation properties. Methods and Findings: Here, we investigated whether these bacterial ferritins can also be used as MRI reporter genes and which of the bacterial ferritins is the most suitable reporter. Bacterial ferritins were overexpressed in probiotic E. coli Nissle 1917. Cultures of these bacteria were analyzed and those generating highest MRI contrast were further investigated in tumor bearing mice. Among members of three classes of bacterial ferritin tested, bacterioferritin showed the most promise as a reporter gene. Although all three proteins accumulated similar amounts of iron when overexpressed individually, bacterioferritin showed the highest contrast change. By site-directed mutagenesis we also show that the heme iron, a unique part of the bacterioferritin molecule, is not critical for MRI contrast change. Tumor-specific induction of bacterioferritin-expression in colonized tumors resulted in contrast changes within the bacteria-colonized tumors. Conclusions: Our data suggest that colonization and gene expression by live vectors expressing bacterioferritin can be monitored by MRI due to contrast changes.}, 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} } @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{HertleinSturmKircheretal.2011, author = {Hertlein, Tobias and Sturm, Volker and Kircher, Stefan and Basse-L{\"u}sebrink, Thomas and Haddad, Daniel and Ohlsen, Knut and Jakob, Peter}, title = {Visualization of Abscess Formation in a Murine Thigh Infection Model of \(Staphylococcus\) \(aureus\) by (19)F-Magnetic Resonance Imaging (MRI)}, series = {PLoS ONE}, volume = {6}, journal = {PLoS ONE}, number = {3}, doi = {10.1371/journal.pone.0018246}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142846}, pages = {e18246}, year = {2011}, abstract = {Background: During the last years, (19)F-MRI and perfluorocarbon nanoemulsion (PFC) emerged as a powerful contrast agent methodology to track cells and to visualize inflammation. We applied this new modality to visualize deep tissue abscesses during acute and chronic phase of inflammation caused by Staphylococcus aureus infection. Methodology and Principal Findings: In this study, a murine thigh infection model was used to induce abscess formation and PFC or CLIO (cross linked ironoxides) was administered during acute or chronic phase of inflammation. 24 h after inoculation, the contrast agent accumulation was imaged at the site of infection by MRI. Measurements revealed a strong accumulation of PFC at the abscess rim at acute and chronic phase of infection. The pattern was similar to CLIO accumulation at chronic phase and formed a hollow sphere around the edema area. Histology revealed strong influx of neutrophils at the site of infection and to a smaller extend macrophages during acute phase and strong influx of macrophages at chronic phase of inflammation. Conclusion and Significance: We introduce (19)F-MRI in combination with PFC nanoemulsions as a new platform to visualize abscess formation in a murine thigh infection model of S. aureus. The possibility to track immune cells in vivo by this modality offers new opportunities to investigate host immune response, the efficacy of antibacterial therapies and the influence of virulence factors for pathogenesis.}, language = {en} } @article{BelairBaudChabasetal.2011, author = {Belair, C{\´e}dric and Baud, Jessica and Chabas, Sandrine and Sharma, Cynthia M and Vogel, J{\"o}rg and Staedel, Cathy and Darfeuille, Fabien}, title = {Helicobacter pylori interferes with an embryonic stem cell micro RNA cluster to block cell cycle progression}, series = {Silence : a Journal of RNA regulation}, volume = {2}, journal = {Silence : a Journal of RNA regulation}, number = {7}, doi = {10.1186/1758-907X-2-7}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-140438}, pages = {1-16}, year = {2011}, abstract = {Background MicroRNAs, post-transcriptional regulators of eukaryotic gene expression, are implicated in host defense against pathogens. Viruses and bacteria have evolved strategies that suppress microRNA functions, resulting in a sustainable infection. In this work we report that Helicobacter pylori, a human stomach-colonizing bacterium responsible for severe gastric inflammatory diseases and gastric cancers, downregulates an embryonic stem cell microRNA cluster in proliferating gastric epithelial cells to achieve cell cycle arrest. Results Using a deep sequencing approach in the AGS cell line, a widely used cell culture model to recapitulate early events of H. pylori infection of gastric mucosa, we reveal that hsa-miR-372 is the most abundant microRNA expressed in this cell line, where, together with hsa-miR-373, it promotes cell proliferation by silencing large tumor suppressor homolog 2 (LATS2) gene expression. Shortly after H. pylori infection, miR-372 and miR-373 synthesis is highly inhibited, leading to the post-transcriptional release of LATS2 expression and thus, to a cell cycle arrest at the G1/S transition. This downregulation of a specific cell-cycle-regulating microRNA is dependent on the translocation of the bacterial effector CagA into the host cells, a mechanism highly associated with the development of severe atrophic gastritis and intestinal-type gastric carcinoma. Conclusions These data constitute a novel example of host-pathogen interplay involving microRNAs, and unveil the couple LATS2/miR-372 and miR-373 as an unexpected mechanism in infection-induced cell cycle arrest in proliferating gastric cells, which may be relevant in inhibition of gastric epithelium renewal, a major host defense mechanism against bacterial infections.}, language = {en} } @phdthesis{Wencker2022, author = {Wencker, Freya Dorothea Ruth}, title = {The methionine biosynthesis operon in \(Staphylococcus\) \(aureus\): Role of concerted RNA decay in transcript stability and T-box riboswitch turnover}, doi = {10.25972/OPUS-20712}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-207124}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {Methionine is the first amino acid of every newly synthesised protein. In combination with its role as precursor for the vital methyl-group donor S-adenosylmethionine, methionine is essential for every living cell. The opportunistic human pathogen Staphylococcus aureus is capable of synthesising methionine de novo, when it becomes scarce in the environment. All genes required for the de novo biosynthesis are encoded by the metICFE-mdh operon, except for metX. Expression is controlled by a hierarchical network with a methionyl-tRNA-specific T-box riboswitch (MET-TBRS) as centrepiece, that is also referred to as met leader (RNA). T-box riboswitches (TBRS) are regulatory RNA elements located in the 5'-untranslated region (5'-UTR) of genes. The effector molecule of T-box riboswitches is uncharged cognate tRNA. The prevailing mechanism of action is premature termination of transcription of the nascent RNA in the absence of the effector (i.e. uncharged cognate tRNA) due to formation of a hairpin structure, the Terminator stem. In presence of the effector, a transient stabilisation of the alternative structure, the Antiterminator, enables transcription of the downstream genes ('read-through'). Albeit, after the read-through the thermodynamically more stable Terminator eventually forms. The Terminator and the Antiterminator are two mutually exclusive structures. Previous work of the research group showed that in staphylococci the MET-TBRS ensures strictly methionine-dependent control of met operon expression. Uncharged methionyl-tRNA that activates the system is only present in sufficient amounts under methionine-deprived conditions. In contrast to other bacterial TBRS, the staphylococcal MET-TBRS has some characteristic features regarding its length and predicted secondary structure whose relevance for the function are yet unkown. Aim of the present thesis was to experimentally determine the structure of the met leader RNA and to investigate the stability of the met operon-specific transcripts in the context of methionine biosynthesis control. Furthermore, the yet unknown function of the mdh gene within the met operon was to be determined. In the context of this thesis, the secondary structure of the met leader was determined employing in-line probing. The structural analysis revealed the presence of almost all highly conserved T-box riboswitch structural characteristics. Furthermore, three additional stems, absent in all T-box riboswitches analysed to date, could be identified. Particularly remarkable is the above average length of the Terminator stem which renders it a potential target of the double-strand-specific endoribonuclease III (RNase III). The RNase III-dependent cleavage of the met leader could be experimentally verified by the use of suitable mutants. Moreover, the exact cleavage site within the Terminator was determined. The unusual immediate separation of the met leader from the met operon mRNA via the RNase III cleavage within the Terminator stem induces the rapid degradation of the met leader RNA and, most likely, that of the 5'-region of the met mRNA. The met mRNA is degraded from its 5'-end by the exoribonuclease RNase J. The stability of the met mRNA was found to vary over the length of the transcript with an instable 5'-end (metI and metC) and a longer half-life towards the 3'-end (metE and mdh). The varying transcript stability is reflected by differences in the available cellular protein levels. The obtained data suggest that programmed mRNA degradation is another level of regulation in the complex network of staphylococcal de novo methionine biosynthesis control. In addition, the MET-TBRS was studied with regard to a future use as a drug target for novel antimicrobial agents. To this end, effects of a dysregulated methionine biosynthesis on bacterial growth and survival were investigated in met leader mutants that either caused permanent transcription of the met operon ('ON') or prevented operon transcription ('OFF'), irrespective of the methionine status in the cell. Methionine deprivation turned out to be a strong selection pressure, as 'OFF' mutants acquired adaptive mutations within the met leader to restore met operon expression that subsequently re-enabled growth. The second part of the thesis was dedicated to the characterisation of the Mdh protein that is encoded by the last gene of the met operon and whose function is unknown yet. At first, co-transcription and -expression with the met operon could be demonstrated. Next, the Mdh protein was overexpressed and purified and the crystal structure of Mdh was solved to high resolution by the Kisker research group (Rudolf-Virchow-Zentrum W{\"u}rzburg). Analysis of the structure revealed the amino acid residues crucial for catalytic activity, and zinc was identified as a co-factor of Mdh. Also, Mdh was shown to exist as a dimer. However, identification of the Mdh substrate was, in the context of this thesis, (still) unsuccessful. Nevertheless, interactions of Mdh with enzymes of the met operon could be demonstrated by employing the bacterial two-hybrid system. This fact and the high conservation of mdh/Mdh on nucleotide and amino acid level among numerous staphylococcal species suggests an important role of Mdh within the methionine metabolism that should be a worthwhile subject of future research.}, subject = {Staphylococcus aureus}, language = {en} } @article{WheelerBarquistKingsleyetal.2016, author = {Wheeler, Nicole E. and Barquist, Lars and Kingsley, Robert A. and Gardner, Paul P.}, title = {A profile-based method for identifying functional divergence of orthologous genes in bacterial genomes}, series = {Bioinformatics}, volume = {32}, journal = {Bioinformatics}, number = {23}, doi = {10.1093/bioinformatics/btw518}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-186502}, pages = {3566-3574}, year = {2016}, abstract = {Motivation: Next generation sequencing technologies have provided us with a wealth of information on genetic variation, but predi cting the functional significance of this variation is a difficult task. While many comparative genomics studies have focused on gene flux and large scale changes, relatively little attention has been paid to quantifying the effects of single nucleotide polymorphisms and indels on protein function, particularly in bacterial genomics. Results: We present a hidden Markov model based approach we call delta-bitscore (DBS) for identifying orthologous proteins that have diverged at the amino acid sequence level in a way that is likely to impact biological function. We benchmark this approach with several widely used datasets and apply it to a proof-of-concept study of orthologous proteomes in an investigation of host adaptation in Salmonella enterica. We highlight the value of the method in identifying functional divergence of genes, and suggest that this tool may be a better approach than the commonly used dN/dS metric for identifying functionally significant genetic changes occurring in recently diverged organisms.}, language = {en} } @article{SunkavalliAguilarSilvaetal.2017, author = {Sunkavalli, Ushasree and Aguilar, Carmen and Silva, Ricardo Jorge and Sharan, Malvika and Cruz, Ana Rita and Tawk, Caroline and Maudet, Claire and Mano, Miguel and Eulalio, Ana}, title = {Analysis of host microRNA function uncovers a role for miR-29b-2-5p in Shigella capture by filopodia}, series = {PLoS Pathogens}, volume = {13}, journal = {PLoS Pathogens}, number = {4}, doi = {10.1371/journal.ppat.1006327}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-158204}, pages = {e1006327}, year = {2017}, abstract = {MicroRNAs play an important role in the interplay between bacterial pathogens and host cells, participating as host defense mechanisms, as well as exploited by bacteria to subvert host cellular functions. Here, we show that microRNAs modulate infection by Shigella flexneri, a major causative agent of bacillary dysentery in humans. Specifically, we characterize the dual regulatory role of miR-29b-2-5p during infection, showing that this microRNA strongly favors Shigella infection by promoting both bacterial binding to host cells and intracellular replication. Using a combination of transcriptome analysis and targeted high-content RNAi screening, we identify UNC5C as a direct target of miR-29b-2-5p and show its pivotal role in the modulation of Shigella binding to host cells. MiR-29b-2-5p, through repression of UNC5C, strongly enhances filopodia formation thus increasing Shigella capture and promoting bacterial invasion. The increase of filopodia formation mediated by miR-29b-2-5p is dependent on RhoF and Cdc42 Rho-GTPases. Interestingly, the levels of miR-29b-2-5p, but not of other mature microRNAs from the same precursor, are decreased upon Shigella replication at late times post-infection, through degradation of the mature microRNA by the exonuclease PNPT1. While the relatively high basal levels of miR-29b-2-5p at the start of infection ensure efficient Shigella capture by host cell filopodia, dampening of miR-29b-2-5p levels later during infection may constitute a bacterial strategy to favor a balanced intracellular replication to avoid premature cell death and favor dissemination to neighboring cells, or alternatively, part of the host response to counteract Shigella infection. Overall, these findings reveal a previously unappreciated role of microRNAs, and in particular miR-29b-2-5p, in the interaction of Shigella with host cells.}, 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{UlbrichtNickelWeidenbachetal.2020, author = {Ulbricht, Andrea and Nickel, Lisa and Weidenbach, Katrin and Vargas Gebauer, Herman and Kießling, Claudia and F{\"o}rstner, Konrad U. and Schmitz, Ruth A.}, title = {The CARF protein MM_0565 affects transcription of the casposon-encoded cas1-solo gene in Methanosarcina mazei G{\"o}1}, series = {Biomolecules}, volume = {10}, journal = {Biomolecules}, number = {8}, issn = {2218-273X}, doi = {10.3390/biom10081161}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-211097}, year = {2020}, abstract = {Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) loci are found in bacterial and archaeal genomes where they provide the molecular machinery for acquisition of immunity against foreign DNA. In addition to the cas genes fundamentally required for CRISPR activity, a second class of genes is associated with the CRISPR loci, of which many have no reported function in CRISPR-mediated immunity. Here, we characterize MM_0565 associated to the type I-B CRISPR-locus of Methanosarcina mazei G{\"o}1. We show that purified MM_0565 composed of a CRISPR-Cas Associated Rossmann Fold (CARF) and a winged helix-turn-helix domain forms a dimer in solution; in vivo, the dimeric MM_0565 is strongly stabilized under high salt stress. While direct effects on CRISPR-Cas transcription were not detected by genetic approaches, specific binding of MM_0565 to the leader region of both CRISPR-Cas systems was observed by microscale thermophoresis and electromobility shift assays. Moreover, overexpression of MM_0565 strongly induced transcription of the cas1-solo gene located in the recently reported casposon, the gene product of which shows high similarity to classical Cas1 proteins. Based on our findings, and taking the absence of the expressed CRISPR locus-encoded Cas1 protein into account, we hypothesize that MM_0565 might modulate the activity of the CRISPR systems on different levels.