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Institute
- Institut für Molekulare Infektionsbiologie (345) (remove)
Sonstige beteiligte Institutionen
- Genelux Corporation, San Diego Science Center, 3030 Bunker Hill Street, Suite 310, San Diego, California 92109, USA (1)
- Helmholtz Center for RNA-based Infection Research (1)
- Institut für Molekulare Infektionsbiologie (MIB) der Universität Würzburg (1)
- MRB Forschungszentrum für Magnet-Resonanz-Bayern e.V., Am Hubland, D-97074 Würzburg (1)
- Research Center for Infectious Diseases (ZINF), University of Wuerzburg, Wuerzburg, Germany, (1)
- Research Center of Infectious Diseases (ZINF) of the University of Wurzburg, Germany (1)
- Universitätsklinikum Münster (1)
- Zentrum für Infektionsforschung (ZINF): Nachwuchsgruppe 2 (1)
Nitrogen-regulated pathogenesis describes the expression of virulence attributes as direct response to the quantity and quality of an available nitrogen source. As consequence of nitrogen availability, the opportunistic human fungal pathogen Candida albicans changes its morphology and secretes aspartic proteases [SAPs], both well characterized virulence attributes. C. albicans, contrarily to its normally non-pathogenic relative Saccharomyces cerevisiae, is able to utilize proteins, which are considered as abundant and important nitrogen source within the human host. To assimilate complex proteinaceous matter, extracellular proteolysis is followed by uptake of the degradation products through dedicated peptide transporters (di-/tripeptide transporters [PTRs] and oligopeptide transporters [OPTs]). The expression of both traits is transcriptionally controlled by Stp1 - the global regulator of protein utilization - in C. albicans. The aim of the present study was to elucidate the regulation of virulence attributes of the pathogenic fungus C. albicans by nitrogen availability in more detail. Within a genome wide binding profile of Stp1, during growth with proteins, more than 600 Stp1 target genes were identified, thereby confirming its role in the usage of proteins, but also other nitrogenous compounds as nitrogen source. Moreover, the revealed targets suggest an involvement of Stp1 in the general adaption to nutrient availability as well as in the environmental stress response. With the focus on protein utilization and nitrogen-regulated pathogenesis, the regulation of the major secreted aspartic protease Sap2 - additionally one of the prime examples of allelic heterogeneity in C. albicans - was investigated in detail. Thereby, the heterogezygous SAP2 promoter helped to identify an unintended genomic alteration as the true cause of a growth defect of a C. albicans mutant. Additionally, the promoter region, which was responsible for the differential activation of the SAP2 alleles, was delimited. Furthermore, general Sap2 induction was demonstrated to be mediated by distinct cis-acting elements that are required for a high or a low activity of SAP2 expression. For the utilization of proteins as nitrogen source it is also crucial to take up the peptides that are produced by extracellular proteolysis. Therefore, the function and importance of specific peptide transporters was investigated in C. albicans mutants, unable to use peptides as nitrogen source (opt1Δ/Δ opt2Δ/Δ opt3Δ/Δ opt4Δ/Δ opt5Δ/Δ ptr2Δ/Δ ptr22Δ/Δ septuple null mutants). The overexpression of individual transporters in these mutants revealed differential substrate specificities and expanded the specificity of the OPTs to dipeptides, a completely new facet of these transporters. The peptide-uptake deficient mutants were further used to elucidate, whether indeed proteins and peptides are an important in vivo nitrogen source for C. albicans. It was found that during competitive colonization of the mouse intestine these mutants exhibited wild-type fitness, indicating that neither proteins nor peptides are primary nitrogen sources required to efficiently support growth of C. albicans in the mouse gut. Adequate availability of the preferred nitrogen source ammonium represses the utilization of proteins and other alternative nitrogen sources, but also the expression of virulence attributes, like Sap secretion and nitrogen-starvation induced filamentation. In order to discriminate, whether ammonium availability is externally sensed or determined inside the cell by C. albicans, the response to exterior ammonium concentrations of ammonium-uptake deficient mutants (mep1Δ/Δ mep2Δ/Δ null mutants) was investigated. This study showed that presence of an otherwise suppressing ammonium concentration did not inhibit Sap2 proteases secretion and arginine-induced filamentation in these mutants. Conclusively, ammonium availability is primarily determined inside the cell in order to control the expression of virulence traits. In sum, the present work contributes to the current understanding of how C. albicans regulates expression of virulence-associated traits in response to the presence of available nitrogen sources - especially proteins and peptides - in order to adapt its lifestyle within a human host.
Base J, beta-d-glucosyl-hydroxymethyluracil, is an epigenetic modification of thymine in the nuclear DNA of flagellated protozoa of the order Kinetoplastida. J is enriched at sites involved in RNA polymerase ( RNAP) II initiation and termination. Reduction of J in Leishmania tarentolae via growth in BrdU resulted in cell death and indicated a role of J in the regulation of RNAP II termination. To further explore J function in RNAP II termination among kinetoplastids and avoid indirect effects associated with BrdU toxicity and genetic deletions, we inhibited J synthesis in Leishmania major and Trypanosoma brucei using DMOG. Reduction of J in L. major resulted in genome-wide defects in transcription termination at the end of polycistronic gene clusters and the generation of antisense RNAs, without cell death. In contrast, loss of J in T. brucei did not lead to genome-wide termination defects; however, the loss of J at specific sites within polycistronic gene clusters led to altered transcription termination and increased expression of downstream genes. Thus, J regulation of RNAP II transcription termination genome-wide is restricted to Leishmania spp., while in T. brucei it regulates termination and gene expression at specific sites within polycistronic gene clusters.
The bacterial pathogen Legionella pneumophila replicates intracellularly in protozoa, but can also cause severe pneumonia, called Legionnaires' disease. The bacteria invade and proliferate in the alveolar macrophages of the human lung. L. pneumophila bacteria exhibit a biphasic life cycle: replicative bacteria are avirulent; in contrast, transmissive bacteria express virulence traits and flagella. Primarily aim of this thesis was to evaluate the impact of the regulatory proteins FleQ, FleR, and RpoN in flagellar gene regulation. Phenotypic analysis, Western blot and electron microscopy of regulatory mutants in the genes coding for FleQ, RpoN and FleR demonstrated that flagellin expression is strongly repressed and that these mutants are non-flagellated in transmissive phase. Transcriptomic studies of these putative flagellar gene expression regulators demonstrated that fleQ controls the expression of numerous flagellar biosynthetic genes. Together with RpoN, FleQ controls transcription of 14 out of 31 flagellar class II genes, coding for the basal body, hook, and regulatory proteins. Unexpectedly, 7 out of 15 late flagellar genes class III and IV) are expressed dependent on FleQ but independent of RpoN. Thus, in contrast to the commonly accepted view that enhancer binding proteins as FleQ always interact with RpoN to initiate transcription, our results strongly indicate that FleQ of L. pneumophila regulates gene expression RpoN-dependent as well as RpoN-independent. Moreover, transcriptome analysis of a fleR mutant strain elucidated that FleR does not regulate the flagellar class III genes as previously suggested. Instead FleR regulates together with RpoN numerous protein biosynthesis and metabolic genes. Based on these experimental results our modified model for the transcriptional regulation of flagellar genes in L. pneumophila is that flagellar class II genes are controlled by FleQ and RpoN, while flagellar class III and IV genes are controlled in a fleQ-dependent but rpoN-independent manner. Although all L. pneumophila strains share the same complex life style, various pathotypes have evolved. This is reflected by the genomes, which contain e.g. genomic islands. The genomic island Trb-1 of L. pneumophila Corby, carries all genes necessary for a type-IV conjugation system, an integrase gene and a putative oriT site. The second aim of this thesis was to investigate the implication of this genomic island in conjugative DNA transfer. Using conjugation assays we showed that the oriT site located on Trb-1 is functional and contributes to conjugation between different L. pneumophila strains. As this is the first oriT site of L. pneumophila known to be functional our results provide evidence that conjugation is a major mechanism for the evolution of new pathotypes in L. pneumophila.
The Two-Component System (TCS) AbrA1/A2 from Streptomyces coelicolor M145 is a negative regulator of antibiotic production and morphological differentiation. In this work we show that it is able to auto-regulate its expression, exerting a positive induction of its own operon promoter, and that its activation is dependent on the presence of iron. The overexpression of the abrA2 response regulator (RR) gene in the mutant DabrA1/A2 results in a toxic phenotype. The reason is an excess of phosphorylated AbrA2, as shown by phosphoablative and phosphomimetic AbrA2 mutants. Therefore, non-cognate histidine kinases (HKs) or small phospho-donors may be responsible for AbrA2 phosphorylation in vivo. The results suggest that in the parent strain S. coelicolor M145 the correct amount of phosphorylated AbrA2 is adjusted through the phosphorylation-dephosphorylation activity rate of the HK AbrA1. Furthermore, the ABC transporter system, which is part of the four-gene operon comprising AbrA1/A2, is necessary to de-repress antibiotic production in the TCS null mutant. Finally, in order to test the possible biotechnological applications of the DabrA1/A2 strain, we demonstrate that the production of the antitumoral antibiotic oviedomycin is duplicated in this strain as compared with the production obtained in the wild type, showing that this strain is a good host for heterologous antibiotic production. Thus, this genetically modified strain could be interesting for the biotechnology industry.
Abstract
Sulphur is an essential element that all pathogens have to absorb from their surroundings in order to grow inside their infected host. Despite its importance, the relevance of sulphur assimilation in fungal virulence is largely unexplored. Here we report a role of the bZIP transcription factor MetR in sulphur assimilation and virulence of the human pathogen Aspergillus fumigatus. The MetR regulator is essential for growth on a variety of sulphur sources; remarkably, it is fundamental for assimilation of inorganic S-sources but dispensable for utilization of methionine. Accordingly, it strongly supports expression of genes directly related to inorganic sulphur assimilation but not of genes connected to methionine metabolism. On a broader scale, MetR orchestrates the comprehensive transcriptional adaptation to sulphur-starving conditions as demonstrated by digital gene expression analysis. Surprisingly, A. fumigatus is able to utilize volatile sulphur compounds produced by its methionine catabolism, a process that has not been described before and that is MetR-dependent. The A. fumigatus MetR transcriptional activator is important for virulence in both leukopenic mice and an alternative mini-host model of aspergillosis, as it was essential for the development of pulmonary aspergillosis and supported the systemic dissemination of the fungus. MetR action under sulphur-starving conditions is further required for proper iron regulation, which links regulation of sulphur metabolism to iron homeostasis and demonstrates an unprecedented regulatory crosstalk. Taken together, this study provides evidence that regulation of sulphur assimilation is not only crucial for A. fumigatus virulence but also affects the balance of iron in this prime opportunistic pathogen.
Author Summary
Invasive pulmonary aspergillosis (IPA) is a life-threatening disease that affects primarily immunosuppressed patients. During the last decades the incidence of this disease that is accompanied by high mortality rates has increased. Since opportunistic pathogenic fungi, unlike other pathogens, do not express specific virulence factors, it is becoming more and more clear that the elucidation of fungal metabolism is an essential task to understand fungal pathogenicity and to identify novel antifungal targets. In this work we report genetic inactivation of the sulphur transcription regulator MetR in Aspergillus fumigatus and subsequent study of the resulting phenotypes and transcriptional deregulation of the mutant. Here we show that regulation of sulphur assimilation is an essential process for the manifestation of IPA. Moreover, a regulatory connection between sulphur metabolism and iron homeostasis, a further essential virulence determinant of A. fumigatus, is demonstrated in this study for the first time. A deeper knowledge of sulphur metabolism holds the promise of increasing our understanding of fungal virulence and might lead to improved antifungal therapy.
S fimbriae are able to recognize receptor molecules containing sialic acid and are produced by pathogenic E. coli strains causing urinary tract infection and menigitis. In order to characterize the corresponding genetic determinant, termed S fimbrial adhesin ( sfa) gene duster, we have cloned the S-specific genes from a urinary pathogen and from a meningitis isolate. Nine genes are involved in the production of S fimbriae, two of these, sfaB and sfaC code for regulatory proteins being necessary for the expression of S fimbriae. Two promoters, PB and Pc, are located in front of these genes. Transcription of the sfa determinant is influenced by activation of the promotersvia SfaB and SfaC, the action of the H-NS protein and an RNaseE-specific mRNA processing. In addition, a third promoter, P A• located in front of the major subunit gene sfaA, can be activated under special circumstances. Four genes of the sfa determinant code for the subunit-specific proteins, SfaA (16 kda), SfaG (17 kda), SfaS (14 kda) and SfaH (29 kda). It was demonstrated that the protein SfaA is the major subunit protein while SfaS is identical to the sialic-acid-specific adhesin of S fimbriae. The introduction of specific mutations into sfaS revealed that a region of six amino acids of the adhesin which includes two lysine and one arginine residues is involved in the receptor specific interaction of S fimbriae. Additionally, it has been shown that SfaS is necessary for the induction of fimbriation while SfaH plays a role in the stringency of binding of S fimbriae to erythrocytes.
In this study the effect of recombinant human interleukin 2 (rec.hIL-2) on the proliferation and maturation of B lymphocytes was investigated. It was found that the presence of rec.hIL 2 results in proliferation of mitogen (LPS)-activated B cell blasts. In addition, it is shown that highly enriched murine B cells can be induced by rec.hIL-2 to proliferate and to develop into antibody-secreting cells (PFC) in the presence of antigen (SRBC). When tested for its effect on B cell preparations enriched for resting (small) or activated (blasted) B lymphocytes, it was found that rec.hIL 2 provides signals for both B cell populations to develop into PFC. In contrast, induction of proliferation by the same lymphokine source was only seen in blasted B cells. The data indicate that IL 2 is involved in the generation of B effector cells by directly acting on their precursors thereby providing differentiation as well as proliferation signals.
Human B cells appropriately activated by a B cell mitogen are rendered susceptible to human Interleukin 2 (IL-2) as demonstrated with recombinant human IL-2 (rec. h IL-2). They show increased proliferation and drastically enhanced immunoglobulin secretion. Susceptibility to IL-2 is accompanied with the expression of the IL-2 receptor (Tac antigen) on B cells. The data suggest that IL-2 is one of the lymphokines directly involved in the activation of B lymphocytes.