}, language = {en} } @phdthesis{Troge2012, author = {Troge, Anja}, title = {Studien am Flagellensystem des Escherichia coli Stammes Nissle 1917 (EcN) im Hinblick auf seine Funktion als Probiotikum}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-74201}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Escherichia coli Nissle 1917 (EcN) geh{\"o}rt zu den am besten untersuchten und charakterisierten probiotischen Bakterienst{\"a}mmen. Seit Beginn des letzten Jahrhunderts wird er als Medikament eingesetzt, um verschiedene Darmerkrankungen wie z.B. Diarrh{\"o}e, entz{\"u}ndliche Darmerkrankungen und Verstopfung zu behandeln. Die Flagelle des EcN vermittelt Beweglichkeit und kann die Produktion von humanem β-Defensin 2 (hBD2) durch Epithelzellen induzieren. Somit ist dieses Organell direkt in die probiotische Funktion des EcN involviert. Es konnte gezeigt werden, dass die Flagellen anderer Bakterien, wie z.B. dem probiotischen Stamm Bacillus cereus CH oder den pathogenen St{\"a}mmen Pseudomonas aeruginosa und Clostridium difficile, die Adh{\"a}sion an intestinalen Mucus, welcher von Epithelzellen sekretiert wird, vermitteln. Allerdings blieb unklar, welcher Teil der Flagelle an welche Mucuskomponente bindet. Die F{\"a}higkeit effizient an Wirtgewebe zu adh{\"a}rieren wird als wichtiges Attribut eines probiotischen Stammes angesehen. Ex vivo Adh{\"a}sionsstudien mit Kryoschnitten humaner Darmbiopsien haben gezeigt, dass die Flagelle des EcN in die effiziente Adh{\"a}sion an humanes Darmgewebe involviert sein muss. Aus diesem Grund wurde in dieser Arbeit die Funktion der Flagelle des EcN als Adh{\"a}sin untersucht. Zun{\"a}chst wurde die hyperflagellierte Variante EcN ATHF isoliert und durch verschiedene Experimente, z.B. Schw{\"a}rmagartests und Elektronenmikroskopie, charakterisiert. Weitere ex vivo Adh{\"a}sionsstudien mit EcN ATHF zeigten eine h{\"o}here Adh{\"a}sionseffizienz dieser hyperflagellierten Variante und best{\"a}tigten damit die Rolle der Flagelle bei der effizienten Adh{\"a}sion von EcN an die Kryoschnitte der humanen Darmbiopsien. Interessanterweise fungierte die Flagelle in in vitro Studien mit den humanen Epithelzellen Caco-2 und T24 nicht als Adh{\"a}sin. Diese Unterschiede zwischen den in vitro und ex vivo Studien f{\"u}hrten zu der Annahme, dass die Flagelle des EcN in vivo die Adh{\"a}sion an Mucus vermittelt, welcher von den Caco-2- und T24-Zellen nicht produziert wird, aber in den Kryoschnitten der Darmbiopsien nachgewiesen wurde. Diese Vermutung wurde durch in vitro Adh{\"a}sionsstudien mit der Mucin-produzierenden Epithelzelllinie LS174-T best{\"a}tigt, da die Flagellen f{\"u}r eine effektive Adh{\"a}sion an diese Zellen essentiell waren. Zudem reduzierte die Pr{\"a}inkubation flagellierter EcN-St{\"a}mme mit Mucin2 ihre Adh{\"a}sionseffizienz an Kryoschnitte humaner Darmbiopsien. Um die direkte Interaktion zwischen Flagellen des EcN Wildtyps und Mucus zu zeigen, wurde ein ELISA etabliert. Es konnte eine direkte konzentrationsabh{\"a}ngige Interaktion zwischen isolierten Flagellen des EcN Wildtyps und Mucin2, bzw. humanem Mucus (Kolon) beobachtet werden. Interessanterweise konnte keine Interaktion zwischen isolierten Flagellen des EcN Wildtyps und murinem Mucus (Duodenum, Ileum, Caecum, Colon) festgestellt werden. Dies weist darauf hin, dass die Mucuszusammensetzung zwischen verschiedenen Spezies variiert. Verschiedene Kohlenhydrate, welche bekannte Mucusbestandteile sind, wurden auf ihre Interaktion mit der Flagelle von EcN getestet und Gluconat wurde als ein Rezeptor identifiziert. Die Pr{\"a}inkubation isolierter Flagellen mit Gluconat reduzierte ihre Interaktion mit Mucin2, bzw. humanem Mucus signifikant. Zudem wurde die oberfl{\"a}chenexponierte Dom{\"a}ne D3 des Flagellins, der Hauptuntereinheit der Flagelle, als m{\"o}glicher Interaktionspartner von Mucin2, bzw. humanem Mucus ausgeschlossen. Flagellen, die aus einer Dom{\"a}ne D3 Deletionsmutante isoliert wurden, zeigten sogar eine effizientere Bindung an Mucin2, bzw. humanen Mucus. Weiterhin konnte gezeigt werden, dass {\"A}nderungen des pH-Wertes signifikante Effekte auf die Interaktion zwischen Mucus und isolierten Flagellen hatten, vermutlich aufgrund von Konformations{\"a}nderungen. Zusammenfassend wurde in dieser Arbeit die Flagelle als neues und scheinbar wichtigstes Adh{\"a}sin in vivo f{\"u}r den probiotischen Stamm EcN identifiziert. Hierf{\"u}r wurden sowohl eine hyperflagellierte Variante, eine ΔfliC Mutante, sowie der dazugeh{\"o}rige komplementierte Stamm verwendet. EcN ist zudem der erste probiotische Stamm f{\"u}r den eine direkte Bindung der Flagellen an humanen Mucus nachgewiesen werden konnte. Die Mucuskomponente Gluconat konnte dabei als wichtiger Rezeptor identifiziert werden. Da einige pathogene Bakterien ihre Flagelle zur Adh{\"a}sion an Wirtsgewebe nutzen, k{\"o}nnte dieses Organell EcN dazu bef{\"a}higen, mit Pathogenen um die erfolgreiche Kolonisierung des Darms zu konkurrieren, was als wichtige Eigenschaft eines Probiotikums betrachtet wird.}, subject = {Escherichia coli}, language = {de} } @phdthesis{Aminake2012, author = {Aminake, Makoah Nigel}, title = {Towards malaria combination therapy: Characterization of hybrid molecules for HIV/malaria combination therapy and of thiostrepton as a proteasome-targeting antibiotic with a dual mode of action}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-71841}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Malaria and HIV are among the most important global health problems of our time and together are responsible for approximately 3 million deaths annually. These two diseases overlap in many regions of the world including sub-Saharan Africa, Southeast Asia and South America, leading to a higher risk of co-infection. In this study, we generated and characterized hybrid molecules to target P. falciparum and HIV simultaneously for a potential HIV/malaria combination therapy. Hybrid molecules were synthesized by covalent fusion between azidothymidine (AZT) and dihydroartemisinin (DHA), tetraoxane or chloroquine (CQ); and a small library was generated and tested for antiviral and antimalarial activity. Our data suggest that dihyate is the most potent molecule in vitro, with antiplasmodial activity comparable to that of DHA (IC50 = 26 nM, SI > 3000), a moderate activity against HIV (IC50 = 2.9 µM; SI > 35) and safe to HeLa cells at concentrations used in the assay (CC50 > 100 µM). Pharmacokinetic studies further revealed that dihyate is metabolically unstable and is cleaved following an O-dealkylation once in contact with cytochrome P450 enzymes. The later further explains the uneffectiveness of dihyate against the CQ-sensitive P. berghei N strain in mice when administered by oral route at 20 mg/kg. Here, we report on a first approach to develop antimalarial/anti-HIV hybrid molecules and future optimization efforts will aim at producing second generation hybrid molecules to improve activity against HIV as well as compound bioavailability. With the emergence of resistant parasites against all the counterpart drugs of artemisinin derivatives used in artemisinin based combination therapies (ACTs), the introduction of antibiotics in the treatment of malaria has renewed interest on the identification of antibiotics with potent antimalarial properties. In this study we also investigated the antiplasmodial potential of thiostrepton and derivatives, synthesized using combinations of tail truncation, oxidation, and addition of lipophilic thiols to the terminal dehydroamino acid. We showed that derivatives SS231 and SS234 exhibit a better antiplasmodial activity (IC50 = 1 µM SI > 59 and SI > 77 respectively) than thiostrepton (IC50 = 8.95 µM, SI = 1.7). The antiplasmodial activity of these derivatives was observed at concentrations which are not hemolytic and non-toxic to human cell lines. Thiostrepton and derivatives appeared to exhibit transmission blocking properties when administered at their IC50 or IC90 concentrations and our data also showed that they attenuate proteasome activity of Plasmodium, which resulted in an accumulation of ubiquitinated proteins after incubation with their IC80 concentrations. Our results indicate that the parasite's proteasome could be an attractive target for therapeutic intervention. In this regard, thiostrepton derivatives are promising candidates by dually acting on two independent targets, the proteasome and the apicoplast, with the capacity to eliminate both intraerythrocytic asexual and transmission stages of the parasite. To further support our findings, we evaluated the activity of a new class of antimalarial and proteasome inhibitors namely peptidyl sulfonyl fluorides on gametocyte maturation and analogues AJ34 and AJ38 were able to completely suppress gametocytogenesis at IC50 concentrations (0.23 µM and 0.17 µM respectively) suggesting a strong transmission blocking potential. The proteasome, a major proteolytic complex, responsible for the degradation and re-cycling of non-functional proteins has been studied only indirectly in P. falciparum. In addition, an apparent proteasome-like protein with similarity to bacterial ClpQ/hslV threonine-peptidases was predicted in the parasite. Antibodies were generated against the proteasome subunits alpha type 5 (α5-SU), beta type 5 (β5-SU) and pfhslV in mice and we showed that the proteasome is expressed in both sexual and asexual blood stages of P. falciparum, where they localize in the nucleus and in the cytoplasm. However, expression of PfhslV was only observed in trophozoites and shizonts. The trafficking of the studied proteasome subunits was further investigated by generating parasites expressing GFP tagged proteins. The expression of α5-SU-GFP in transgenic parasite appeared to localize abundantly in the cytoplasm of all blood stages, and no additional information was obtained from this parasite line. In conclusion, our data highlight two new tools towards combination therapy. Hybrid molecules represent promising tools for the cure of co-infected individuals, while very potent antibiotics with a wide scope of activities could be useful in ACTs by eliminating resistant parasites and limiting transmission of both, resistances and disease.}, subject = {Malaria}, language = {en} } @phdthesis{Schubert2011, author = {Schubert, Sabrina}, title = {Funktionelle Analyse des „Multidrug-Resistance"-Regulators MRR1 im humanpathogenen Hefepilz Candida albicans}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-70916}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2011}, abstract = {Der Hefepilz Candida albicans geh{\"o}rt zu den fakultativ pathogenen Infektionserregern und ist Teil der nat{\"u}rlichen Mikroflora der Schleimh{\"a}ute des Verdauungs- und Urogenitaltraktes der meisten gesunden Menschen. Ist das Gleichgewicht der Flora gest{\"o}rt, kann es zu oberfl{\"a}chlichen Mykosen kommen, wie z.B. der oropharyngealen Candidiasis (Mundsoor), die in der Regel durch die Gabe eines Antimykotikums in wenigen Tagen zu behandeln sind. In seltenen F{\"a}llen kann es auch zu schwerwiegenden Infektionsverl{\"a}ufen bis hin zu lebensbedrohlichen systemischen Mykosen kommen. Haupts{\"a}chlich immunsupprimierte Patienten, wie z.B. AIDS-Patienten oder Personen, die k{\"u}rzlich einer Organ- oder Knochenmarkstransplantation unterzogen wurden, leiden h{\"a}ufig an oberfl{\"a}chlichen C. albicans-Infektionen. Insbesondere bei wiederkehrenden Infektionen ist der Pilz in der Lage, gegen das h{\"a}ufig verabreichte Medikament Fluconazol eine Resistenz zu entwickeln. Ein wichtiger Mechanismus dieser Resistenzentwicklung ist die {\"U}berexpression von Effluxpumpen, die das Medikament aus der Zelle heraustransportieren. Zwei Arten von Effluxpumpen, die eine Rolle in der Resistenzentwicklung in C. albicans spielen, konnten bisher identifiziert werden, die ABC (ATP binding cassette)-Transporter Cdr1 und Cdr2 sowie der MFS (major facilitator superfamily)-Transporter Mdr1. Der Zinc-Cluster Transkriptionsfaktor Mrr1 spielt eine wichtige Rolle in der Regulation der MDR1-E¬ffluxpumpe. Er kontrolliert die MDR1-Expression in Anwesenheit induzierender Substanzen und sogenannte "gain-of-function" Mutationen in MRR1 konnten als die Ursache der konstitutiven MDR1-Hochregulierung und der "Multidrug-Resistance" in C. albicans identifiziert werden. In dieser Arbeit konnte ein Ortholog zu MRR1 aus C. albicans in Candida dubliniensis, einer zu C. albicans nahe verwandten Hefe, identifiziert werden. Es wurde gezeigt, dass in den untersuchten klinischen und in vitro generierten Fluconazol-resistenten C. dubliniensis-St{\"a}mmen ebenfalls gain-of-funcion Mutationen in MRR1 die MDR1-{\"U}berexpression und eine Resistenz bewirken. Die Ergebnisse demonstrieren, dass der Transkriptionsfaktor Mrr1 eine wichtige Rolle in der Entwicklung der Resistenz in diesen humanpathogenen Pilzen spielt. Bisher ist nicht bekannt, wie der Zinc-Cluster Transkriptionsfaktor MRR1 durch induzierende Substanzen oder gain-of-function Mutationen aktiviert wird. Um zu verstehen, wie die Mrr1- Aktivit{\"a}t reguliert wird, wurden in dieser Arbeit durch Deletionsstudien funktionelle Dom{\"a}nen des Transkriptionsfaktors identifiziert. Um einen besseren Einblick in die Regulation der MDR1-vermittelten Resistenz in C. albicans zu bekommen, wurde in dieser Arbeit die gegenseitige Abh{\"a}ngigkeit von Mrr1 und Cap1 bzw. Upc2 in Bezug auf die MDR1-Expression untersucht. Es wurden ChIP-on-chip Analysen und Transkriptionsprofile mit aktiviertem Mrr1 durchgef{\"u}hrt, um direkte Targets von Mrr1 zu identifizieren. Mit der vorliegenden Arbeit wurde ein wichtiger Beitrag zum Verst{\"a}ndnis der Entwicklung der Multidrug-Resistenz in C. albicans geleistet. E¬ffluxpumpen und deren Regulatoren stellen in der Bek{\"a}mpfung von C. albicans-Infektionen ein interessantes Angriffsziel f{\"u}r die Entwicklung neuer Medikamente und die Weiterentwicklung bereits vorhandender Antimykotika dar.}, subject = {Candida albicans}, language = {de} } @article{ZdziarskiBrzuszkiewiczWulltetal.2010, author = {Zdziarski, Jaroslaw and Brzuszkiewicz, Elzbieta and Wullt, Bjorn and Liesegang, Heiko and Biran, Dvora and Voigt, Birgit and Gronberg-Hernandez, Jenny and Ragnarsdottir, Bryndis and Hecker, Michael and Ron, Eliora Z. and Daniel, Rolf and Gottschalk, Gerhard and Hacker, Joerg and Svanborg, Catharina and Dobrindt, Ulrich}, title = {Host Imprints on Bacterial Genomes-Rapid, Divergent Evolution in Individual Patients}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-68594}, year = {2010}, abstract = {Bacteria lose or gain genetic material and through selection, new variants become fixed in the population. Here we provide the first, genome-wide example of a single bacterial strain's evolution in different deliberately colonized patients and the surprising insight that hosts appear to personalize their microflora. By first obtaining the complete genome sequence of the prototype asymptomatic bacteriuria strain E. coli 83972 and then resequencing its descendants after therapeutic bladder colonization of different patients, we identified 34 mutations, which affected metabolic and virulence-related genes. Further transcriptome and proteome analysis proved that these genome changes altered bacterial gene expression resulting in unique adaptation patterns in each patient. Our results provide evidence that, in addition to stochastic events, adaptive bacterial evolution is driven by individual host environments. Ongoing loss of gene function supports the hypothesis that evolution towards commensalism rather than virulence is favored during asymptomatic bladder colonization.