Bacterial small RNAs (sRNAs) are key elements of regulatory networks that modulate gene expression. The sRNA RydC of Salmonella sp. and Escherichia coli is an example of this class of riboregulators. Like many other sRNAs, RydC bears a 'seed' region that recognises specific transcripts through base-pairing, and its activities are facilitated by the RNA chaperone Hfq. The crystal structure of RydC in complex with E. coli Hfq at 3.48 angstrom resolution illuminates how the protein interacts with and presents the sRNA for target recognition. Consolidating the protein-RNA complex is a host of distributed interactions mediated by the natively unstructured termini of Hfq. Based on the structure and other data, we propose a model for a dynamic effector complex comprising Hfq, small RNA, and the cognate mRNA target.
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. epidermülis 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.
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 Lü~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.
Background: Gametogenesis and fertilization play crucial roles in malaria transmission. While male gametes are thought to be amongst the simplest eukaryotic cells and are proven targets of transmission blocking immunity, little is known about their molecular organization. For example, the pathway of energy metabolism that power motility, a feature that facilitates gamete encounter and fertilization, is unknown.
Methods: Plasmodium berghei microgametes were purified and analysed by whole-cell proteomic analysis for the first time. Data are available via ProteomeXchange with identifier PXD001163.
Results: 615 proteins were recovered, they included all male gamete proteins described thus far. Amongst them were the 11 enzymes of the glycolytic pathway. The hexose transporter was localized to the gamete plasma membrane and it was shown that microgamete motility can be suppressed effectively by inhibitors of this transporter and of the glycolytic pathway.
Conclusions: This study describes the first whole-cell proteomic analysis of the malaria male gamete. It identifies glycolysis as the likely exclusive source of energy for flagellar beat, and provides new insights in original features of Plasmodium flagellar organization.
Monoallelic expression within a gene family is found in pathogens exhibiting antigenic variation and in mammalian olfactory neurons. Trypanosoma brucei, a lethal parasite living in the human bloodstream, expresses variant surface glycoprotein (VSG) from 1 of 15 bloodstream expression sites (BESs) by virtue of a multifunctional RNA polymerase I. The active BES is transcribed in an extranucleolar compartment termed the expression site body (ESB), whereas silent BESs, located elsewhere within the nucleus, are repressed epigenetically. The regulatory mechanisms, however, are poorly understood. Here we show that two essential subunits of the basal class I transcription factor A (CITFA) predominantly occupied the promoter of the active BES relative to that of a silent BES, a phenotype that was maintained after switching BESs in situ. In these experiments, high promoter occupancy of CITFA was coupled to high levels of both promoter-proximal RNA abundance and RNA polymerase I occupancy. Accordingly, fluorescently tagged CITFA-7 was concentrated in the nucleolus and the ESB. Because a ChIP-seq analysis found that along the entire BES, CITFA-7 is specifically enriched only at the promoter, our data strongly indicate that monoallelic BES transcription is activated by a mechanism that functions at the level of transcription initiation.
As matchmaker between mRNA and sRNA interactions, the RNA chaperone Hfq plays a key role in riboregulation of many bacteria. Often, the global influence of Hfq on the transcriptome is reflected by substantially altered proteomes and pleiotropic phenotypes in hfq mutants. Using quantitative proteomics and co-immunoprecipitation combined with RNA-sequencing (RIP-seq) of Hfq-bound RNAs, we demonstrate the pervasive role of Hfq in nutrient acquisition, metabolism and motility of the plant pathogen Agrobacterium tumefaciens. 136 of 2544 proteins identified by iTRAQ (isobaric tags for relative and absolute quantitation) were affected in the absence of Hfq. Most of them were associated with ABC transporters, general metabolism and motility. RIP-seq of chromosomally encoded Hfq 3xFlag revealed 1697 mRNAs and 209 non-coding RNAs (ncRNAs) associated with Hfq. 56 ncRNAs were previously undescribed. Interestingly, 55% of the Hfq-bound ncRNAs were encoded antisense (as) to a protein-coding sequence suggesting that A. tumefaciens Hfq plays an important role in asRNA-target interactions. The exclusive enrichment of 296 mRNAs and 31 ncRNAs under virulence conditions further indicates a role for post-transcriptional regulation in A. tumefaciens-mediated plant infection. On the basis of the iTRAQ and RIP-seq data, we assembled a comprehensive model of the Hfq core regulon in A. tumefaciens.
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.
Background
Prokaryotes have relatively small genomes, densely-packed with protein-encoding sequences. RNA sequencing has, however, revealed surprisingly complex transcriptomes and here we report the transcripts present in the model hyperthermophilic Archaeon, Thermococcus kodakarensis, under different physiological conditions.
Results
Sequencing cDNA libraries, generated from RNA isolated from cells under different growth and metabolic conditions has identified >2,700 sites of transcription initiation, established a genome-wide map of transcripts, and consensus sequences for transcription initiation and post-transcription regulatory elements. The primary transcription start sites (TSS) upstream of 1,254 annotated genes, plus 644 primary TSS and their promoters within genes, are identified. Most mRNAs have a 5'-untranslated region (5'-UTR) 10 to 50 nt long (median = 16 nt), but ~20% have 5'-UTRs from 50 to 300 nt long and ~14% are leaderless. Approximately 50% of mRNAs contain a consensus ribosome binding sequence. The results identify TSS for 1,018 antisense transcripts, most with sequences complementary to either the 5'- or 3'-region of a sense mRNA, and confirm the presence of transcripts from all three CRISPR loci, the RNase P and 7S RNAs, all tRNAs and rRNAs and 69 predicted snoRNAs. Two putative riboswitch RNAs were present in growing but not in stationary phase cells. The procedure used is designed to identify TSS but, assuming that the number of cDNA reads correlates with transcript abundance, the results also provide a semi-quantitative documentation of the differences in T. kodakarensis genome expression under different growth conditions and confirm previous observations of substrate-dependent specific gene expression. Many previously unanticipated small RNAs have been identified, some with relative low GC contents (≤50%) and sequences that do not fold readily into base-paired secondary structures, contrary to the classical expectations for non-coding RNAs in a hyperthermophile.
Conclusion
The results identify >2,700 TSS, including almost all of the primary sites of transcription initiation upstream of annotated genes, plus many secondary sites, sites within genes and sites resulting in antisense transcripts. The T. kodakarensis genome is small (~2.1 Mbp) and tightly packed with protein-encoding genes, but the transcriptomes established also contain many non-coding RNAs and predict extensive RNA-based regulation in this model Archaeon.
Virotherapy using oncolytic vaccinia virus (VACV) strains is one promising new strategy for canine cancer therapy. In this study we describe the establishment of an in vivo model of canine soft tissue sarcoma (CSTS) using the new isolated cell line STSA-1 and the analysis of the virus-mediated oncolytic and immunological effects of two different Lister VACV LIVP1.1.1 and GLV-1h68 strains against CSTS. Cell culture data demonstrated that both tested VACV strains efficiently infected and destroyed cells of the canine soft tissue sarcoma line STSA-1. In addition, in our new canine sarcoma tumor xenograft mouse model, systemic administration of LIVP1.1.1 or GLV-1h68 viruses led to significant inhibition of tumor growth compared to control mice. Furthermore, LIVP1.1.1 mediated therapy resulted in almost complete tumor regression and resulted in long-term survival of sarcoma-bearing mice. The replication of the tested VACV strains in tumor tissues led to strong oncolytic effects accompanied by an intense intratumoral infiltration of host immune cells, mainly neutrophils. These findings suggest that the direct viral oncolysis of tumor cells and the virus-dependent activation of tumor-associated host immune cells could be crucial parts of anti-tumor mechanism in STSA-1 xenografts. In summary, the data showed that both tested vaccinia virus strains and especially LIVP1.1.1 have great potential for effective treatment of CSTS.
Preclinical development of an immunotherapy against antibiotic-resistant Staphylococcus aureus
(2017)
The Gram-positive bacterium Staphylococcus aureus is the leading cause of nosocomial infections. In particular, diseases caused by methicillin-resistant S. aureus (MRSA) are associated with higher morbidity, mortality and medical costs due to showing resistance to several classes of established antibiotics and their ability to develop resistance mechanisms against new antibiotics rapidly. Therefore, strategies based on immunotherapy approaches have the potential to close the gap for an efficient treatment of MRSA.
In this thesis, a humanized antibody specific for the immunodominant staphylococcal antigen A (IsaA) was generated and thoroughly characterized as potential candidate for an antibody based therapy. A murine monoclonal antibody was selected for humanization based on its binding characteristics and the ability of efficient staphylococcal killing in mouse infection models. The murine antibody was humanized by CDR grafting and mouse and humanized scFv as well as scFv-Fc fragments were constructed for comparative binding studies to analyse the successful humanization. After these studies, the full antibody with the complete Fc region was constructed as isotype IgG1, IgG2 and IgG4, respectively to assess effector functions, including antibody-dependent killing of S. aureus. The biological activity of the humanized antibody designated hUK-66 was analysed in vitro with purified human PMNs and whole blood samples taken from healthy donors and patients at high risk of S. aureus infections, such as those with diabetes, end-stage renal disease, or artery occlusive disease (AOD).
Results of the in vitro studies show, that hUK-66 was effective in antibody-dependent killing of S. aureus in blood from both healthy controls and patients vulnerable to S. aureus infections. Moreover, the biological activity of hUK-66 and hUK-66 combined with a humanized anti-alpha-toxin antibody (hUK-tox) was investigated in vivo using a mouse pneumonia model. The in vivo results revealed the therapeutic efficacy of hUK-66 and the antibody combination of hUK-66 and hUK-tox to prevent staphylococcal induced pneumonia in a prophylactic set up.
Based on the experimental data, hUK-66 represents a promising candidate for an antibody-based therapy against antibiotic resistant MRSA.
Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) of clonal complex CC398 typically carry various antimicrobial resistance genes, many of them located on plasmids. In the bovine LA-MRSA isolate Rd11, we previously identified plasmid pAFS11 in which resistance genes are co-localized with a novel ica-like gene cluster, harboring genes required for polysaccharide intercellular adhesin (PIA)-mediated biofilm formation. The ica genes on pAFS11 were acquired in addition to a pre-existing ica locus on the S. aureus Rd11 chromosomal DNA. Both loci consist of an icaADBC operon and icaR, encoding a corresponding icaADBC repressor. Despite carrying two biofilm gene copies, strain Rd11 did not produce PIA and transformation of pAFS11 into another S. aureus strain even slightly diminished PIA-mediated biofilm formation. By focusing on the molecular background of the biofilm-negative phenotype of pAFS11-carrying S. aureus, we identified the pAFS11-borne ica locus copy as functionally fully active. However, transcription of both plasmid- and core genome-derived icaADBC operons were efficiently suppressed involving IcaR. Surprisingly, although being different on the amino acid sequence level, the two IcaR repressor proteins are mutually replaceable and are able to interact with the icaA promoter region of the other copy. We speculate that this regulatory crosstalk causes the biofilm-negative phenotype in S. aureus Rd11. The data shed light on an unexpected regulatory interplay between pre-existing and newly acquired DNA traits in S. aureus. This also raises interesting general questions regarding functional consequences of gene transfer events and their putative implications for the adaptation and evolution of bacterial pathogens.
Pf38 is a surface protein of the malarial parasite Plasmodium falciparum. In this study, we produced and purified recombinant Pf38 and a fusion protein composed of red fluorescent protein and Pf38 (RFP-Pf38) using a transient expression system in the plant Nicotiana benthamiana. To our knowledge, this is the first description of the production of recombinant Pf38. To verify the quality of the recombinant Pf38, plasma from semi-immune African donors was used to confirm specific binding to Pf38. ELISA measurements revealed that immune responses to Pf38 in this African subset were comparable to reactivities to AMA-1 and \(MSP1_{19}\). Pf38 and RFP-Pf38 were successfully used to immunise mice, although titres from these mice were low (on average 1:11.000 and 1:39.000, respectively). In immune fluorescence assays, the purified IgG fraction from the sera of immunised mice recognised Pf38 on the surface of schizonts, gametocytes, macrogametes and zygotes, but not sporozoites. Growth inhibition assays using \(\alpha Pf38\) antibodies demonstrated strong inhibition \((\geq 60 \% ) \) of the growth of blood-stage P. falciparum. The development of zygotes was also effectively inhibited by \(\alpha Pf38\) antibodies, as determined by the zygote development assay. Collectively, these results suggest that Pf38 is an interesting candidate for the development of a malaria vaccine.
Background: Despite the limited success after decades of intensive research and development efforts, vaccination still represents the most promising strategy to significantly reduce the disease burden in malaria endemic regions. Besides the ultimate goal of inducing sterile protection in vaccinated individuals, the prevention of transmission by so-called transmission blocking vaccines (TBVs) is being regarded as an important feature of an efficient malaria eradication strategy. Recently, Plasmodium falciparum GAP50 (PfGAP50), a 44.6 kDa transmembrane protein that forms an essential part of the invasion machinery (glideosome) multi-protein complex, has been proposed as novel potential transmission-blocking candidate. Plant-based expression systems combine the advantages of eukaryotic expression with a up-scaling potential and a good product safety profile suitable for vaccine production. In this study we investigated the feasibility to use the transient plant expression to produce PfGAP50 suitable for the induction of parasite specific inhibitory antibodies.
Results: We performed the transient expression of recombinant PfGAP50 in Nicotiana benthamiana leaves using endoplasmatic reticulum (ER) and plastid targeting. After IMAC-purification the protein yield and integrity was investigated by SDS-PAGE and Western Blot. Rabbit immune IgG derived by the immunization with the plastidtargeted variant of PfGAP50 was analyzed by immune fluorescence assay (IFA) and zygote inhibition assay (ZIA). PfGAP50 could be produced in both subcellular compartments at different yields IMAC (Immobilized Metal Affinity Chromatography) purification from extract yielded up to 4.1 mu g/g recombinant protein per fresh leaf material for ER-retarded and 16.2 mu g/g recombinant protein per fresh leave material for plasmid targeted PfGAP50, respectively. IgG from rabbit sera generated by immunization with the recombinant protein specifically recognized different parasite stages in immunofluorescence assay. Furthermore up to 55 % inhibition in an in vitro zygote inhibition assay could be achieved using PfGAP50-specific rabbit immune IgG.
Conclusions: The results of this study demonstrate that the plant-produced PfGAP50 is functional regarding the presentation of inhibitory epitopes and could be considered as component of a transmission-blocking malaria vaccine formulation.