}, subject = {Proteomanalyse}, language = {en} } @article{SasseSchilligDierolfetal.2011, author = {Sasse, Christoph and Schillig, Rebecca and Dierolf, Franziska and Weyler, Michael and Schneider, Sabrina and Mogavero, Selene and Rogers, David P. and Morschh{\"a}user, Joachim}, title = {The Transcription Factor Ndt80 Does Not Contribute to Mrr1-, Tac1-, and Upc2-Mediated Fluconazole Resistance in Candida albicans}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-69201}, year = {2011}, abstract = {The pathogenic yeast Candida albicans can develop resistance to the widely used antifungal agent fluconazole, which inhibits ergosterol biosynthesis, by the overexpression of genes encoding multidrug efflux pumps or ergosterol biosynthesis enzymes. Zinc cluster transcription factors play a central role in the transcriptional regulation of drug resistance. Mrr1 regulates the expression of the major facilitator MDR1, Tac1 controls the expression of the ABC transporters CDR1 and CDR2, and Upc2 regulates ergosterol biosynthesis (ERG) genes. Gain-of-function mutations in these transcription factors result in constitutive overexpression of their target genes and are responsible for fluconazole resistance in many clinical C. albicans isolates. The transcription factor Ndt80 contributes to the drug-induced upregulation of CDR1 and ERG genes and also binds to the MDR1 and CDR2 promoters, suggesting that it is an important component of all major transcriptional mechanisms of fluconazole resistance. However, we found that Ndt80 is not required for the induction of MDR1 and CDR2 expression by inducing chemicals. CDR2 was even partially derepressed in ndt80D mutants, indicating that Ndt80 is a repressor of CDR2 expression. Hyperactive forms of Mrr1, Tac1, and Upc2 promoted overexpression of MDR1, CDR1/CDR2, and ERG11, respectively, with the same efficiency in the presence and absence of Ndt80. Mrr1- and Tac1-mediated fluconazole resistance was even slightly enhanced in ndt80D mutants compared to wild-type cells. These results demonstrate that Ndt80 is dispensable for the constitutive overexpression of Mrr1, Tac1, and Upc2 target genes and the increased fluconazole resistance of strains that have acquired activating mutations in these transcription factors.}, subject = {Candida albicans}, language = {en} } @article{PernitzschSharma2012, author = {Pernitzsch, Sandy R. and Sharma, Cynthia M.}, title = {Transcriptome Complexity and Riboregulation in the Human Pathogen Helicobacter pylori}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-75096}, year = {2012}, subject = {Medizin}, language = {en} } @phdthesis{Schnitzer2012, author = {Schnitzer, Johannes K.}, title = {Mechanism of dendritic cell-based vaccination against Leishmania major}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-74865}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Die Impfung mittels Antigen-beladener dendritischer Zellen [DZ] ist mittlerweile eine gut etablierte Technik, die dann zum Einsatz kommt, wenn Standard-Impftechniken versagen, vor Krankheiten zu sch{\"u}tzen beziehungsweise diese zu heilen. Die Effizienz dieser Technik konnte bereits f{\"u}r diverse Infektionskrankheiten und Krebserkrankungen in experimentellen Tiermodellen sowie am Menschen gezeigt werden. Hierbei ist die M{\"o}glichkeit zur wohldefinierten Manipulation und Antigenbeladung der DZ ein großer Vorteil gegen{\"u}ber den konventionellen Ans{\"a}tzen. Jedoch ist vor allem bei der Anwendung im klinischen Bereich die Pr{\"a}paration, Herstellung und Manipulation dieser autologen DZ mit einem erheblichen technischen, zeitlichen sowie finanziellen Aufwand verbunden. Hinsichtlich einer Pr{\"a}ventivimpfung gegen eine pandemische Infektionskrankheit, die in haupts{\"a}chlich unterentwickelten L{\"a}ndern vorkommt, wird dieser Aufwand sicherlich ein Hindernis darstellen. Daher muss f{\"u}r solche F{\"a}lle ein maßgeschneiderter Impfstoff entwickelt werden, der sich am Vorbild des effektiven DZ-basierten Impfstoffs orientiert. F{\"u}r die Impfung gegen die Leishmania Parasiten besteht so ein DZ-basierter Impfstoff bereits. Dessen Wirkung, eine T-Zell Antwort vom Typ Th1 zu induzieren, wurde bereits in mehreren Ver{\"o}ffentlichungen demonstriert. Zus{\"a}tzlich hat aber eine unserer Studien gezeigt, dass das typische Th1-bezogene Zytokin IL-12 zur Differenzierung naiver T-Zellen nicht von den injizierten DZ bereitgestellt werden muss, sondern von der geimpften Maus. Dies gab erste Hinweise auf eine st{\"a}rkere Beteiligung des Wirts-Immunsystems als zuvor angenommen. Daher sollte hier vertieft der Mechanismus dieser DZ-basierten Impfung untersucht werden, wobei modifizierte Impfstoff-Ans{\"a}tze zum Einsatz kommen sollten. Dabei wurden die Fragen nach der vom Impfstoff transportierten Information und dem Empf{\"a}nger dieser Information ber{\"u}cksichtigt. Das aktuelle Paradigma zur DZ-basierten Impfung besagt, dass transferierte DZ im direkten Kontakt mittels dreier Signale T-Zellen stimulieren und aktivieren. Daf{\"u}r m{\"u}ssen diese DZ mit dem entsprechenden Antigen beladen und aktiviert worden sein um das Antigen-Peptide mittels MHC Molek{\"u}l im Kontext der Co-Stimulation pr{\"a}sentieren zu k{\"o}nnen. Jedoch zeigt diese Studie hier, dass weder eine Aktivierung der DZ noch die Pr{\"a}sentation des Antigens mittels passender MHC Molek{\"u}le notwendig ist f{\"u}r die Induktion einer protektiven Immunantwort gegen Leishmania Parasiten. Aufgeschlossene, mit Antigen beladene DZ m{\"u}ssen nicht vor dem Transfer mit CpG ODN aktiviert worden sein, um entsprechende Immunit{\"a}t zu verleihen. Ebenso hat der MHC Typ in diesem Falle auch keinen Einfluss auf die Effektivit{\"a}t des Impfstoffs. Da im Weiteren aufgeschlossene mit Leishmania-Antigen beladene Makrophagen nach Impfung die gleiche Wirkung erzielen, wie vorangegangene DZ-basierte Impfstoffe, k{\"o}nnen keine DZ spezifischen Mechanismen Schl{\"u}sselkomponenten der Induktion einer protektiven Immunit{\"a}t sein. Dar{\"u}ber hinaus konnte gezeigt werden, dass die DZ der geimpften M{\"a}use, eine maßgebliche Rolle bei der Verarbeitung transferierter Signale spielen. Suspensionen aufgeschlossener DZ stellen eine Kombination aus freigesetzten l{\"o}slichen Molek{\"u}len sowie Membranvesikeln dar, die sich nach dem Aufschluss gebildet haben. Nach Auftrennung dieser beiden Fraktionen konnte gezeigt werden, dass ausschließlich die Membran-Fraktion nach Verimpfung eine geeignete Immunantwort zum Schutz vor Leishmania Parasiten induzieren kann. Als Vorteil dieser Aufreinigung erweist sich zudem die stabile Lagerm{\"o}glichkeit bei -80°C. Somit ist klar gezeigt, dass die Immunit{\"a}t-verleihende Einheit dieser Impfstoffvarianten in der Membran-Fraktion liegt. Verfolgt man die Induktion Th1-zugeh{\"o}riger Zytokine in in vivo Experimenten so ergibt sich im Falle der Gesamtsuspension aufgeschlossener, mit Leishmania-Antigen beladener DZ ein klares Bild. Diese Suspension erzeugt das volle Spektrum der DZ-basierten Impfung gegen Leishmania Parasiten. Es kann sowohl Produktion von IL-12 und IL-2 als auch eine antigenspezifische T-Zell Proliferation nach Stimulation von Splenozyten mit der entsprechenden Suspension verzeichnet werden. Außerdem produzieren Splenozyten von entsprechend geimpften M{\"a}usen nach Stimulation mit Leishmania-Antigen erhebliche Mengen des entscheidenden Zytokins IFNγ. Obwohl jedoch die Verimpfung aufgereinigter Membranvesikel dieses Ansatzes im Tierversuch zu biologisch sowie statistisch signifikanten Ergebnissen f{\"u}hrt, lassen sich die entsprechend Th1-bezogenen Zytokine im in vivo Ansatz nur in geringen Maße nachweisen. Ob dies jedoch f{\"u}r einen in vivo unbemerkten Aktivit{\"a}tsverlust des Vakzins oder f{\"u}r andere lymphatische Organe als Ort der T-Zell Instruktion spricht, ist noch unbekannt und muss noch gekl{\"a}rt werden.}, subject = {Leishmania major}, language = {en} } @phdthesis{Masic2012, author = {Masic, Anita}, title = {Signaling via Interleukin-4 Receptor alpha chain during dendritic cell-mediated vaccination is required to induce protective immunity against Leishmania major in susceptible BALB/c mice}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-75508}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Cutaneous leishmaniasis is endemic in tropical and subtropical regions of the world. Effective vaccination strategies are urgently needed because of the emergence of drug-resistant parasites and severe side effects of chemotherapy. The research group of Heidrun Moll previously established a DC-based vaccination strategy to induce complete and long-lasting immunity to experimental leishmaniasis using LmAg-loaded and CpG ODN-activated DC as a vaccine carrier. Prevention of tissue damages at the site of L. major inoculation can be achieved if the BALB/c mice were systemically given LmAg-loaded BMDC that had been exposed to CpG ODN. The interest in further exploring the role of IL-4 aroused as previous studies allowed establishing that IL-4 was involved in the redirection of the immune response towards a type 1 profile. Thus, wt BALB/c mice or DC-specific CD11ccreIL-4Rα-/lox BALB/c mice were given either wt or IL-4Rα-deficient LmAg-loaded BMDC exposed or not to CpG ODN prior to inoculation of 2 x 105 stationary phase L. major promastigotes into the BALB/c footpad. The results provide evidence that IL4/IL-4Rα-mediated signaling in the vaccinating DC is required to prevent tissue damages at the site of L. major inoculation, as properly conditioned wt DC but not IL-4Rα-deficient DC were able to confer resistance. Furthermore, uncontrolled L. major population size expansion was observed in the footpad and the footpad draining LN in CD11ccreIL-4Rα-/lox mice immunized with CpG ODN-exposed LmAg-loaded IL-4Rα-deficient DC, indicating the influence of IL-4R-mediated signaling in host DC to control parasite replication. In addition, no footpad damage was observed in BALB/c mice that were systemically immunized with LmAg-loaded wt DC doubly exposed to CpG ODN and recombinant IL-4. Discussing these findings allow the assumption that triggering the IL4/IL4Rα signaling pathway could be a precondition when designing vaccines aimed to prevent damaging processes in tissues hosting intracellular microorganisms.}, subject = {Leishmania major}, 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} } @article{HessStritzkerHaertletal.2011, author = {Hess, Michael and Stritzker, Jochen and H{\"a}rtl, Barbara and Sturm, Julia and Gentschev, Ivaylo and Szalay, Aladar}, title = {Bacterial glucuronidase as general marker for oncolytic virotherapy or other biological therapies}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-69163}, year = {2011}, abstract = {Background: Oncolytic viral tumor therapy is an emerging field in the fight against cancer with rising numbers of clinical trials and the first clinically approved product (Adenovirus for the treatment of Head and Neck Cancer in China) in this field. Yet, until recently no general (bio)marker or reporter gene was described that could be used to evaluate successful tumor colonization and/or transgene expression in other biological therapies. Methods: Here, a bacterial glucuronidase (GusA) encoded by biological therapeutics (e.g. oncolytic viruses) was used as reporter system. Results: Using fluorogenic probes that were specifically activated by glucuronidase we could show 1) preferential activation in tumors, 2) rena l excretion of the activated fluorescent compounds and 3) reproducible detection of GusA in the serum of oncolytic vaccinia virus treated, tumor bearing mice in several tumor models. Time course studies revealed that reliable differentiation between tumor bearing and healthy mice can be done as early as 9 days post injection of the virus. Regarding the sensitivity of the newly developed assay system, we could show that a single infected tumor cell could be reliably detected in this assay. Conclusion: GusA therefore has the potential to be used as a general marker in the preclinical and clinical evaluation of (novel) biological therapies as well as being useful for the detection of rare cells such as circulating tumor cells}, subject = {Virologie}, language = {en} } @phdthesis{Angermeier2011, author = {Angermeier, Hilde Gabriele}, title = {Molecular and ecological investigations of Caribbean sponge diseases}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-56855}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2011}, abstract = {W{\"a}hrend gewinnbringende Assoziationen von Schw{\"a}mmen mit Mikroorganismen in den letzten Jahren viel Aufmerksamkeit erhalten haben, wurde weit weniger in die Interaktion von Schw{\"a}mmen mit m{\"o}glicherweise pathogenen Mikroben investiert. Somit war es das Ziel dieser Studie zwei ausgew{\"a}hlte Karibische Schwammkrankheiten namens „Sponge Orange Band" und „Sponge White Patch" mittels {\"o}kologischer und molekularer Methoden zu untersuchen. Die Sponge Orange Band (SOB) Erkrankung bef{\"a}llt den bedeutenden karibischen Fass-Schwamm Xestospongia muta, der zu den bakterienhaltigen (HMA) Schw{\"a}mmen gez{\"a}hlt wird, w{\"a}hrend die Sponge White Patch (SWP) Erkrankung den h{\"a}ufig vorkommenden Seil-Schwamm Amphimedon compressa betrifft, der zu den bakterienarmen (LMA) Schw{\"a}mmen geh{\"o}rt. F{\"u}r beide Karibischen Schwammkrankheiten konnte ich einen Krankheitsverlauf beschreiben, der mit massiver Gewebszerst{\"o}rung und dem Verlust charakteristischer mikrobieller Signaturen einhergeht. Obwohl ich zeigen konnte, dass zus{\"a}tzliche Bakterienarten die gebleichten Schwammbereiche kolonisieren, lieferten meine Infektionsversuche in beiden F{\"a}llen keinen Beweis f{\"u}r die Beteiligung eines mikrobiellen Pathogens als Krankheitserreger. Somit liegen die eigentlichen Ausl{\"o}ser der Erkrankungen Sponge Orange Band als auch Sponge White Patch noch immer im Dunkeln.}, subject = {Meeresschw{\"a}mme}, language = {en} } @article{KuehnPradel2010, author = {Kuehn, Andrea and Pradel, Gabriele}, title = {The Coming-Out of Malaria Gametocytes [Review Article]}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-68196}, year = {2010}, abstract = {The tropical disease malaria, which results in more than one million deaths annually, is caused by protozoan parasites of the genus Plasmodium and transmitted by blood-feeding Anopheline mosquitoes. Parasite transition from the human host to the mosquito vector is mediated by gametocytes, sexual stages that are formed in human erythrocytes, which therefore play a crucial part in the spread of the tropical disease. The uptake by the blood-feeding mosquito triggers important molecular and cellular changes in the gametocytes, thus mediating the rapid adjustment of the parasite from the warm-blooded host to the insect host and subsequently initiating reproduction. The contact with midgut factors triggers gametocyte activation and results in their egress from the enveloping erythrocyte, which then leads to gamete formation and fertilization. This review summarizes recent findings on the role of gametocytes during transmission to themosquito and particularly focuses on the molecular mechanisms underlying gametocyte activation and emergence from the host erythrocyte during gametogenesis.}, subject = {Malaria}, language = {en} } @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{Seo2012, author = {Seo, Ean Jeong}, title = {Construction of recombinant E. coli Nissle 1917 (EcN) strains for the expression and secretion of defensins}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72005}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {Der probiotische Escherichia coli Stamm Nissle 1917 (EcN) ist eines der wenigen Probiotika, die als aktive Komponente eines Medikaments in mehreren L{\"a}ndern zugelassen sind. Am besten ist die Wirksamkeit des EcN f{\"u}r die Remissionserhaltung von an Colitis Ulcerosa leidenden Patienten dokumentiert. Diese F{\"a}higkeit ist vermutlich darauf zur{\"u}ckzuf{\"u}hren, dass EcN in der Lage ist die Produktion des humanen beta-Defensins 2 (HBD2) mittels seiner Flagelle zu Induzieren. In dieser Studie wurden rekombinante EcN St{\"a}mme konstruiert, die ein Defensin zu produzieren verm{\"o}gen. Zu diesem Zweck wurden Kodon-optimierte Defensingene in Expressionsplasmidvektoren kloniert, die entweder die Proform mit der Signalsequenz oder die reife Defensinform des humanen -Defensins 5 (HD5) oder des humanen -Defensins 2 (HBD2) unter der Kontrolle des T7-Promotors kodieren. Die Synthese dieser Defensine wurde mittels Western-Blot nach der Induktion der Expression und der Lyse der rekombinanten EcN St{\"a}mme demonstriert. Das rekombinante reife HBD2 mit einem N-terminalen His-Tag konnte mittels Ni-S{\"a}ulen-Chromatographie aufgereinigt werden. Das so gewonnene HBD2 zeigte antimikrobielle Aktivit{\"a}t gegen E. coli, Salmonella enterica Serovar Typhimurium und Listeria monocytogenes. In einem zweiten Ansatz wurde der Teil des HBD2-Gens mit dem yebF-Gen fusioniert, der das reife HBD2 kodiert. Das resultierende Fusionsprotein YebFMHBD2 wurde von dem entsprechenden EcN Stamm nach Induktion der Expression sekretiert. Die Pr{\"a}senz von YebFMHBD2 im Medium war nicht das Ergebnis von Zellyse wie Western-Blots spezifisch f{\"u}r die -Galaktosidase und das Maltose-Bindeprotein mit dem Kultur{\"u}berstand zeigten. Dieser Kultur{\"u}berstand inhibierte das Wachstum von E. coli, Salmonella enterica Serovar Typhimurium und Listeria monocytogenes nach Dialyse und Aufkonzentration sowohl in Agardiffusionsassays als auch in Fl{\"u}ssigcokultur. Damit konnte gezeigt werden, dass EcN ein f{\"u}r die Produktion von bestimmten humanen Defensinen geeignetes Probiotikum darstellt. EcN ist bei der Behandlung von Morbus Crohn Patienten nicht aktiv. Dies ist vermutlich in der genetisch bedingten Unf{\"a}higkeit zur ausreichenden Defensinproduktion solcher Individuen begr{\"u}ndet. Als ein erster Schritt in der Entwicklung von alternativen Ans{\"a}tzen zur Behandlung Morbus Crohn Patienten wurden in dieser Arbeit EcN St{\"a}mme konstruiert, die in der Lage sind HD5 oder HBD2 zu produzieren.