The white-opaque switch is a bistable, epigenetic transition affecting multiple traits in Candida albicans including mating, immunogenicity, and niche specificity. To compare how the two cell states respond to external cues, we examined the fitness, phenotypic switching, and filamentation properties of white cells and opaque cells under 1,440 different conditions at 25°C and 37°C. We demonstrate that white and opaque cells display striking differences in their integration of metabolic and thermal cues, so that the two states exhibit optimal fitness under distinct conditions. White cells were fitter than opaque cells under a wide range of environmental conditions, including growth at various pHs and in the presence of chemical stresses or antifungal drugs. This difference was exacerbated at 37°C, consistent with white cells being the default state of C. albicans in the mammalian host. In contrast, opaque cells showed greater fitness than white cells under select nutritional conditions, including growth on diverse peptides at 25°C. We further demonstrate that filamentation is significantly rewired between the two states, with white and opaque cells undergoing filamentous growth in response to distinct external cues. Genetic analysis was used to identify signaling pathways impacting the white-opaque transition both in vitro and in a murine model of commensal colonization, and three sugar sensing pathways are revealed as regulators of the switch. Together, these findings establish that white and opaque cells are programmed for differential integration of metabolic and thermal cues and that opaque cells represent a more metabolically specialized cell state than the default white state.
Phenotypic heterogeneity at the cellular level in response to various stresses, e.g., antibiotic treatment has been reported for a number of bacteria. In a clonal population, cell-to-cell variation may result in phenotypic heterogeneity that is a mechanism to survive changing environments including antibiotic therapy. Stenotrophomonas rnaltophilia has been frequently isolated from cystic fibrosis patients, can cause numerous infections in other organs and tissues, and is difficult to treat due to antibiotic resistances. S. maltophilia K279a produces the Li and L2 beta-lactamases in response to beta-lactam treatment. Here we report that the patient isolate S. rnaltophilia K279a diverges into cellular subpopulations with distinct but reversible morphotypes of small and big colonies when challenged with ampicillin. This observation is consistent with the formation of elongated chains of bacteria during exponential growth phase and the occurrence of mainly rod-shaped cells in liquid media. RNA-seq analysis of small versus big colonies revealed differential regulation of at least seven genes among the colony morphotypes. Among those, bleu and bla(L2) were transcriptionally the most strongly upregulated genes. Promoter fusions of b/a(L1) and b/a(L2) genes indicated that expression of both genes is also subject to high levels of phenotypic heterogeneous expression on a single cell level. Additionally, the comE homolog was found to be differentially expressed in homogenously versus heterogeneously bla(L2) expressing cells as identified by RNA(seq) analysis. Overexpression of cornE in S. maltophilia K279a reduced the level of cells that were in a bla(L2)-ON mode to 1% or lower. Taken together, our data provide strong evidence that S. maltophilia K279a populations develop phenotypic heterogeneity in an ampicillin challenged model. This cellular variability is triggered by regulation networks including b/a(L1), b/a(L2), and comE.
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.
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.
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.
ABSTRACT Bacteria organize many membrane-related signaling processes in functional microdomains that are structurally and functionally similar to the lipid rafts of eukaryotic cells. An important structural component of these microdomains is the protein flotillin, which seems to act as a chaperone in recruiting other proteins to lipid rafts to facilitate their interaction. In eukaryotic cells, the occurrence of severe diseases is often observed in combination with an overproduction of flotillin, but a functional link between these two phenomena is yet to be demonstrated. In this work, we used the bacterial model Bacillus subtilis as a tractable system to study the physiological alterations that occur in cells that overproduce flotillin. We discovered that an excess of flotillin altered specific signal transduction pathways that are associated with the membrane microdomains of bacteria. As a consequence of this, we detected significant defects in cell division and cell differentiation. These physiological alterations were in part caused by an unusual stabilization of the raft-associated protease FtsH. This report opens the possibility of using bacteria as a working model to better understand fundamental questions related to the functionality of lipid rafts.
IMPORTANCE The identification of signaling platforms in the membrane of bacteria that are functionally and structurally equivalent to eukaryotic lipid rafts reveals a level of sophistication in signal transduction and membrane organization unexpected in bacteria. It opens new and promising venues to address intricate questions related to the functionality of lipid rafts by using bacteria as a more tractable system. This is the first report that uses bacteria as a working model to investigate a fundamental question that was previously raised while studying the role of eukaryotic lipid rafts. It also provides evidence of the critical role of these signaling platforms in orchestrating diverse physiological processes in prokaryotic cells.
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.
Background: Recent data suggest that cancer stem cells (CSCs) play an important role in cancer, as these cells possess enhanced tumor-forming capabilities and are responsible for relapses after apparently curative therapies have been undertaken. Hence, novel cancer therapies will be needed to test for both tumor regression and CSC targeting. The use of oncolytic vaccinia virus (VACV) represents an attractive anti-tumor approach and is currently under evaluation in clinical trials. The purpose of this study was to demonstrate whether VACV does kill CSCs that are resistant to irradiation and chemotherapy.
Methods: Cancer stem-like cells were identified and separated from the human breast cancer cell line GI-101A by virtue of increased aldehyde dehydrogenase 1 (ALDH1) activity as assessed by the ALDEFLUOR assay and cancer stem cell-like features such as chemo-resistance, irradiation-resistance and tumor-initiating were confirmed in cell culture and in animal models. VACV treatments were applied to both ALDEFLUOR-positive cells in cell culture and in xenograft tumors derived from these cells. Moreover, we identified and isolated CD44\(^+\)CD24\(^+\)ESA\(^+\) cells from GI-101A upon an epithelial-mesenchymal transition (EMT). These cells were similarly characterized both in cell culture and in animal models.
Results: We demonstrated for the first time that the oncolytic VACV GLV-1h68 strain replicated more efficiently in cells with higher ALDH1 activity that possessed stem cell-like features than in cells with lower ALDH1 activity. GLV-1h68 selectively colonized and eventually eradicated xenograft tumors originating from cells with higher ALDH1 activity. Furthermore, GLV-1h68 also showed preferential replication in CD44\(^+\)CD24\(^+\)ESA\(^+\) cells derived from GI-101A upon an EMT induction as well as in xenograft tumors originating from these cells that were more tumorigenic than CD44\(^+\)CD24\(^-\)ESA\(^+\) cells.
Conclusions: Taken together, our findings indicate that GLV-1h68 efficiently replicates and kills cancer stem-like cells. Thus, GLV-1h68 may become a promising agent for eradicating both primary and metastatic tumors, especially tumors harboring cancer stem-like cells that are resistant to chemo and/or radiotherapy and may be responsible for recurrence of tumors.
Nucleotide sequence of the sfaA gene coding for the S fimbrial protein subunit of Escherichia coli
(1987)
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.
F 1 C fimbriae allow uropathogenic Escherichia coli to adhere to specific epithelial surfaces. This adhesive property is probably due to the presence of minor fimbrial components in F1C fimbriae. The foe gene cluster encoding F1C fimbriae has been cloned, as described previously. Here we present the nucleotide sequence (2081 bp) coding for the F 1 C minor fimbria I subunits. The structural genes code for polypeptides of 175 (FocF), 166 (FocG), and 300 (FocH) amino acids. The deduced amino acids of the F 1 C minor subunits were compared with the reported sequences of the minor subunits of other types of fimbriae. The data show that the Foc minor subunits are highly homologous to the corresponding Sfa proteins, whereas homology to the minor subunits of type 1 and P fimbriae is much lower.