}, subject = {Escherichia coli}, 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{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{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{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} } @phdthesis{Simon2012, author = {Simon, Nina Monica}, title = {Molecular interactions of the malaria parasite Plasmodium falciparum during the sexual reproduction in the mosquito midgut}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72403}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2012}, abstract = {The sexual phase of Plasmodium falciparum begins with the differentiation of intraerythrocytic sexual stages, termed gametocytes, in the human host. Mature gametocytes circulate in the peripheral blood and are taken up by the mosquito during the blood meal. These stages are essential for the spread of the malaria disease and form gametes in the mosquito midgut within minutes. A highly conserved family of six secreted proteins has been identified in Plasmodium falciparum. They comprise multiple adhesive domains and are termed PfCCp1 through PfCCp5, and PfFNPA. It was revealed in this work that PfCCp multi-domain adhesion proteins form protein complexes in gametocytes and on the surface of newly emerged macrogametes by adhesion domain-mediated binding. Co-Immunoprecipitation assays with activated gametocyte lysates show interactions between PfCCp proteins and indicate surface association via Pfs230 and Pfs25. Pfs230 is connected with the plasma membrane of the parasite by its interaction partner Pfs48/45. This protein is linked to the plasma membrane by a GPI anchor and presumably retains the multi-protein complex on the surface of newly emerged macrogametes in the mosquito midgut. A WD40 domain containing protein was identified to be part of this protein complex. It might serve as platform for the assembly of the multi protein complex or mediate the interplay among proteins, as suggested from known functions of the WD40 domain repeats. During egress from the host erythrocyte, the emerging gametes become vulnerable to factors of the human complement, which is taken up with the blood meal. In this thesis it was found that the complement system is active for about one hour post feeding. Macrogametes defend against complement-mediated lysis by co-opting the human complement regulators Factor H and FHL-1 from the blood-meal. These serum proteins bind via its SCR domains 5-7 to the surface of macrogametes. Once bound, they trigger complement inactivation of the alternative pathway, which prevents induction of complement lysis on the surface of the malaria parasite. Antibodies against Factor H are able to impair the sexual development in vitro and are able to block transmission to the mosquito. Interaction studies on endogenous proteins and immobilized recombinant proteins revealed the PfGAP50 protein as binding partner of Factor H and FHL-1. This protein was hitherto described as a glideosome-associated protein in invasive parasite stages, but has not yet been characterized in gametes. First localization studies indicate a relocation of PfGAP50 from the inner membrane complex to the surface of macrogametes. Malaria still persists as one of the deadliest infectious diseases worldwide. Investigations on the essential transmissive stages, gametocytes and gametes of Plasmodium falciparum, stood in the background of research for a long time. This work deciphered details on protein interactions on the surface of the malaria parasite and provides first information about coactions between the parasite and the human complement in the mosquito midgut.}, subject = {Plasmodium falciparum}, language = {en} } @phdthesis{Menzel2011, author = {Menzel, Thomas Michael}, title = {Studien zum Wirkungsmechanismus neuer antiinfektiver Bisnaphthalimide gegen Staphylococcus aureus und Transkriptomanalysen zur Auswirkung von Antibiotika auf S. epidermidis}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-56362}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2011}, abstract = {Die Therapie von bakteriellen Infektionen beruht heutzutage zum Großteil auf dem Einsatz von Antibiotika. Die schnelle Entwicklung und rasche Verbreitung von resistenten St{\"a}mmen mancher Erreger gegen diese Antibiotika stellt ein enormes Problem f{\"u}r das Gesundheitswesen dar. Da momentan zur Antibiotikatherapie keine Alternativen bestehen, kommt der Erforschung neuer potenzieller Wirkstoffe eine sehr große Bedeutung zu. In einem Screening-Verfahren lagen die minimalen Hemmkonzentrationen einiger bisquart{\"a}rer Bisnaphthalimide gegen Staphylococcus aureus und S. epidermidis im Bereich von 0,6 bis 2,5 µg/ml. Die Substanz mit den geringsten minimalen Hemmkonzentrationen war MT02. Daraufhin wurde das Wirkungsspektrum von MT02 gegen Bakterien detaillierter untersucht und gefunden, dass die Substanz vorwiegend gegen Gram-positive Erreger und nicht gegen Gram-negative Bakterien wirksam ist. Zytotoxizit{\"a}tstests ergaben eine geringe bis nicht nachweisbare Toxizit{\"a}t gegen verschiedene Zelllinien im Bereich von 73 bis mehr als 150 µg/ml. Um die Wirkungsweise von MT02 genauer zu untersuchen wurden zun{\"a}chst DNA-Microarray-Untersuchungen an S. aureus durchgef{\"u}hrt. Deren Ergebnisse ließen einen Einfluss der Substanz auf viele Gene des DNA-Metabolismus erkennen. Inkorporationsstudien mittels radioaktiver Ganzzellmarkierung best{\"a}tigten die Auswirkung von MT02 auf den DNA-Stoffwechsel. Durch kompetitive Inkubation wurde festgestellt, dass MT02 in der Lage ist Ethidiumbromid von DNA zu verdr{\"a}ngen bzw. dessen Bindung zu verhindern. Genauere Untersuchungen mittels Oberfl{\"a}chen-Plasmon-Resonanz ergaben, dass MT02 konzentrationsabh{\"a}ngig, reversibel und sequenzunspezifisch an DNA bindet. Die thermodynamischen Dissoziationskonstanten lagen im Mittel bei ca. 4 x 10-8 mol/l und beschrieben somit eine relativ starke Bindung von MT02 an DNA. Neben diesem prim{\"a}ren Wirkungsmechanismus der DNA-Bindung gaben mehrere Befunde Hinweise auf einen sekund{\"a}ren Wirkmechanismus, der die Zellwand-Struktur bzw. Zellwand-Biosynthese beinhaltet. Eine MT02-resistente Mutante von S. aureus HG001 konnte durch vielfaches Passagieren in MT02-haltigem Medium generiert werden. Diese erzeugte bei Wachstum mit hohen Konzentrationen an MT02 einen roten Ph{\"a}notyp. Die Natur dieses roten Farbstoffes konnte bislang nicht aufgekl{\"a}rt werden, jedoch gibt es Hinweise, dass dieser auf Abbauprodukte von MT02 zur{\"u}ckzuf{\"u}hren ist. In einem weiteren Projekt wurde mittels Transkriptionsstudien die Auswirkung von verschiedenen bekannten Antibiotika sowie von neuen Wirkstoffen auf das Transkriptom von S. epidermidis untersucht. Die Ergebnisse dieser Studien k{\"o}nnen durch vergleichende Analysen als Grundlage f{\"u}r die Einordnung des Wirkmechanismus neuer Substanzen dienen.}, subject = {MRSA}, language = {de} } @article{WeibelRaabYuetal.2011, author = {Weibel, Stephanie and Raab, Viktoria and Yu, Yong A. and Worschech, Andrea and Wang, Ena and Marincola, Francesco M. and Szalay, Aladar A.}, title = {Viral-mediated oncolysis is the most critical factor in the late-phase of the tumor regression process upon vaccinia virus infection}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-68691}, year = {2011}, abstract = {Background: In principle, the elimination of malignancies by oncolytic virotherapy could proceed by different mechanisms - e.g. tumor cell specific oncolysis, destruction of the tumor vasculature or an anti-tumoral immunological response. In this study, we analyzed the contribution of these factors to elucidate the responsible mechanism for regression of human breast tumor xenografts upon colonization with an attenuated vaccinia virus (VACV). Methods: Breast tumor xenografts were analyzed 6 weeks post VACV infection (p.i.; regression phase) by immunohistochemistry and mouse-specific expression arrays. Viral-mediated oncolysis was determined by tumor growth analysis combined with microscopic studies of intratumoral virus distribution. The tumor vasculature was morphologically characterized by diameter and density measurements and vessel functionality was analyzed by lectin perfusion and extravasation studies. Immunological aspects of viral-mediated tumor regression were studied in either immune-deficient mouse strains (T-, B-, NK-cell-deficient) or upon cyclophosphamide-induced immunosuppression (MHCII+-cell depletion) in nude mice. Results: Late stage VACV-infected breast tumors showed extensive necrosis, which was highly specific to cancer cells. The tumor vasculature in infected tumor areas remained functional and the endothelial cells were not infected. However, viral colonization triggers hyperpermeability and dilatation of the tumor vessels, which resembled the activated endothelium in wounded tissue. Moreover, we demonstrated an increased expression of genes involved in leukocyte-endothelial cell interaction in VACV-infected tumors, which orchestrate perivascular inflammatory cell infiltration. The immunohistochemical analysis of infected tumors displayed intense infiltration of MHCII-positive cells and colocalization of tumor vessels with MHCII+/CD31+ vascular leukocytes. However, GI-101A tumor growth analysis upon VACV-infection in either immunosuppressed nude mice (MHCII+-cell depleted) or in immune-deficient mouse strains (T-, B-, NK-cell-deficient) revealed that neither MHCII-positive immune cells nor T-, B-, or NK cells contributed significantly to VACV-mediated tumor regression. In contrast, tumors of immunosuppressed mice showed enhanced viral spreading and tumor necrosis. Conclusions: Taken together, these results indicate that VACV-mediated oncolysis is the primary mechanism of tumor shrinkage in the late regression phase. Neither the destruction of the tumor vasculature nor the massive VACV-mediated intratumoral inflammation was a prerequisite for tumor regression. We propose that approaches to enhance viral replication and spread within the tumor microenvironment should improve therapeutical outcome.}, subject = {Virusinfektion}, language = {en} } @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{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{SimonNerzPresteretal.1985, author = {Simon, M. M. and Nerz, G. and Prester, M. and Moll, Heidrun}, title = {Immunoregulation by mouse T cell clones III. Cloned H-Y-specific cytotoxic T cells secrete a soluble mediator(s) that inhibits cytotoxic responses by acting on both Lyt-2\(^-\) and L3T4\(^-\)- lymphocytes}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-31625}, year = {1985}, abstract = {In this study we report that cloned Thy-l +, L3T4-, Lyt-l-, Lyt-2+, H-Y-specific and H-2Db-restricted cytotoxic T ce11 lines (CTLL) when indueed by lectin or antigen secrete a soluble mediator(s) (SF) that inhibits proliferation and generation of cytotoxic lymphocytes (CTL) in mixed lymphocyte cultures (MLC). The biological activity was separable by gel filtration and appeared as a broad peak in the moleeular mass range between 10000 and 50000 kDa. It was found that the suppressive activity released by CTLL neither strictly correlates with their cytotoxic potential nor with their ability to produce immune interferon or Iymphotoxin. SF was shown to elicitits activity in an antigen-nonspeeific manner in that it suppressed the maturation of T lymphocytes responding to both, the appropriate H-Y antigen as weH as to unrelated H_2d alloantigens or to the hapten 2,4,6-trinitrophenyl (TNP). The effect of SF on CTL responses was most pronounced in early phases of primary or secondary MLC. When analyzed for its inhibitory activity on precursor ceHs in populations selected for either Lyt-2- or L3T4- lymphocytes, it was found that SF interfered with the maturation of both subsets. The inhibition of CTL responses elicited by SF could not be reversed by the addition of exogenous interleukin 2. The findtng that SF also inhi. bited the proliferation of some but not a11 antigen-dependent cloned T ceHs with helper or eytc'toxic potential provides evidence that the faetor also may regulate effector lymphl)cytes. In addition, the results support the assumption that SF exerts its effect direetly on the responder rather than the stimulator population, and demonstrate that the development of CTL from their preeursor eeHs is contro11ed at least in part by the eytotoxic effeetor cells themselves via a soluble factor(s) that interferes with distinct stages of T ce11 maturation. These findings again emphasize the expression of multiple functions by CTL and indieate their possible role du ring the course of an immune response by their capability to eliminate target cells and to secrete a soluble product(s) that mediates feedback contro!.}, subject = {Infektionsbiologie}, language = {en} } @phdthesis{Agarwal2010, author = {Agarwal, Shruti}, title = {Functional characterization of four CDK-like kinases and one Calmodulin-dependent kinase of the human malaria parasite, Plasmodium falciparum}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-48522}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2010}, abstract = {Malaria still persists as one of the deadliest infectious disease in addition to AIDS and tuberculosis. lt is a leading cause of high mortality and morbidity rates in the developing world despite of groundbreaking research on global eradication of the disease initiated by WHO, about half a century ago. Lack of a commercially available vaccine and rapid spread of drug resistance have hampered the attempts of extinguishing malaria, which still leads to an annual death toll of about one million people. Resistance to anti-malarial compounds thus renders search for new target proteins imperative. The kinome of the human malaria parasite Plasmodium falciparum comprises representatives of most eukaryotic protein kinase groups, including kinases which regulate proliferation and differentiation processes. Several reports till date have suggested involvement of parasite kinases in the human host and as well as in the mosquito vector. Kinases essential for life cycle stages of the parasite represent promising targets for anti-malarial compounds thus, provoking characterization of additional malarial kinases. Despite extensive research on most plasmodial enzymes, very little information is available regarding the four identified members of the cyclin dependent kinase like kinase (CLK) family. Thus, the present thesis dealt with the functional characterization of four members of the PfCLK kinase family of the parasite denoted as PfCLK-1/Lammer, PfCLK-2, PfCLK-3 and PfCLK-4 with a special focus on the first two kinases. Additionally, one Ca2+/Calmodulin dependent putative kinase-related protein, PfPKRP, presumed to be involved in sexual stage development of the parasite, was investigated for its expression in the life cycle of the parasite. In other eukaryotes, CLK kinases regulate mRNA splicing through phosphorylation of Serine/Arginine-rich proteins. Transcription analysis revealed abundance of PfCLK kinase genes throughout the asexual blood stages and in gametocytes. By reverse genetics approach it was demonstrated that all four kinases are essential for completion of the asexual replication cycle of P. falciparum. PfCLK 1/Lammer possesses two nuclear localization signals and PfCLK-2 possesses one of these signals upstream of the C-terminal catalytic domains. Protein level expression and sub-cellular localization of the two kinases was determined by generation of antiserum directed against the kinase domains of the respective kinase. Indirect immunofluorescence, Western blot and electron microscopy data confirm that the kinases are primarily localized in the parasite nucleus, and in vitro assays show that both enzymes are associated with phosphorylation activity. Finally, mass spectrometric analysis of co immunoprecipitated proteins shows interactions of the two PfCLK kinases with proteins, which have putative nuclease, phosphatase or helicase functions. PfPKRP on the other hand is predominantly expressed during gametocyte differentiation as identified from transcriptional analysis. Antiserum directed against the catalytic domain of PfPKRP detected the protein expression profile in both asexual and gametocyte parasite lysates. Via immunofluorescence assay, the kinase was localized in the parasite cytoplasm in a punctuated manner, mostly in the gametocyte stages. Reverse genetics resulted in the generation of PfPKRP gene-disruptant parasites, thus demonstrating that unlike CLK kinases, PfPKRP is dispensable for asexual parasite survival and hence might have crucial role in sexual development of the parasite. On one hand, characterization of PfCLK kinases exemplified the kinases involved in parasite replication cycle. Successful gene-disruption and protein expression of PfPKRP kinase on the other hand, demonstrated a role of the kinase in sexual stage development of the parasite. Both kinase families therefore, represent potential candidates for anti-plasmodial compounds.