Ongoing resistance developments against antibiotics that also affect last-resort antibiotics require novel antibacterial compounds. Strategies to discover such novel structures have been dimerization or hybridization of known antibacterial agents. We found novel antibacterial agents by dimerization of indols and hybridization with carbazoles. They were obtained in a simple one-pot reaction as bisindole tetrahydrocarbazoles. Further oxidation led to bisindole carbazoles with varied substitutions of both the indole and the carbazole scaffold. Both the tetrahydrocarbazoles and the carbazoles have been evaluated in various S. aureus strains, including MRSA strains. Those 5-cyano substituted derivatives showed best activities as determined by MIC values. The tetrahydrocarbazoles partly exceed the activity of the carbazole compounds and thus the activity of the used standard antibiotics. Thus, promising lead compounds could be identified for further studies.
Defeat of the antibiotic resistance of pathogenic bacteria is one great challenge today and for the future. In the last century many classes of effective antibacterials have been developed, so that upcoming resistances could be met with novel drugs of various compound classes. Meanwhile, there is a certain lack of research of the pharmaceutical companies, and thus there are missing developments of novel antibiotics. Gram-positive bacteria are the most important cause of clinical infections. The number of novel antibacterials in clinical trials is strongly restricted. There is an urgent need to find novel antibacterials. We used synthetic chemistry to build completely novel hybrid molecules of substituted indoles and benzothiophene. In a simple one-pot reaction, two novel types of thienocarbazoles were yielded. Both indole substituted compound classes have been evaluated as completely novel antibacterials against the Staphylococcus and Enterococcus species. The evaluated partly promising activities depend on the indole substituent type. First lead compounds have been evaluated within in vivo studies. They confirmed the in vitro results for the new classes of small-molecule antibacterials.
\(Enterococcus\) species cause increasing numbers of infections in hospitals. They contribute to the increasing mortality rates, mostly in patients with comorbidities, who suffer from severe diseases. \(Enterococcus\) resistances against most antibiotics have been described, including novel antibiotics. Therefore, there is an ongoing demand for novel types of antibiotics that may overcome bacterial resistances. We discovered a novel class of antibiotics resulting from a simple one-pot reaction of indole and \(o\)-phthaldialdehyde. Differently substituted indolyl benzocarbazoles were yielded. Both the indole substitution and the positioning at the molecular scaffold influence the antibacterial activity towards the various strains of \(Enterococcus\) species with the highest relevance to nosocomial infections. Structure-activity relationships are discussed, and the first lead compounds were identified as also being effective in the case of a vancomycin resistance.
Increasing antibacterial drug resistance threatens global health, unfortunately, however, efforts to find novel antibacterial agents have been scaled back by the pharmaceutical industry due to concerns about a poor return on investment. Nevertheless, there is an urgent need to find novel antibacterial compounds to combat antibacterial drug resistance. The synthesis of novel drugs from natural sources is mostly cost-intensive due to those drugs’ complicated structures. Therefore, it is necessary to find novel antibacterials by simple synthesis to become more attractive for industrial production. We succeeded in the discovery of four antibacterial compound (sub)classes accessible in a simple one-pot reaction based on fluorinated benzothiophene-indole hybrids. They have been evaluated against various S. aureus and MRSA strains. Structure- and substituent-dependent activities have been found within the (sub)classes and promising lead compounds have been identified. In addition, bacterial pyruvate kinase was found to be the molecular target of the active compounds. In conclusion, simple one-pot synthesis of benzothiophene-indoles represents a promising strategy for the search of novel antimicrobial compounds.
The eukaryotic unicellular pathogen Plasmodium falciparum tightly regulates gene expression, both during development and in adaptation to dynamic host environments. This regulation is evident in the mutually exclusive expression of members of clonally variant virulence multigene families. While epigenetic regulators have been selectively identified at active or repressed virulence genes, their specific recruitment remains a mystery. In recent years, noncoding RNAs (ncRNAs) have emerged as lynchpins of eukaryotic gene regulation; by binding to epigenetic regulators, they provide target specificity to otherwise non-specific enzyme complexes. Not surprisingly, there is great interest in understanding the role of ncRNA in P. falciparum, in particular, their contribution to the mutually exclusive expression of virulence genes. The current repertoire of P. falciparum ncRNAs includes, but is not limited to, subtelomeric ncRNAs, virulence gene-associated ncRNAs and natural antisense RNA transcripts. Continued improvement in high-throughput sequencing methods is sure to expand this repertoire. Here, we summarize recent advances in P. falciparum ncRNA biology, with an emphasis on ncRNA-mediated epigenetic modes of gene regulation.
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.
Next-generation humanized NSG-SGM3 mice are highly susceptible to Staphylococcus aureus infection
(2023)
Humanized hemato-lymphoid system mice, or humanized mice, emerged in recent years as a promising model to study the course of infection of human-adapted or human-specific pathogens. Though Staphylococcus aureus infects and colonizes a variety of species, it has nonetheless become one of the most successful human pathogens of our time with a wide armory of human-adapted virulence factors. Humanized mice showed increased vulnerability to S. aureus compared to wild type mice in a variety of clinically relevant disease models. Most of these studies employed humanized NSG (NOD-scid IL2Rgnull) mice which are widely used in the scientific community, but show poor human myeloid cell reconstitution. Since this immune cell compartment plays a decisive role in the defense of the human immune system against S. aureus, we asked whether next-generation humanized mice, like NSG-SGM3 (NOD-scid IL2Rgnull-3/GM/SF) with improved myeloid reconstitution, would prove to be more resistant to infection. To our surprise, we found the contrary when we infected humanized NSG-SGM3 (huSGM3) mice with S. aureus: although they had stronger human immune cell engraftment than humanized NSG mice, particularly in the myeloid compartment, they displayed even more pronounced vulnerability to S. aureus infection. HuSGM3 mice had overall higher numbers of human T cells, B cells, neutrophils and monocytes in the blood and the spleen. This was accompanied by elevated levels of pro-inflammatory human cytokines in the blood of huSGM3 mice. We further identified that the impaired survival of huSGM3 mice was not linked to higher bacterial burden nor to differences in the murine immune cell repertoire. Conversely, we could demonstrate a correlation of the rate of humanization and the severity of infection. Collectively, this study suggests a detrimental effect of the human immune system in humanized mice upon encounter with S. aureus which might help to guide future therapy approaches and analysis of virulence mechanisms.