}, subject = {Plasmodium falciparum}, language = {en} } @phdthesis{Hartmann2010, author = {Hartmann, Thomas}, title = {Nitrogen metabolism in Aspergillus fumigatus with emphasis on the oligopeptide transporter (OPT) gene family}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-54027}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2010}, abstract = {The saprophytic filamentous fungus Aspergillus fumigatus has been gaining importance as an opportunistic human pathogen over the past decades. Advances in modern medicine have created a growing group of patients susceptible to infection with A. fumigatus, often contracting potentially deadly invasive aspergillosis. The virulence of this pathogen appears to be a multifactorial trait, a combination of physiological characteristics that enables the fungus to infect immunocompromised humans. This work concentrates on the nitrogen metabolism of A. fumigatus, which is essential for meeting the nutritional needs inside the human host. Using DNA microarrays, the transcriptional response during growth on three different secondary nitrogen sources was examined, which revealed the metabolic versatility of A. fumigatus, especially when challenged with proteins as the sole source of nitrogen. In-depth transcriptional profiling of the eight-member oligopeptide transporter (OPT) gene family underlined the importance of oligopeptide transport for growth on complex nitrogen sources like BSA or collagen. Heterologous expression of the opt genes in Saccharomyces cerevisiae showed their functionality as oligopeptide transporters, and characterized their substrate specificity. Using a Cre/loxP based genetic tool, a complete deletion of all opt genes in A. fumigatus was achieved. The resultant strain exhibited diminished growth on medium where the oligopeptide GPGG was the sole nitrogen source, but did not show any other in vitro phenotype. The opt deletion strain was not attenuated in virulence in a murine model of pulmonary aspergillosis, suggesting that the OPT gene family is not necessary for successful infection. The connection of oligopeptide transport and extracellular proteolytic activity was investigated by deleting the genes encoding Dpp4 and Dpp5, two dipeptidyl peptidases, or PrtT, the transcriptional regulator of major secreted proteases, in the complete opt deletion background. In contrast to the deletion of dpp4 and dpp5, which did not result in any additional phenotype, the absence of prtT led to a drastic growth defect on porcine lung agar. This suggests a synergistic action of extracellular proteolytic digest of proteins and transport of oligopeptide degradation products into the cell. Finally, this work established the bacterial β-Rec/six site-specific recombination system as a novel genetic tool for targeted gene deletion in A. fumigatus.}, subject = {Aspergillus fumigatus}, language = {en} } @phdthesis{Reichardt2010, author = {Reichardt, Elisabeth}, title = {Untersuchungen zur Verbreitung von Virulenzfaktoren extraintestinal pathogener Escherichia coli-St{\"a}mme bei Isolaten boviner Mastitiden}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-53884}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2010}, abstract = {In dieser Arbeit werden die Ergebnisse der molekular-epidemiologischen Analyse von Virulenzgenen im Genom von insgesamt 222 Escherichia coli (E. coli)-Isolaten dargestellt, die von Mastitis-F{\"a}llen bei Rindern isoliert wurden. Mit Hilfe der Multiplex-Polymerase-Kettenreaktion wurde die Verbreitung von 42 potentiellen Virulenzfaktor-Genen extraintestinal pathogener E. coli (ExPEC) analysiert. Neben der quantitativen Bestimmung des Vorkommens jedes Einzelgens wurde in dieser Arbeit eine differenzierte Auswertung von Genkombinationen bei E. coli Mastitis-Isolaten vorgenommen. Diese ermittelten genetischen Muster werden zur 1. Pr{\"a}valenz der in der Gesamtheit der Isolate, 2. Pr{\"a}valenz in den phylogenetischen ECOR-Gruppen, 3. akut klinischen und chronischen Mastitis-Episoden und 4. dem Vorkommen spezifisch tierpathogener Adh{\"a}sine korreliert. Die Mastitis-Isolate konnten aufgrund der Virulenzmarkerverteilung und Phylogenie keinem bestimmten charakteristischen Pathotyp zugeordnet werden. Die {\"u}berwiegende Mehrzahl der Mastitis-Isolate zeigte aufgrund einer geringen Pr{\"a}valenz Virulenz-assoziierter Gene sowie der Zugeh{\"o}rigkeit zu den phylogenetischen Entwicklungslinien A und B1 ein geringes Virulenzpotential extraintestinal pathogener E. coli. Die Mehrzahl der St{\"a}mme enthielt eine singul{\"a}re Virulenzdeterminante (83 St{\"a}mme; 37,4 \%), eine Zweierkombination (69 St{\"a}mme; 31,1 \%) oder eine Dreierkombination von Virulenzgenen (34 St{\"a}mme; 15,3 \%). Vier Gene f{\"u}r Virulenzfaktoren in Kombination zeigten sich lediglich in sieben St{\"a}mmen (3,1 \%). Insbesondere die Anwesenheit von 5 bis 18 differenten Virulenzgenen pro Genom traten nur mit einer geringen Frequenz in zusammen 16 Isolaten (7,2 \%) auf. Das absolut h{\"a}ufigste Virulenz-assoziierte Gen, das nachgewiesen wurde, war fimH, das f{\"u}r die mannosespezifische Adh{\"a}sinuntereinheit der Typ1-Fimbrien kodiert. Insgesamt gaben 88,7 \% aller 222 untersuchten St{\"a}mme ein positives Signal in der Multiplex-PCR, und zwar 89,9 \% der 199 klinischen Isolate sowie 85,7 \% der Isolate chronischer Mastitiden. In etwa der H{\"a}lfte aller untersuchten St{\"a}mme trat auch das Gen traT auf, das Serumresistenz vermittelt (43,7 \%). Die Genkombination fimH-traT wurde in wechselnden Konstellationen in insgesamt 83 St{\"a}mmen (37,3 \%) gefunden. Sie ist damit die h{\"a}ufigste Virulenzgenkombination in den untersuchten E. coli-Genomen mit multiplen Virulenzdeterminanten. Da bei Rinder-Mastitis besonders in den schweren F{\"a}llen systemische Verl{\"a}ufe f{\"o}rdernde Faktoren wie Serumresistenz eine bedeutende Rolle spielen, k{\"o}nnte hier eine Selektion auf genetische Kopplung von traT mit fimH vorliegen. Deutlich geringere Pr{\"a}valenzen wiesen die Virulenzgene f{\"u}r α-H{\"a}molysin (hlyA, 10,8 \%), den Yersiniabactinrezeptor (fyuA, 12,2 \%) sowie das ebenfalls an der Serumresistenz beteiligte Gen iss (8,5 \%) auf. Nur 13 (5,8 \%) der 222 E. coli-Isolate besaßen keines der untersuchten Virulenzgene. Das Fehlen bekannter Virulenzgene in diesen St{\"a}mmen deutet darauf hin, dass weitere unber{\"u}cksichtigte Faktoren eine Rolle bei der Virulenz von Mastitisisolaten spielen k{\"o}nnten oder der Status des Wirtsorganismus in diesen F{\"a}llen ausschlaggebend f{\"u}r eine erfolgreiche Infektion des Euters sein k{\"o}nnte. Offensichtlich sind die meisten der untersuchten E. coli- Virulenzfaktoren f{\"u}r die Pathogenese der Rindermastitis von untergeordneter Bedeutung. Von den 222 Isolaten z{\"a}hlten insgesamt 137 St{\"a}mme zur phylogenetischen Linie (ECOR-Gruppe) A, 62 zur ECOR-Gruppe B1, 20 zur ECOR-Gruppe B2 und 14 zur Gruppe D. Die St{\"a}mme, die zu den phylogenetischen Entwicklungslinien A, B1 und D geh{\"o}ren, unterschieden sich hinsichtlich der Pr{\"a}valenz der Virulenzfaktormuster nicht vom Gesamtbild. Lediglich Isolate der ECOR-Gruppe B2 wiesen eine f{\"u}r sie typische H{\"a}ufung von Virulenzgenclustern auf. Das relativ geringe Vorkommen bzw. weitgehende Fehlen (5 von 8) von Adh{\"a}singenen spezifisch tierpathogener E. coli l{\"a}sst darauf schließen, dass bislang beschriebenen Rinder-pathogenen E. coli keine Bedeutung als Verursacher einer Rindermastitis zukommt. F{\"u}r die Analyse der chronischen Verlaufsform der Mastitis standen nur 21 Isolate zur Verf{\"u}gung, die keinen hinreichend gesicherten Vergleich zu den F{\"a}llen mit akuter klinischer Mastitis (199 St{\"a}mme insgesamt) erlauben. Die auff{\"a}llige Zunahme des H{\"a}molysingens hlyA (23,8 \%) gegen{\"u}ber 9,5 \% in den klinischen Isolaten (und 10,8 \% in allen St{\"a}mmen) m{\"u}sste in k{\"u}nftigen Untersuchungen invasiven Verhaltens der ExPEC beobachtet werden. Die Ergebnisse dieser Arbeit legen nahe, dass eine bovine Mastitis durch verschiedene E. coli-Varianten hervorgerufen werden kann und ein großes Potential extraintestinaler Virulenzfaktoren dazu nicht erforderlich ist. Entscheidend ist eine durch das fimH-Gen vermittelte Adh{\"a}sion, in der H{\"a}lfte der untersuchten F{\"a}lle unterst{\"u}tzt durch das Serumresistenz vermittelnde Gen traT.}, subject = {Escherichia coli}, language = {de} } @phdthesis{Zdziarski2008, author = {Zdziarski, Jaroslaw Maciej}, title = {Bacterial Genome Plasticity and its Role for Adaptation and Evolution of Asymptomatic Bacteriuria (ABU) Escherichia coli Strains}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-32879}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2008}, abstract = {Asymptomatic bacteriuria (ABU) represents the long term bacterial colonization of the urinary tract, frequently caused by Escherichia coli (E. coli), without typical symptoms of a urinary tract infection (UTI). To investigate characteristics of ABU E. coli isolates in more detail, the geno- and phenotypes of eleven ABU isolates have been compared. Moreover, consecutive in vivo re-isolates of the model ABU strain 83972 were characterized with regard to transcriptomic, proteomic and genomic alterations upon long term in vivo persistence in the human bladder. Finally, the effect of the human host on bacterial adaptation/evolution was assessed by comparison of in vitro and in vivo-propagated strain 83972. ABU isolates represent a heterologous group of organisms. The comparative analysis of different ABU isolates elucidated the remarkable genetic and phenotypic flexibility of E. coli isolates. These isolates could be allocated to all four major E. coli phylogenetic lineages as well as to different clonal groups. Accordingly, they differed markedly in genome content, i.e., the genome size as well as the presence of typical UPEC virulence-associated genes. Multi locus sequence typing suggested that certain ABU strains evolved from UPEC variants that are able to cause symptomatic UTI by genome reduction. Consequently, the high E. coli genome plasticity does not allow a generalized view on geno- and phenotypes of individual isolates within a clone. Reductive evolution by point mutations, DNA rearrangements and deletions resulted in inactivation of genes coding for several UPEC virulence factors, thus supporting the idea that a reduced bacterial activation of host mucosal inflammation promotes the ABU lifestyle of these E. coli isolates. Gene regulation and genetic diversity are strategies which enable bacteria to live and survive under continuously changing environmental conditions. To study adaptational changes upon long term growth in the bladder, consecutive re-isolates of model ABU strain 83972 derived from a human colonisation study and from an in vitro long term cultivation experiment were analysed with regard to transcriptional changes and genome rearrangements. In this context, it could be demonstrated that E. coli, when exposed to different host backgrounds, is able to adapt its metabolic networks resulting in an individual bacterial colonisation strategy. Transcriptome and proteome analyses demonstrated distinct metabolic strategies of nutrients acquisition and energy production of tested in vivo re-isolates of strain 83972 that enabled them to colonise their host. Utilisation of D-serine, deoxy- and ribonucleosides, pentose and glucuronate interconversions were main up-regulated pathways providing in vivo re-isolates with extra energy for efficient growth in the urinary bladder. Moreover, this study explored bacterial response networks to host defence mechanisms: The class III alcohol dehydrogenase AdhC, already proven to be involved in nitric oxide detoxification in pathogens like Haemophilus influenzae, was shown for the first time to be employed in defending E. coli against the host response during asymptomatic bacteriuria. Consecutive in vivo and in vitro re-isolates of strain 83972 were also analysed regarding their genome structure. Several changes in the genome structure of consecutive re-isolates derived from the human colonisation study implied the importance of bacterial interactions with the host during bacterial microevolution. In contrast, the genome structure of re-isolates from the in vitro long term cultivation experiment, where strain 83972 has been propagated without host contact, was not affected. This suggests that exposure to the immune response promotes genome plasticity thus being a driving force for the development of the ABU lifestyle and evolution within the urinary tract.}, subject = {Escherichia coli}, language = {en} } @article{MollFuchsBlanketal.1993, author = {Moll, Heidrun and Fuchs, Harald and Blank, Christine and R{\"o}llinghoff, Martin}, title = {Langerhans cells transport Leishmania major from the infected skin to the draining lymph node for presentation to antigen-specific T cells}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-46023}, year = {1993}, abstract = {No abstract available}, subject = {Immunologie}, 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} } @article{SolbachMollRoellinghoff1991, author = {Solbach, Werner and Moll, Heidrun and R{\"o}llinghoff, Martin}, title = {Lymphocytes play the music but the macrophage calls the tune}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-45889}, year = {1991}, abstract = {No abstract available}, subject = {Immunologie}, language = {en} } @article{MollBoesingSchneider1988, author = {Moll, Heidrun and B{\"o}sing-Schneider, Rita}, title = {The transplantation barrier of nude mice}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-46045}, year = {1988}, abstract = {Syngeneic memory cells can be stimulated to yield a secondary immune response after their transfer into irradiated euthymie recipients as well as into young thymusless nude mice. It is shown that nude mice older than twelve weeks of age are not permissive towards memory cell activation as it is found in non-irradiated euthymie animals. This barrier to isogeneie or congeneic cells seems to be caused by a pool of cyclophosphamide-sensitive cells. Since young nude mice could be rendered as unpermissive as older nude mice by pretreatment with either PNA-agglutinable thymus cells or nylon-wool passed spleen cells, it is suggested that an increased number of precursor T cells in older nude mice might induce this effect. Further experiments with monoclonal antibodies against the Lyt-l, Lyt-2, and L3T4 marker on T cells indicate that T -helper/inducer activity might be required to establish the "isogeneie barrief" in nude mice.}, language = {de} } @article{PfefferSchoelGulleetal.1991, author = {Pfeffer, K. and Schoel, H. and Gulle, H. and Moll, Heidrun and Kromer, S. and Kaufmann, S. H. E. and Wagner, H.}, title = {Analysis of primary T cell responses to intact and fractionated microbial pathogens}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-46916}, year = {1991}, abstract = {Freshly isolated human T lymphocytes were tested for their response to mycobacteria, mycobacteriallysates, 2 dimensional (2D) PAGE separated mycobacteriallysates, leishmania and defined leishmanial antigen preparations. While,o T cells proliferated vigourously in the presence of mycobacteria and mycobacteria derived lysates, a significant stimulation from 2 D gel separated lysates was not detected. In addition '10 T cells failed to respond towards leishmania or leishmanial components. In the ab T cell compartment some donors, presumably according to their state of immunity against mycobacteria, responded to mycobacteria, mycobacterial lysates and 2 D gel separated mycobacterial lysates. Neither freshly isolated '10 T cells nor ab T cells from naive donors did mount a significant immune response against leishmania.}, 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} } @article{WillBlankRoellinghoffetal.1992, author = {Will, Antje and Blank, Christine and R{\"o}llinghoff, Martin and Moll, Heidrun}, title = {Murine epidermal Langerhans cells are potent stimulators of an antigen-specific T cell response to Leishmania major, the cause of cutaneous leishmaniasis}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-45872}, year = {1992}, abstract = {Cutaneous leishmaniasis is initiated by the bite of an infected sandfly and inoculation of Leishmania major parasites into the mammalian skin. Macrophages are known to playa central role in the course of infection because they are the prime host cells and funetion as antigen-presenting eells (APC) for induetion of the eell-mediated immune response. However, in addition to maerophages in the dermis. the skin eontains epidermal Langerhans eells (LC) which ean present antigen (Ag) to T cells. Therefore, using a murine model of cutaneous leishmaniasis, we analyzed the ability of epidermal cells to induce a T eell response to L.major. The results demonstrated that freshly isolated LC, but not cuItured LC, are highly active in presenting L.major Ag in vitro to T cells from primed mice and to a L.major-specific T cell clone. Furthermore, freshly isolated LC had the ability to retain L.major Ag in immunogenic form for at least 2 days. Their efficiency was much greater than that of irradiated spleen cells, a standard population of APC. LC stimulated both T cell proliferation and production of the Iymphokines interleukin (IL)-2 and IL-4. The response was Ag specific and could be induced by lysate of L. major parasites and by live organisms. The data suggest that epidermal LC are important APC in eutaneous leishmaniasis. They may perform a critical funetion by eapturing L.major Ag in the skin and presenting it either to quiescent T eells circulating through the draining lymph node or locally to T effector cells infiltrating the cutaneous lesion.}, subject = {Immunologie}, language = {de} } @article{Moll1993, author = {Moll, Heidrun}, title = {Epidermal Langerhans cells are critical for immunoregulation of cutaneous leishmaniasis}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-61323}, year = {1993}, abstract = {In leishmaniasis, macrophages are known to play a central role as modulators of the specific immune activity. In this article, Heidrun Moll presents evidence for the critical involvement of another component of the skin immune system, the epidermal Langerhans cell. She proposes that Langerhans cells take up parasites in the skin and transport them to the draining lymph node for presentation to T cells and initiation of the specific immune response.}, subject = {Biologie}, language = {en} } @article{MollBinoederBogdanetal.1990, author = {Moll, Heidrun and Bin{\"o}der, Kerstin and Bogdan, Christian and Solbach, Werner and R{\"o}llinghoff, Martin}, title = {Production of tumour necrosis factor during murine cutaneous leishmaniasis}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-61291}, year = {1990}, abstract = {We have assessed the role of tumour necrosis factor-a (TNF) during cutaneous leishmaniasis and demonstrated that significant levels of TNF were released by spleen cells from infected mice after in cirro restimulation with Leishmania major promastigotes. Spleen cells from both genetically resistant and genetically susceptible mice were equally capable of producing TNF. After challenge with bacterial endotoxin, TNF activity could also be demonstrated in the serum of L. mujor-infected mice and the titres correlated with the course of cutaneous disease in susceptible and resistant mice. TNF did not exert a direct leishmanicidal effect in uitro. Furthermore, our study indicated that macrophages are the source of L. major-induced TNF activity and that its elicitation is dependent on the presence of T cells. These findings suggest that TNF acts in concert with other cytokines produced during L. major infection and that its role depends on the composition of T cell subsets and cytokines present.}, subject = {Immunologie}, language = {en} } @article{MollRoellinghoff1990, author = {Moll, Heidrun and R{\"o}llinghoff, Martin}, title = {Resistance to murine cutaneous leishmaniasis is mediated by T\(_H\)1 cells, but disease-promoting CD4\(^+\) cells are different from T\(_H\)2 cells}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-61305}, year = {1990}, abstract = {No abstract available}, subject = {Biologie}, language = {en} } @article{MollMuellerGillitzeretal.1991, author = {Moll, Heidrun and M{\"u}ller, Christoph and Gillitzer, Reinhard and Fuchs, Harald and R{\"o}llinghoff, Martin and Simon, Markus M. and Kramer, Michael D.}, title = {Expression of T-cell-associated serine proteinase-1 during murine Leishmania major infection correlates with susceptibility to disease}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-61311}, year = {1991}, abstract = {The expression of T-cell-associated serine proteinase 1 (MTSP-1) in vivo during Leishmania major infection was analyzed in genetically resistant C57BL/6 mice and in genetically susceptible BALB/c mice. Using a monoclonal antibody as well as an RNA probe specific for MTSP-1 to stain tissue sections, we found T cells expressing MTSP-1 in skin lesions and spleens of mice of both strains. In skin lesions, MTSP-1-positive T cells could be detected as early as 3 days after infection. Most importantly, the frequency of T cells expressing MTSP-1 was significantly higher in susceptible BALB/c mice than in resistant C57BL/6 mice. These findings suggest that MTSP-1 is associated with disease-promoting T cells and that it may be an effector molecule involved in the pathogenesis of cutaneous leishmaniasis.}, subject = {Biologie}, language = {en} } @article{HughesHackerDueveletal.1987, author = {Hughes, C. and Hacker, J{\"o}rg and D{\"u}vel, H. and Goebel, W}, title = {Chromosomal deletions and rearrangements cause coordinate loss of hemolysis, fimbriation and serum resistance in an uropathogenic strain of Escherichia coli}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59470}, year = {1987}, abstract = {No abstract available}, 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{HughesHackerRobertsetal.1983, author = {Hughes, C. and Hacker, J{\"o}rg and Roberts, A. and Goebel, W}, title = {Hemolysin production as a virulence marker in symptomatic and asymptomatic urinary tract infections caused by Escherichia coli}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59346}, year = {1983}, abstract = {Potential virulence, as defined by combined Ievels of adhesion to urinary epithelial cells, serum resistance, and mouse toxicity, was assessed for Escherichia coli strains causing symptomatic and asymptomatic urinary tract infections in relation to the carriage of hemolysin and other suspected virulence determinants. Hemolysin production (Hly), associated with certain 0 (04, 06, 018, and 075), K (5), and hemagglutination (VI and VII) antigenic types but not colicin V production (Cva), was evident in 83 and 60\% ofisolates in groups possessing high potential virulence andin only 11 and 6\% of those with low virulence. Strains of particular 0-types were not more virulent per se, but among the serotypes, specific combinations of virulence factors appeared decisive, e.g., 018 HAVI B/D/G Hly+ K5+t- and 018 HAIIIIIVBN Hly- Cva +t- Kl +t- strains were, respectively, of high and low potential virulence. Isolates with high potential virulence were found to a similar extent in symptomatic and asymptomatic infections.}, subject = {Infektionsbiologie}, 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{SchefferKoenigHackeretal.1985, author = {Scheffer, J. and K{\"o}nig, W. and Hacker, J{\"o}rg and Goebel, W.}, title = {Bacterial adherence and hemolysin production from Escherichia coli induces histamine and leukotriene release from various cells}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59361}, year = {1985}, abstract = {We investigated the role of bacterial adherence and hemolysin production from Escherichia coli parent and genetically cloned strains as to their eft'ects on bistaJidne release from rat mast cells and leukotriene generation from human polymorphonuclear granulocytes. These mediators were involved in the induction of inftammatory disease processes and led, for example, to enhancement of vascular permeability, chemotaxis (leukotriene 84 [LTB4]), chemoaggregation, lysosomal enzyme release, and smooth muscle contraction, (LTC4, LTD4 , and LTE4). Washed bacteria (E. coli K-12 Ms+ my=; E. coli 536 Ms+ MR= my=) as weil as their culture supematants were analyzed. Washed E. coli K-12 (Hiy+), unlike Hly- strains, induced high amounts of histamine release from rat mast cells and chemotactic activity from human polymorphonuclear granulocytes. Significant leukotriene releasewas obtained with washed E. coli K-12 my+ strains and their bacterial culture supematants. Leukotriene induction was dependent on the amount of hemolysin activity present in the supematant. However, additional soluble factors should also be considered. The presence of hemolysin appeared to aceeierate and enhance the rate of phagocytosis of bacteria by neutrophUs. When E. coli 536 (MS+ MR= Hly=) strains were analyzed, the simultaneous presence of MR+ pili and hemolysin production led to an increase in histamine release as compared with MR- my+ strains. The genetically cloned MR+ my+ E. coli 536 strain induced higher amounts of IeukotrieDes as compared with the wUd-type strain. Our data soggest a potent role for adhesins and hemolysin as virulence factors in inducing the release of inftammatory mediators.}, subject = {Infektionsbiologie}, language = {en} } @article{MarreHacker1987, author = {Marre, R. and Hacker, J{\"o}rg}, title = {Role of S and common type I-fimbriae of Escherichia coli in experimental upper and lower urinary tract infection}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59468}, year = {1987}, abstract = {No abstract available}, subject = {Infektionsbiologie}, language = {en} } @article{SchmollHackerGoebel1987, author = {Schmoll, T. and Hacker, J{\"o}rg and Goebel, W.}, title = {Nucleotide sequence of the sfaA gene coding for the S fimbrial protein subunit of Escherichia coli}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59480}, year = {1987}, abstract = {The sfaA gene of the uropathogenic Escherichia coli 06 strain 536, which is responsible for the determination of the S fimbrial protein subunit, was sequenced. The structural gene codes for a polypeptide of 180 amino acids including a 24-residue N-terminal signal sequence. A size of 15.95 kDa was calculated for the processed SfaA protein. The nucleotide and deduced amino acid sequences show significant homology to those of the F1C fimbria and, to a lesser extent, of the mannose- sensitive hemagglutinating fimbria (FimA, PilA). Only week homology toP fimbriae subunits (F72 , Pap) was found.}, subject = {Infektionsbiologie}, language = {en} } @article{OttSchmollGoebeletal.1987, author = {Ott, M. and Schmoll, T. and Goebel, W. and Van Die, I. and Hacker, J{\"o}rg}, title = {Comparison of the genetic determinant coding for the S-fimbrial adhesin (sfa) of Escherichia coli to other chromosomally encoded fimbrial determinants}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59499}, year = {1987}, abstract = {DNA probes specific for different regions of the S-fimbrial adhesin (sja) determinant were constructed and hybridized with DNA sequences coding for P (F8 and F13), mannose-sensitive hemagglutinating type 1 (FlA), and FlC fimbriae. While the sfa and F1C DNA determinants exhibited homology along their entire lengths, the P-fimbrial and type 1-fimbrial determinants exhibited homology to regions of the sfa duster responsible for the control of transcription and, to a minor extent, to regions coding for proteins involved in biogenesis and/or adhesion of the fimbriae and for the N-terminal part of the fimbrillin subunit.}, subject = {Infektionsbiologie}, language = {en} } @article{ParkkinenKorhonenPereetal.1988, author = {Parkkinen, J. and Korhonen, T. K. and Pere, A. and Hacker, J{\"o}rg and Soinila, S.}, title = {Binding sites in the rat brain for Escherichia coli S fimbriae associated with neontal meningitis}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59500}, year = {1988}, abstract = {Escherichia coli strains that cause sepsis and meningitis in neonatal infants carry S fimbriae that bind to sialyl galactoside units of cell surface glycoproteins. To investigate the possible role of S fimbriae in determining the tissue tropism of neonatal menlngitis, we have studied the preselice of binding sites for S fimbriae in different tissues of the neonatal rat which is susceptible to meningitis caused by S-fimbriated E. coli. Purified S fimbriae were incubated on cryostat sections of different rat oipns and their bindina was assessed by indirect immunofluorescence. In the bnin of the neonatal rat, S fimbriae specifically bound to the luminal surfaces of the vascular endothelium and of the epithelium lining the choroid plexuses and bnin ventricles. The · bindlog W.s completely inhibited by the trisaccharide NeuAca2-3Ga)ßl-4Gic, a receptor analogue of S fimbriae, and by a preceding neuraminidase treatment of the sections. A recombinant E. coli strain expressina S fimbriae adhered in large numbers to the same tissue sites in the neonatal brain sections as did the purified fimbriae, · whereas the nonfimbriated host strahi and a recombiiuuit strain expresslog P fi.mbriae did not adhere to brain tissues. The results soggest that adhesion of S-fimbriated bacteria to the binding sites observed in the neonatai bnin has a pathogenetic roJe durlog bacterial Invasion from cii'culation into the cerebrospinal fluid.}, subject = {Infektionsbiologie}, language = {en} } @article{OttHoschuetzkyJannetal.1988, author = {Ott, M. and Hosch{\"u}tzky, H. and Jann, K. and Van Die, I. and Hacker, J{\"o}rg}, title = {Gene clusters for S fimbrial adhesin (sfa) and F1C Fimbriae (foc) of Escherichia coli: Comparative aspects of structure and function}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59519}, year = {1988}, abstract = {Fimbrial 8dhesins en8ble b8cteria to 8ttach t9 eucaryotic ceU~. The genetic determin8nts for S fimbrial 8dhesins (sja) an.d for FlC ("pseudotype I") fimbri8e ifoc) were compared. Sfa and FlC represent functionally distinct 8dbesins in tbeir receptor specificities. Nevertheless, 8 high degree of bomology between both determin8nts was found on the basis of DNA-DNA hybridizations. Characteristic difl'erences in the restriCtion maps of tbe corresponding gene clusters, bowever, were visible in regions coding for the fimbrial subunits and for the S-specific 8dhesin. While a plasmid carrying the geneiic deternlinant for FlC fimbri8e was 8ble to complement transposon-induced sfa mutants, 8 plasmid carrying tbe genetic determin8nt for 8 tbird 8dht\$in type, termed P fimbriae, was un8ble to do so. Proximal sfa-specific sequences carrying the S fimbrial st'"uctural gene were fused to sequences representing tbe di\$tal part of the foc gene cluster to form 8 hybrid cluster, and tbe foc proxim~ region coding for tbe structural protein was Iigated to sfa distal sequences to form 8 second hybrid. Botb hybrid clones produced intact fimbriae. Anti-FlC monoclonal8ntibodies (MAbs) only recognized clones which produced FlC fimbriae, and an ~ti-S 8dhesin MAb marked clones whicb expressed the S adhesin. Bowever, one of four other anti-S fimbri8e-specific MAbs reacted witb both fimbrial structures, S and FlC, indicating 8 common epitope on both antigens. The results presented bere ~upport tbe view th8t sfa and foc determinants code for fimbri8e tb8t 8re simil8r in several aspects, wbile the P fimbri8e are members of 8 more distantly rel8ted group.}, subject = {Infektionsbiologie}, language = {en} } @article{PawelzikHeesemannHackeretal.1988, author = {Pawelzik, M. and Heesemann, J. and Hacker, J{\"o}rg and Opferkuch, W.}, title = {Cloning and characterization of a new type of fimbria (S/F1C-related fimbria) expressed by an Escherichia coli O75:K1:H7 blood culture isolate}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59529}, year = {1988}, abstract = {The Escherichia coli blood culture isolate BK658 (07S:K1:H7) expresses F1A and F1B fimbriae as weil as a third fimbrial type which reacts with anti-S-fimbrial antiserum but fails to show S-specific binding properlies (i.e., agglutination of bovine erythrocytes). To characterize these fimbriae, we cloned the respective genetic determinant in E. coli K-12. The resulting recombinant clone HB101(pMMP658-6) expresses fimbriae of 1.2-p.m length and a diameter of approximately 7 nm. The determinant codes for the fimbrillin subunit, a protein of 17 kUodaltons in size, and for at least five other proteins of 87, 31, 23, 14.3, and 13.8 kUodaltons. By restriction analysis and by DNA-DNA hybridization, it could be shown that the cloned fimbrial determinant of strain BK658 exhibits a high degree of sequence homology to the gene clusters coding for S fimbrial adhesins (sfa) and F1C fimbriae (/oc). By using the Western blot (immunoblot) technique and a quantitative enzyme-linked immunosorbent assay, it could be further demonstrated that the cloned fimbriae of BK658, S fimbriae, and FlC fimbriae share cross-reactive epitopes as weil as antigenic determinants specific for each fimbrial type. No antigenic cross-reactivity with F1C fimbriae could be detected. The results indicate a genetical and serological relatedness of the cloned fimbriae toS fimbriae and F1C fimbriae. Therefore, this new type of fimbriae is preliminarily termed SIF1C-related fimbriae (Sfr).