Recent progresses and developments in molecular biology provide a wealth of new but insufficiently characterised data. This fund comprises amongst others biological data of genomic DNA, protein sequences, 3-dimensional protein structures as well as profiles of gene expression. In the present work, this information is used to develop new methods for the characterisation and classification of organisms and whole groups of organisms as well as to enhance the automated gain and transfer of information. The first two presented approaches (chapters 4 und 5) focus on the medically and scientifically important enterobacteria. Its impact in medicine and molecular biology is founded in versatile mechanisms of infection, their fundamental function as a commensal inhabitant of the intestinal tract and their use as model organisms as they are easy to cultivate. Despite many studies on single pathogroups with clinical distinguishable pathologies, the genotypic factors that contribute to their diversity are still partially unknown. The comprehensive genome comparison described in Chapter 4 was conducted with numerous enterobacterial strains, which cover nearly the whole range of clinically relevant diversity. The genome comparison constitutes the basis of a characterisation of the enterobacterial gene pool, of a reconstruction of evolutionary processes and of comprehensive analysis of specific protein families in enterobacterial subgroups. Correspondence analysis, which is applied for the first time in this context, yields qualitative statements to bacterial subgroups and the respective, exclusively present protein families. Specific protein families were identified for the three major subgroups of enterobacteria namely the genera Yersinia and Salmonella as well as to the group of Shigella and E. coli by applying statistical tests. In conclusion, the genome comparison-based methods provide new starting points to infer specific genotypic traits of bacterial groups from the transfer of functional annotation. Due to the high medical importance of enterobacterial isolates their classification according to pathogenicity has been in focus of many studies. The microarray technology offers a fast, reproducible and standardisable means of bacterial typing and has been proved in bacterial diagnostics, risk assessment and surveillance. The design of the diagnostic microarray of enterobacteria described in chapter 5 is based on the availability of numerous enterobacterial genome sequences. A novel probe selection strategy based on the highly efficient algorithm of string search, which considers both coding and non-coding regions of genomic DNA, enhances pathogroup detection. This principle reduces the risk of incorrect typing due to restrictions to virulence-associated capture probes. Additional capture probes extend the spectrum of applications of the microarray to simultaneous diagnostic or surveillance of antimicrobial resistance. Comprehensive test hybridisations largely confirm the reliability of the selected capture probes and its ability to robustly classify enterobacterial strains according to pathogenicity. Moreover, the tests constitute the basis of the training of a regression model for the classification of pathogroups and hybridised amounts of DNA. The regression model features a continuous learning capacity leading to an enhancement of the prediction accuracy in the process of its application. A fraction of the capture probes represents intergenic DNA and hence confirms the relevance of the underlying strategy. Interestingly, a large part of the capture probes represents poorly annotated genes suggesting the existence of yet unconsidered factors with importance to the formation of respective virulence phenotypes. Another major field of microarray applications is gene expression analysis. The size of gene expression databases rapidly increased in recent years. Although they provide a wealth of expression data, it remains challenging to integrate results from different studies. In chapter 6 the methodology of an unsupervised meta-analysis of genome-wide A. thaliana gene expression data sets is presented, which yields novel insights in function and regulation of genes. The application of kernel-based principal component analysis in combination with hierarchical clustering identified three major groups of contrasts each sharing overlapping expression profiles. Genes associated with two groups are known to play important roles in Indol-3 acetic acid (IAA) mediated plant growth and development as well as in pathogen defence. Yet uncharacterised serine-threonine kinases could be assigned to novel functions in pathogen defence by meta-analysis. In general, hidden interrelation between genes regulated under different conditions could be unravelled by the described approach. HMMs are applied to the functional characterisation of proteins or the detection of genes in genome sequences. Although HMMs are technically mature and widely applied in computational biology, I demonstrate the methodical optimisation with respect to the modelling accuracy on biological data with various distributions of sequence lengths. The subunits of these models, the states, are associated with a certain holding time being the link to length distributions of represented sequences. An adaptation of simple HMM topologies to bell-shaped length distributions described in chapter 7 was achieved by serial chain-linking of single states, while residing in the class of conventional HMMs. The impact of an optimisation of HMM topologies was underlined by performance evaluations with differently adjusted HMM topologies. In summary, a general methodology was introduced to improve the modelling behaviour of HMMs by topological optimisation with maximum likelihood and a fast and easily implementable moment estimator. Chapter 8 describes the application of HMMs to the prediction of interaction sites in protein domains. As previously demonstrated, these sites are not trivial to predict because of varying degree in conservation of their location and type within the domain family. The prediction of interaction sites in protein domains is achieved by a newly defined HMM topology, which incorporates both sequence and structure information. Posterior decoding is applied to the prediction of interaction sites providing additional information of the probability of an interaction for all sequence positions. The implementation of interaction profile HMMs (ipHMMs) is based on the well established profile HMMs and inherits its known efficiency and sensitivity. The large-scale prediction of interaction sites by ipHMMs explained protein dysfunctions caused by mutations that are associated to inheritable diseases like different types of cancer or muscular dystrophy. As already demonstrated by profile HMMs, the ipHMMs are suitable for large-scale applications. Overall, the HMM-based method enhances the prediction quality of interaction sites and improves the understanding of the molecular background of inheritable diseases. With respect to current and future requirements I provide large-scale solutions for the characterisation of biological data in this work. All described methods feature a highly portable character, which allows for the transfer to related topics or organisms, respectively. Special emphasis was put on the knowledge transfer facilitated by a steadily increasing wealth of biological information. The applied and developed statistical methods largely provide learning capacities and hence benefit from the gain of knowledge resulting in increased prediction accuracies and reliability.
ABSTRACT
The highly conserved heterotrimeric protein kinase SNF1 is important for metabolic adaptations in the pathogenic yeast Candida albicans. A key function of SNF1 is to inactivate the repressor protein Mig1 and thereby allow the expression of genes that are required for the utilization of alternative carbon sources when the preferred carbon source, glucose, is absent or becomes limiting. However, how SNF1 controls Mig1 activity in C. albicans has remained elusive. Using a phosphoproteomics approach, we found that Mig1 is phosphorylated at multiple serine residues. Replacement of these serine residues by nonphosphorylatable alanine residues strongly increased the repressor activity of Mig1 in cells lacking a functional SNF1 complex, indicating that additional protein kinases are involved in the regulation of Mig1. Unlike wild-type Mig1, whose levels strongly decreased when the cells were grown on sucrose or glycerol instead of glucose, the levels of a mutant Mig1 protein lacking nine phosphorylation sites remained high under these conditions. Despite the increased protein levels and the absence of multiple phosphorylation sites, cells with a functional SNF1 complex could still sufficiently inhibit the hyperactive Mig1 to enable wild-type growth on alternative carbon sources. In line with this, phosphorylated forms of the mutant Mig1 were still detected in the presence and absence of a functional SNF1, demonstrating that Mig1 contains additional, unidentified phosphorylation sites and that downstream protein kinases are involved in the control of Mig1 activity by SNF1.
IMPORTANCE
The SNF1 protein kinase signaling pathway, which is highly conserved in eukaryotic cells, is important for metabolic adaptations in the pathogenic yeast Candida albicans. However, so far, it has remained elusive how SNF1 controls the activity of one of its main effectors, the repressor protein Mig1 that inhibits the expression of genes required for the utilization of alternative carbon sources when glucose is available. In this study, we have identified multiple phosphorylation sites in Mig1 that contribute to its inactivation. Mutation of these sites strongly increased Mig1 repressor activity in the absence of SNF1, but SNF1 could still sufficiently inhibit the hyperactive Mig1 to enable growth on alternative carbon sources. These findings reveal features of Mig1 that are important for controlling its repressor activity. Furthermore, they demonstrate that both SNF1 and additional protein kinases regulate Mig1 in this pathogenic yeast.