}, subject = {Infektionsbiologie}, 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{ZinglerBlumFalkenhagenetal.1993, author = {Zingler, G. and Blum, G. and Falkenhagen, U. and Orskov, I. and Orskov, F. and Hacker, J{\"o}rg and Ott, M}, title = {Clonal differentiation of uropathogenic E. coli isolates of serotype O6:K5 by fimbrial antigen typing and DNA long-range mapping techniques}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59865}, year = {1993}, abstract = {Escherichia coli isolates of serotype 06: K5 are the most common causative agents of cystitis and pyelonephritis in adults. To answer the question, as to whether strains of this particular serotype represent one special clonal group, out of a collection of 34 serotype 06: K5 isolates [Zingler et al. ( 1990) Zentralbl. Bakteriol Mikrobiol Hyg [A] 274:372-381] 15 strains were selected andanalyzed in detail. The flagellar (H) antigen and the outer membrane protein (OMP) pattern were determined. Furtherserum resistance properties and the genetic presence and expression of other virulence factors, including hemolysin, aerobactin, P fimbriae, S/F1C fimbriae and type 1 fimbriae was evaluated. In~laddition the Xbalmacrorestriction pattern of ten representative isolates was elaborated and the fimbrial (F) antigentype ofthe P fimbriae was determined, to obtain the complete 0: K: H: F pattern. These analyses could clearly show that the 06: K5 isolates do not represent one clonal group. The Xbal-macrorestriction profiles were heterogeneaus and marked differences in the hybridization patterns, using virulenceassociated gene probes in Southern hybridization of long-range-separated genomic DNA, were observed among the strains. However, some of strains showed similarities in the genomic profiles, arguing for clonal groupings among the 06: K5 isolates. lnterstingly the strains grouped tagether exhibited the same fimbrial F typethat many indicate a coincidence of this phenotypic trait with clonality.}, 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{LinhardtZiebuhrMeyeretal.1992, author = {Linhardt, F. and Ziebuhr, W. and Meyer, P. and Witte, W. and Hacker, J{\"o}rg}, title = {Pulsed-field gel electrophoresis of genomic restriction fragments as a tool for the epidemiological analysis of Staphylococcus aureus and coagulase-negative Staphylococci}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59811}, year = {1992}, abstract = {Thirtccn StttJ1hylococcus dw·eus and s: <'pid<'l'· midis strains ohtaincd from nnsc and hand nf twn cmployccs and onc paticnt uf a mcdical ward as weil as two S. hemol.\"licus strains wcrc analyscd according to thcir rcstrktion fmgmcnt lcngth pattcrns ( RFLP) hy pulscd-ficld gcl clcctrophorcsis (PFGE) using thc rcslriction cnzymcs SmaJ and s.. .· tll. Spccics idcntification nf thc isolatcs was pcrformcd hy a systcm which includcs :!O hiochcmical rc"ctions. Furthcrmorc. thc antillintic resistancc pattcrns of thc stmins wcrc dctcrmincd. Whilc scvcral isolatcs cxhihitcd idcnticaf antihiotic susccptihilitics and hiochcmical prnfilcs. diffcrences in thc RFLP wcrc ohtaincd. ln thrcc cascs, S. epiderm{\"u}lis strains colonizing thc skin showcd an idcntical rcstriction profilc as isollltcs from thc mucous mcmhrancs of thc samc pcrson. Wc C(mcludcd that thc analysis of staphylococcal strains hy PFGE is an important cpidcmiolngical tnnl with high discrimination power.}, subject = {Infektionsbiologie}, language = {en} } @article{OttBenderLuecketal.1992, author = {Ott, M. and Bender, L. and L{\"u}ck, P. C. and Meyer, P. and Hacker, J{\"o}rg}, title = {Distribution of Legionellae in a hospital water system: prevalence of immunologically and genetically related Legionella pneumophila serogroup 6 isolates}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59827}, year = {1992}, abstract = {A hospital warm water system was monitored for the prcsence and distribution of lcgionellac. Subtyping of ten scletled Legionella pneumophiltl isolates. originating from four different sites in the system by using serogroup spccific antisera in an indircct immunofluorcscence tcst, rcvcalcd that nine of the tcn isolatcs belonged to scrogroup 6, while the remaining one was serogroup I 0. Two monoclonal antibodics (mAbs) spccific for a subgroup of serogroup 6 strains were further used for characterization. None of the strains reactcd with these mAbs. Genome analysis by elaborating Not I profiles using the pulscd field gel electrophoresis (PFGE) technique revealed that nearly all serogroup 6 isolates dcrived from different sites, including a new building connected hy a ring pipe. wcrc identical according to restriction fragment pattems. The patterns were distinguishable from those of the two L. pnewnophi/a serogroup 6 rcfcrencc strains, and ftom that of thc L. pneumophila scrogroup 10 isolate. These data arguc for a relatively homogeneaus L. pneunwpltila serogroup 6 population in the entire watcr system.}, subject = {Infektionsbiologie}, language = {en} } @article{SchrotenSteinigPlogmannetal.1992, author = {Schroten, H. and Steinig, M. and Plogmann, R. and Hanisch, F. G. and Hacker, J{\"o}rg and Herzig, P. and Wahn, V}, title = {S-fimbriae mediated adhesin of Escherichia coli to human buccal epithelial cells is age independent}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59830}, year = {1992}, abstract = {S-fimbriated Escherichia coli, which cause sepsis and meningitis in the newbom, bind to sialic acid-containing glycoprotein structures on the surface of human buccal epithelial cells. The dependence of · this binding on host age was examined. S-fimbriated · E. coli adhered in comparable numbers to cells in newborns, infants, children and adults (23.0 ± 8.6; 23.1 ± 11.5; 24.7 ± 7.9; 28.9 ± 8.8). Thus, the increased susceptibility of neonates to infections caused by S-fimbriated E. coli cannot be explained by enhanced · adhesion to epithelial cells}, subject = {Infektionsbiologie}, language = {en} } @article{MorschhaeuserUhlinHacker1993, author = {Morschh{\"a}user, J. and Uhlin, B. E. and Hacker, J{\"o}rg}, title = {Transcriptional analysis and regulation of the sfa determinant coding for S fimbria of pathogenic E. coli strains}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59844}, year = {1993}, abstract = {The sfa determinant codes for S fimbrial adhesins which constitute adherence factors of pathogenic Escherichia coli strains. Wehave recently shown that the sfa determinant is transcribed from three pr{\"o}moters, pA, pB, and pC. In comparison with the promoters pB and pC, promoter pA, which is located in front of the structural gene sfaA, showed very weak activity. Herewe have determined the exact positions ofthe mRNA start points by primer extension studies. We have also shown that mRNAs of 500, 700 and 1400 bases can be detected using oligonucleotide probes specific for the genes sfaB, sfaC and sfaA. SfaB and SfaC arepositive regulators infiuencing fimbriation and the production of the S-specific adhesin which is encoded by the gene sfaS Iocated in the distal half of the determinant. In addition, it is demonstrated that SfaB and SfaC interfere with the regulatory effect of the histone-like protein H-NS, encoded by a locus termed drdX or osmZ. In a drdx+ strain the regulators are necessary for transcription of the sfa determinant. In contrast, sfa expression is activator-independent in a drdx- strain. In this latter genetic background, a substantial fraction of the sfa transcripts is initiated from promoter pA. On the basis of these data we discuss a model for the regulation of this adhesin-specific determinant.}, subject = {Infektionsbiologie}, language = {en} } @article{OttBenderBlumetal.1991, author = {Ott, M. and Bender, L. and Blum, G. and Schmittroth, M. and Achtmann, M. and Tsch{\"a}pe, H. and Hacker, J{\"o}rg}, title = {Virulence patterns and long range mapping of extraintestinal Escherichia coli K1, K5 and K100 isolates: Use of pulse field gel electrophoresis}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59738}, year = {1991}, abstract = {A total of 127 extraintestinal Escherichia coli strains of the capsule serotypes Kl, KS, and KlOO from human and animal sources were analyzed for DNA sequences specific for the genes for various adhesins (P fimbriae fpap] and P-related sequences fprs], S fimbriae [s/a)/FlC fimbriae [foc], and type I fimbriae lfim]), aerobactin (aer), and hemolysin (hly). The expression of corresponding virulence factors was also tested. Twenty-four selected strains were analyzed by long-range DNA mapping to evaluate their genetic relationships. DNA sequences for the adhesins were often found in strains not expressing them, while strains with hemolysin and aerobactin genes usually did express them. Different isolates of the same serotype orten expressed different virulence patterns. The use of virulence-associated gene probes for Southern hybridization with genomic DNA fragments separated by pulsed-field gel electrophoresis revealed that a highly heterogeneous restriction fragment length and hybridization pattern existed even within strains of the same serotype. Long-range DNA mapping is therefore useful for the evaluation of genetic relatedness among individual isolates and facilitates the performance of .precise molecular epidemiology.}, subject = {Infektionsbiologie}, language = {en} } @article{BenderOttDebesetal.1991, author = {Bender, L. and Ott, M. and Debes, A. and Rdest, U. and Heesemann, J. and Hacker, J{\"o}rg}, title = {Distribution, expression and long range mapping of legiolysin gene (lly) specific DNA sequences in Legionellae}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59744}, year = {1991}, abstract = {The legiolysin gene (lly) cloned from Legionella pneumophila Philadelphia 1 confers the phenotypes of hemolysis and browning of the culture medium. An internal Uy-specific DNA probe was used in Southern hybridizations for the detection of Uy-specific DNA in the genomes of legioneUae and other gram-negative pathogenic bacteria. Under conditi9ns of high stringency, tlie Uy DNA probe specifically reacted with DNA fragments fr9m L. pneumophi{\"u}z isolates; by reducing stringency, hybridization was also observed for all other Legionella strains tested. No hybridization occurred with DNAs isolated from bact~ria of other genera. The Uy genewas mapped by pulsed-field gel electrophoresis to the respective genomic Notl fragments of Legionelltz isolates. By using antilegiolysin monospecific polyclonal antibodies in Western blots (immunoblots), Lly proteins could be detected only in L. pneumophila isolates.}, subject = {Infektionsbiologie}, language = {en} } @article{OttMessnerHeesemannetal.1991, author = {Ott, M. and Messner, P. and Heesemann, J. and Marre, R. and Hacker, J{\"o}rg}, title = {Temperature dependent expression of flagella in Legionella}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59755}, year = {1991}, abstract = {Legionel/a pneumophila, the causative agent of Legionnaires' disease, was analysed by electron microscopy for production of surface structures. Crystalline surface (S-) layers and fimbriae were not detected, but monotrichous flagellation was seen. Polyclonal antibodies specific for the 47 kDa ftagellin subunit of L. pneumophila Philadelphia I were used in Western blots to confirm the presence of flagella subunits in various L. pneumophila strains tested, but the antiserumalso reacted with flagellin subunits of L. micdlulei, L. hackelia (serogroup (SG) l and SG21 and L./ongbetichae (SG2). Flagellation of Legionellae was shown to be temperature regulated. When the growth temperature of virulent and avirulent variants of strain L. pneumophila Philadelphia I was shifted from 30 oc to either 37 or 41 oc, a decrease in the percentage offtagellated bacteria within the populationwas observed.}, subject = {Infektionsbiologie}, language = {en} } @article{OttBenderChirinosetal.1991, author = {Ott, M. and Bender, L. and Chirinos, E. and Ehret, W. and Hacker, J{\"o}rg}, title = {Phenotype versus genotype of the 19 kd peptido-glycan associated protein of Legionella (PplA) among Legionellae and other gram-negative bacteria}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59768}, year = {1991}, abstract = {The protein PpiA (19 kD) cloned from a genomic library of Legionella pneumophila, Philadelphia 1, represents a peptido-glycan associated outer membrane protein in recombinant E. coli K-12 and L. pneumophila. lt exhibits distinct sequence homology to Iipoproteins of Haemophilus influenzae and E. coli. A ppiA specific DNA probe generated by PCR was used in Southern hybridizations of chromosomal DNA of Legionella strains and other Gram-negative pathogens. Under conditions of high stringency, hybridization could only be observed in L. pneumophila isolates, but alt other Legionella strains tested displayed hybridization under lower stringency. No signals appeared after hybridization of chromosomal DNA from a variety of other bacteria. Using anti-PpiA monospecific polyclonal antibodies in Western blots, it was demonstrated that PpiA related proteins of nearly the same size are found in all L. pneumophila isolates and in a variety of, but not alt, the Legionella species analysed here.}, subject = {Infektionsbiologie}, language = {en} } @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{SchrotenHanischPlogmannetal.1992, author = {Schroten, H. and Hanisch, F. G. and Plogmann, R. and Hacker, J{\"o}rg and Uhlenbruck, G. and Wahn, V.}, title = {Inhibition of Adhesion of S-fimbriated Escherichia coli to buccal epithelial cells by human milk fat globule membrane components: a novel aspect of protective function of mucins in the non-immunoglobulin fraction}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59793}, year = {1992}, abstract = {We investigated the presence of factors in human milkthat inhibit Invasion of pathogenic bacteria. The efl'ect of human milk fat globule membrane (HMFGM) components on adhesion of cloned S-fimbriated Escherichia coli to human buccal epithelial cells was analyzed. S fimbriae are a common feature of E. coli strains causing sepsis and meningitis in newborns and are bound to epithelia via sialyl-(a-2-3)galactoside structures. Human milk fat globules (HMFG) could be agglutinated by the above-mentioned bacteria. Agglutination could be inhibited by fetuin, human glycophorin, and a 1-acid glycoprotein. In addition, pretreatment of HMFG with Jlibrio cholerae neuraminidase markedly reduced bacterium-induced agglutinations, indicating the involvement of neuraminic acid-containing glycoproteins. In contrast, Iipid droplets of infant formula or artificiallipid emulsions (Intralipid) could not be agglutinated. HMFG were present in stools of breast-fed neonates as shown by indirect immunofluorescence staining with a monoclonal antibody directed against carbohydrate residues present on HMFGM. These HMFG could be agglutinated by bacteria. HMFG inhibited E. coli adhesion to buccal epithelial cells. To further characterize relevant E. coli binding structures, HMFGM components w~re separated by gel chromatography. The mucin fraction showed the most pronounced inhibitory efrect on adhesion of S-fimbriated E. coli to human buccal epithelial cells. Our data soggest that HMFG inhibit bacterial adhesion in the entire intestine and thereby may provide protection against bacterial infection.}, subject = {Infektionsbiologie}, language = {en} } @article{ZinglerOttBlumetal.1992, author = {Zingler, G. and Ott, M. and Blum, G. and Falkenhagen, U. and Naumann, G. and Sokolowska-K{\"o}hler, W. and Hacker, J{\"o}rg}, title = {Clonal analysis of Escherichia coli serotype O6 strains from urinary tract infections}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59786}, year = {1992}, abstract = {A total of 36 Escherichia coli urinary tract isolates (UTI) of serotype 06, with different combinations of capsule ( K) and flagellin ( H) antigens, were analysed according to the outer membrane pattern (OMP), serum resistance properties, mannose-resistant hemagglutination using various types of erythrocytes, and also for the genetic presence and the expression of Pfimbriae. S fimbriae/F1 C fimbriae, Type 1 fimbriae, aerobactin and hemolysin. Twenty selected strains were further analysed by pulsed field gel electrophoresis (PFGE), elaborating genomic profilas by Xba I cleavage and subsequent Southern hybridization to virulence-associated DNA probes. lt could be shown that 06 UTI isolates represent a highly heterogeneaus group of strains according to the occurrence and combination of these traits. Relatedness an the genetic and the phenotypic Ievei was found for some of the strains exhibiting the same 0: K: H: F serotype. DNA Iang-range mapping further indicated some interesting features, according to the copy number and the genomic linkage of virulence genes.}, subject = {Infektionsbiologie}, language = {en} } @article{SchrotenLethenHanischetal.1992, author = {Schroten, H. and Lethen, A. and Hanisch, F., G. and Plogmann, R. and Hacker, J{\"o}rg and Nobis-Bosch, R. and Wahn, V.}, title = {Inhibition of adhesion of S-fimbriated Escherichia coli to epithelial cells by meconium feces of breast fed and formula fed newborns - mucins are the major inhibitor component}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59804}, year = {1992}, abstract = {We investigated the ability of meconium, feces from human milk-fed (HMF) newborns, and feces from formula-fed (FF) newborns to inhibit adhesion of S-fimbriated E. coli to human buccal epithelial cells. S-fimbriae are a common property of E.·coli strains causing sepsis and meningitis in neonates. Meconium had the highest content of neuraminic acid and the strongest inhibitory effect on bacterial adhesion. HMF also exerted high inhibitory activity while FF was markedly less active: To achieve inhibitory effects comparable to HMF a sixfold amount of FF was required. Glycoproteins from excretions were separated by gel chromatography. Fractions obtained were analyzed for adhesion-inhibiting activity. In all excretions analyzed, the mucin-containing fraction could be identified as the major inhibitory component. Inhibition was probably mediated by specific interaction of this fraction with S-fimbriae, as shown by binding of isolated fimbriae on Western blots after electrophoretic separation of glycoproteins. In conclusion, our data support the view that the mucin-containing fraction from meconium and human milk exerts antibacterial functions by preventing adhesin-mediated binding of pathogenic bacteria to mucosal epithelia. Key Words: S-fimbriated E. coli-Inhibition of adhesion-Meconium- Feces of human milk-fed newborns-Feces of formula-fed newborns-Mucins.}, subject = {Infektionsbiologie}, language = {en} } @article{MunoaHackerJuarez1988, author = {Munoa, F. and Hacker, J{\"o}rg and Juarez, A.}, title = {Characterization of a chromosomal mutant that blocks hemolysin excretion in Escherichia coli}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59534}, year = {1988}, abstract = {We analyzed an Escherichia coli strain which harbours a chromosomal mutation that blocks the hemolysin excretion. Compartmentation studies showed that hemolysin accumulates in the cytoplasm and not in the periplasm. The mutation did not affect the SDS-PAGE protein pattern of the outer membrane, although some alterations were apparent in the periplasmic protein pattern. The mutant strain, E. coli Hsb-1 also failed to export a cloned fimbrial adhesin. The mutation maps in the min. 3.5 of the E. coli genetic map.}, subject = {Infektionsbiologie}, language = {en} } @article{KrallmannWenzelOttHackeretal.1989, author = {Krallmann-Wenzel, U. and Ott, M. and Hacker, J{\"o}rg and Schmidt, G}, title = {Chromosomal mapping of genes encoding mannose-sensitive (type I) and mannose-resistant F8(P) fimbriae of Escherichia coli O18:K5:H5}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59545}, year = {1989}, abstract = {DNA hybridization experiments demonstrated that the gene clusters encoding the F8 fimbriae (fei) as well as the type I fimbriae (pi/) exist in a single copy on the chromosome of E. coli 018:K5 strain 2980. In conjugation experiments with appropriate donors, the chromosomal site of these gene clusters was determined. The pil genes were mapped close to the gene clusters thr and Jeu controlling the biosynthesis of threonine and leucine, respectively. The fei genes were found to be located close to the galactose operon (gal) between the position 17 and 21 of the E. coli chromosomallinkage map.}, subject = {Infektionsbiologie}, language = {en} } @article{MarreHackerWoodetal.1989, author = {Marre, R. and Hacker, J{\"o}rg and Wood, G. and Schmidt, G.}, title = {Oral vaccination of rats with live avirulent Salmonella derivatives expressing adhesive fimbrial agents of uropathogenic Escherichia coli}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59559}, year = {1989}, abstract = {The avirulent Salmonella typhimurium F885 was transformed with a plasmid carrying the cloned S fimbriae genes of a uropathogenic Escherichia coli. The resulting transformant (F885-1) produced efficiently E. coli S fimbriae and was used for live oral vaccination of rats. For comparison rats were immunized subcutaneously with isolated S fimbriae. Both routes of vaccination resulted in a significant lgG antibody response to S fimbriae. In addition live oral vaccination induced a serum lgA response against S fimbriae. After transurethral infection of rats with a S fimbriae producing E. coli a 10-fold reduction of bacterial counts in the kidney was observed in rats orally vaccinated with F885-1 as compared to unvaccinated controls. This study suggests that the avirulent Salmonella F885 may be used as a fimbrial antigen carrier for oral vaccination against renal infections.}, subject = {Infektionsbiologie}, language = {en} } @article{KoenigKoenigSchefferetal.1989, author = {K{\"o}nig, W. and K{\"o}nig, B. and Scheffer, J. and Hacker, J{\"o}rg and Goebel, W.}, title = {Role of cloned virulence factors (mannose-resistant hemagglutination, mannose-resistant adhesins) from uropathogenic Escherichia coli strains in release of inflammatory mediators from neutrophils and mast cells}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59564}, year = {1989}, abstract = {Genetically cloned E. co/i strains expressing cloned virulence factors were studied with regard to their capability to induce inflammatory mediator release from various target cells. Among the strains were E. co/i strains with mannose-resistant haemagglutination (MRH +) and mannose-resistant adhesins, e.g. E. coli 536/21 pANN 80 I /4, E. coli 536/21 pANN 921 and E. coli 536/21 pANN 801-1. In comparison, E. coli 536/21, E. coli 536/21 pGB 30 int and E. coli Kl2, without and with mannosesensitive haemagglutination (MSH±), and adhesins were studied. The properties of the various strains for human PMN with regard to adherence and phagocytosis, chemiluminescence, 5-lipoxygenase activation of arachidonic acid, leukotriene formation, granular enzyme release and release of histamine from rat mast cells were analysed. It is evident that the various 'biochemical processes of cell activation are dissociated events. The highest chemiluminescence response is obtained with strains expressing MSH+, P-M RH+ or S-M RH+; the presence of S-adhesins suppressed the response. Highest leukotriene formation is obtained with E. coli 536/21 pANN 801-4, while E. coli with MSH was inactive. The concomitant presence of haemolysin secretion enhanced mediator release significantly. Our data suggest a potent role for mannose-resistant haemagglutination (MRH), adhesins and haemolysin as virulence factors in inducing the release of inflammatory mediators.}, subject = {Infektionsbiologie}, language = {en} } @article{OttBenderMarreetal.1991, author = {Ott, M. and Bender, L. and Marre, R. and Hacker, J{\"o}rg}, title = {Pulsed field electrophoresis of genomic restriction fragments for the detection of nosocomial Legionella pneumophila in hospital water supplies}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59672}, year = {1991}, abstract = {Ten Legionella pneumophUa strains isolated from dift'erent sources were analyzed according to their restriction fragment patterils obtained by cle~vage of gen.omic DNA With Notl and Sftl and separation by pulsed field electrophoresis. Three L. pneumophila isolate~ from a nosocomial outbreak in L{\"u}~k (Germany) and three other L. prreumophilll stralns independently isolated from a water tap located in the care unit where tbe patients were bospitalized 'xhibited identical restricti9n fragment profiles. Therefore, we concluded that these environment81 spee~ens were the source of the Legionnatres dlsease. Anotber two isolates from patients and two strains from the environment, all unrelated to the outJlreak described, sbowed different cleavage patterns.}, 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{OttHacker1991, author = {Ott, M. and Hacker, J{\"o}rg}, title = {Analysis of the variability of S fimbriae expression in an Escherichia coli pathogen.}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59695}, year = {1991}, abstract = {The uropathogenic Escherichia coli wiJd..:type strain 536 produces S-fimbriae, P-related fimbriae and type I fimbriae. Using immuno-colony dot and ELISA techniques, variants were detected showing an increased degree of S-fimbrial production. It was demonstrated by itrtmunofluorescence microscopy that in noimal (wild-type) and hyperS- fimbriated E. coli populaiions non-fimbriated cells also · exist, and that the percentage of Sfinibrlated and non-fimbriated bacteria was roughly identica1 in either population. Hyper-Sfimbriated variants could be stably maintained. The transition from wild-type to hyper-S-fimbriation, which occurs spontaneously, is markedly higher than vice versa. Southern blot analysis of the S fimbrial adhesin (sfa) determinants of normal and hyper-fimbriated strains revealed no marked difference in the gene structure.}, subject = {Infektionsbiologie}, language = {en} } @article{WintermeyerRdestLudwigetal.1991, author = {Wintermeyer, E. and Rdest, U. and Ludwig, B. and Debes, A. and Hacker, J{\"o}rg}, title = {Characterization of legiolysin (lly); responsible for hemolytic activity, colour production and fluorescence of Legionella pneumophila}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59706}, year = {1991}, abstract = {No abstract available}, subject = {Infektionsbiologie}, 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{LudwigSchmidMarreetal.1991, author = {Ludwig, B. and Schmid, A. and Marre, R. and Hacker, J{\"o}rg}, title = {Cloning, genetic analysis and nucleotide sequence of a determinant coding for a 19 kd peptidoglycan-associated protein (Ppl) of Legionella pneumophila}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59721}, year = {1991}, abstract = {A genomic library of Legionello pneumophihz, the causative agent of Legionnaires disease in humans, was constructed in Escherichill coli K-12, and the recombinant clones were screened by immuno-colony blots with im antiserum raised against heat-killed L. pneumophilo. Twenty-three clones coding for a LegioneUa-specific protein of 19 kDa were isolated. The 19-kDa protein, which represents an outer membrane protein, was found tobe associated with the peptidoglycan layer bothin L. pneumophilo andin the recombinant E. coli clones. This was shown by electrophoresis and Western immunoblot analysis of bacterial cell membrane fractions witb a monospecific polyclonal 19-kDa protein-specific antiserum. Tbe protein was termed peptidoglycan-associated protein of L. pneumophilo (Ppl). The corresponding genetic determinant, ppl, was subcloned on a 1.8-kb Clol fragment. DNA sequence studies revealed that two open reading frames, pplA and pplB, coding for putative proteins of 18~9 and 16.8 kDa, respectively, were located on the Clol fragment. Exonuclease 111 digestion studies confirmed tbat pplA is the gene coding for the peptidoglycan.;.associated 19-kDa protein of L. pneumophilo. The amino acid sequence of PpiA exhibits a high degree of homology to the sequences of the Pal Iipoproteins of E. coli K-12 and liaemophilus injluenvze.}, subject = {Infektionsbiologie}, language = {en} } @article{SchmollOttOugedaetal.1990, author = {Schmoll, T. and Ott, M. and Ougeda, B. and Hacker, J{\"o}rg}, title = {Use of a wild-type gene fusion to determine the influence of environmental conditions on expression of the S fimbrial adhesin in an Escherichia coli pathogen}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59625}, year = {1990}, abstract = {S fimbrial adhesins (Sfa) enable pathogenic Escherichia coli strains to bind to sialic acid-containing eucaryotic receptor molecules. In order to determine the inftuence of culture conditions on the expression of the sfa determinant in a wild-type strain, we fused the gene lacZ, coding for the enzyme ß-galactosidase, to the sfaA gene, responsible for the major protein subunit of S fimbriae. By using a plasmid which carries an R6K origin, the sfaA-Iac hybrid construct was site-specifically integrated into the chromosome of the uropathogenic E. coli strain S36WT. The expression of lacZ, which was under the control of the sfa wild-type promoters, was now equivalent to the sfa expression of strain S36WT. With the help of this particular wild-type construct, it was demonstrated that the sfa determinant is better expressed on solid media than in liquid broth. The growth rate bad a strong inftuence on Sfa expression under aerobic but not under anaerobic conditions. Production of Sfa was further regulated by catabolite repression, osmolarity, and temperature.}, subject = {Infektionsbiologie}, language = {en} } @article{VenturSchefferHackeretal.1990, author = {Ventur, Y. and Scheffer, J. and Hacker, J{\"o}rg and K{\"o}nig, W.}, title = {Effects of adhesins from mannose-resistant Escherichia coli on mediator release from human lymphocytes, monocytes and basophils and from polymorphonuclear granulo-cytes}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59636}, year = {1990}, abstract = {We investigated the roJe of Escherichia coU expressing mannose-resistant hemagglutination and adhesins with regard to the induction of leukotrienes from a suspension of human lymphocytes, monocytes, and basophils (LMBs) compared with human polymorphonuclear granulocytes (PMNs). Genetically cloned E. coli strains expressing various types of mannose-resistant hemagglutination (MRH+) were phagocytosed to a higher degree by monocytes than the nonadherent E. coli strain. The various strains dUfered in their capacity to induce a chemiluminescence response, which showed the same pattern for LMBs and PMNs. Stimulation of LMBs with bacteria alone, unlike granulocytes, did not activate the cells for the release of leukotrienes. However, preincubation of LMBs with bacteria decreased subsequent leukotriene formation when the cells were stimulated with calcium ionophore. The inhibitory eft'ect was dependent on the concentration of bacteria used for preincubation as weil as on the preincubation temperature. The various bacterial strains dift'ered in inhibitory potency for mediator release. Preincubation of LMBs with zymosan, opsonized zymosan, the bacterfal peptide FMLP, and peptidoglycan bad no inhibitory eft'ect or even increased subsequent IeukotrieDe formation. Opsonized bacteria were far less inhibitory than nonopsonized bacteria. In contrast to human LMBs, preincubation of human PMNs with mannose-resistant bacteria led to increased leukotriene 84 generation and reduced w-oxidation of leukotriene 84 • Our data soggest that phagocytes (neutrophils, monocytes) respond in a different way for leukotriene formation after Interaction with mannose-resistant E. coli.}, subject = {Infektionsbiologie}, language = {en} } @article{MarreKreftHacker1990, author = {Marre, R. and Kreft, B. and Hacker, J{\"o}rg}, title = {Genetically engineered S and F1C fimbriae differ in their contribution to adherence of Escherichia coli to cultured renal tubular cells}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59644}, year = {1990}, abstract = {Escherichia coU K-12 strains producing S-fimbrial adhesins, FlC fimbriae, and mutagenized fimbriae were tested in a binding assay with a renal tubular cell line. S-fimbrial adhesins and FlC fimbriae mediated bindlog to tubular cells. The SfaA, SfaG, and SfaS subunits of S fimbriae contributed to attachment. Site-specific mutations in the sfaS gene reduced binding. The Inhibitionprofile of FlC fimbriae resembled that of S fimbriae.}, subject = {Infektionsbiologie}, language = {en} } @article{BenderOttMarreetal.1990, author = {Bender, L. and Ott, M. and Marre, R. and Hacker, J{\"o}rg}, title = {Genome analysis of Legionella spp. by orthogonal field alternation gel electrophoresis (OFAGE)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-59657}, year = {1990}, abstract = {Various Legionella isolates from different sources and origins were analysed by orthogonal field alternation gel electrophoresis of Not I cleaved genomic DNA. The genome of L pneumophila Philadelphia I, the original isolate of the epidemics in 1976, exhibits only five Not I fragments. Two virulent derivatives. derived from L pneumophila Philadelphia I. which were obtained by prolonged passage on artificial cuhure media, did not differ from their isogenic virulent strain according the Not I fragment pattern. By summing the lengths of the Notl fragments, the genome size of L. pneumophila Philadelphia I was calculated as approximately 3.9 Mb. Environmental L pneumophila strains exhibited different Not I pattems, as did Legionella strains not belongi'ng to the species pneumophila. The usefulness of DNA long range mapping of Legionella ssp. with Notl for epidemiology and evaluation of their evolutionary rela· tionships is discussed.}, subject = {Infektionsbiologie}, language = {en} }