The 06 serogroup Escherichia coli strain 536 carries two hemolysin (hly) determinants integrated into the chromosome. The two hly determinants are not completely identical, either functionally or structurally, as demonstrated by spontaneous deletion mutants carrying only one of them and by cloning each of the two determinants separately into cosmid vectors. Each hly determinant is independently deleted at a frequency of 10-4 , leading to variants which exhibit similar levels of internal hemolysin but different amounts of secreted hemolysin. The two hly determinants were also identified in the 04 E. coli strain 519. The three E. coli strains 251, 764, and 768, which belong to the serogroup 018, and the 04 strain 367 harbor a single chromosomal hly determinant, as demonstrated by hybridization with hly-gene-specific probes. However, a hybridization probe derived from a sequence adjacent to the hlyC-proximal end of the plasmid pHlyl52-encoded hly determinant hybridizes with several additional chromosomal bands in hemolytic 018 and 06 E. coli strains and even in E. coli K-12. The size ofthe probe causing the multiple hybridization suggests a 1,500- to 1,800-base pair sequence directly flanking hlyC. Spontaneous hemolysin-negative mutants were isolated from strains 764 and 768, which had lost the entire hly determinant but retained all copies of the hlyC-associated sequence. This sequence is not identical to a previously identified (J. Hacker, S. Knapp, and W. Goebel, J. Bacteriol. 154:1145-1154, 1983) somewhat smaller (about 850 base pairs) sequence flanking the other (hlyBb-proximal) end of the plasmid pHlyl52-encoded hly determinant which, as shown here, exists also in multiple copies in these hemolytic E. coli strains and in at least two copies in E. coli K-12. In contrast to the plasmid-encoded hly determinant which is directly flanked at both ends by these two diJJerent sequences, the chromosomal hly determinants are not immediately flanked by such sequences.
MRSA (Methicillin-resistant Staphylococcus aureus) is the second-leading cause of deaths by antibiotic-resistant bacteria globally, with more than 100,000 attributable deaths annually. Despite the high urgency to develop a vaccine to control this pathogen, all clinical trials with pre-clinically effective candidates failed so far. The recent development of “humanized” mice might help to edge the pre-clinical evaluation closer to the clinical situation and thus close this gap. We infected humanized NSG mice (huNSG: (NOD)-scid IL2R\(_γ\)\(^{null}\) mice engrafted with human CD34+ hematopoietic stem cells) locally with S. aureus USA300 LAC* lux into the thigh muscle in order to investigate the human immune response to acute and chronic infection. These mice proved not only to be more susceptible to MRSA infection than wild-type or “murinized” mice, but displayed furthermore inferior survival and signs of systemic infection in an otherwise localized infection model. The rate of humanization correlated directly with the severity of disease and survival of the mice. Human and murine cytokine levels in blood and at the primary site of infection were strongly elevated in huNSG mice compared to all control groups. And importantly, differences in human and murine immune cell lineages surfaced during the infection, with human monocyte and B cell numbers in blood and bone marrow being significantly reduced at the later time point of infection. Murine monocytes in contrast behaved conversely by increasing cell numbers. This study demonstrates significant differences in the in vivo behavior of human and murine cells towards S. aureus infection, which might help to sharpen the translational potential of pre-clinical models for future therapeutic approaches.
Background: Searching the orthologs of a given protein or DNA sequence is one of the most important and most commonly used Bioinformatics methods in Biology. Programs like BLAST or the orthology search engine Inparanoid can be used to find orthologs when the similarity between two sequences is sufficiently high. They however fail when the level of conservation is low. The detection of remotely conserved proteins oftentimes involves sophisticated manual intervention that is difficult to automate.
Results: Here, we introduce morFeus, a search program to find remotely conserved orthologs. Based on relaxed sequence similarity searches, morFeus selects sequences based on the similarity of their alignments to the query, tests for orthology by iterative reciprocal BLAST searches and calculates a network score for the resulting network of orthologs that is a measure of orthology independent of the E-value. Detecting remotely conserved orthologs of a protein using morFeus thus requires no manual intervention. We demonstrate the performance of morFeus by comparing it to state-of-the-art orthology resources and methods. We provide an example of remotely conserved orthologs, which were experimentally shown to be functionally equivalent in the respective organisms and therefore meet the criteria of the orthology-function conjecture.
Conclusions: Based on our results, we conclude that morFeus is a powerful and specific search method for detecting remotely conserved orthologs.
Ten monoclonal antibodies (McAbs) raised to Schistosoma japonicum eggs could be assigned using several serological and immunochemical techniques to 3 groups. The McAbs, termed A, B and C-McAbs, apparently recognize carbohydrate epitopes that can be located on the same antigen molecule. The antibodies, generally of IgM isotype, are idiotypically related. They are distinct from another IgM McAb (Group D-McAb) the carbohydrate target epitope of which can also be associated with the epitopes of A. B and C-McAbs. The McAbs produce large vacuolated bleb reactions in the circumoval precipitin test (COPT) and target epitopes have different representations in various life cycle stages such as immature and mature eggs, male and female worms (including S. mansoni). Antigens affinity purified on columns containing A, B, C and D-McAbs stimulate proliferation of T cells from egg-sensitized mice and elicit DTH reactions in such mice. This raises the possibility that the target antigens of these carbohydrate-reactive monoclonal antibodies are immunopathologic and involved in egg-induced granuloma formation.
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.
The genetic determinant coding for the Pspecific F8 fimbriae was cloned from · the chromosome of the Escherichia coli wild-type strain 2980 (018: K5: H5: FlC, F8). The F8 determinant was further subcloned into the Pstl site of pBR322 and a restriction map was established. In a Southern hybridization experiment identity between the chromosomally encoded F8 determinant of 2980 and its cloned Counterpart was demonstrated. The cloned F8 fimbriäe and those of the wild type strain consist of a protein subunit of nearly 20 kDa. F8 fimbriated strains were agglutinated by an F8 polyclonal antiserum, caused mannose-resistant hemagglutination and attached to human uroepi thellal cells. The cloned F8 determinant was weil expressed in a variety of host strains.
The fungus Candida albicans is a typical member of the human microbiota, where it usually behaves as a commensal. It can also become pathogenic; often causing minor superficial infections in healthy people, but also potentially fatal invasive systemic infections in immunocompromised people. Unfortunately, there is only a fairly limited set of antifungal drugs, and evolution of drug resistance threatens their efficacy. Greater understanding of the mechanisms that C. albicans uses to survive in and infect the host can uncover candidate targets for novel antifungals. Protein kinases are central to a vast array of signalling pathways which govern practically all aspects of life, and furthermore are relatively straightforward to design drugs against. As such, investigation and characterization of protein kinases in C. albicans as well as their target proteins and the pathways they govern are important targets for research. AMP-activated kinases are well conserved proteins which respond to energy stress; they are represented in yeasts by the heterotrimeric SNF1 complex, which responds primarily to the absence of glucose. In this work, the SNF1 pathway was investigated with two primary goals: identify novel targets of this protein kinase and elucidate why SNF1 is essential. Two approaches were used to identify novel targets of SNF1. In one, suppressor mutants were evolved from a strain in which SNF1 activity is reduced, which exhibits defects in carbon source utilization and cell wall integrity. This revealed a suppressor mutation within SNF1 itself, coding for the catalytic subunit of the complex – SNF1Δ311-316. The second approach screened a library of artificially activated zinc cluster transcription factors, identifying Czf1 as one such transcription factor which, upon artificial activation, restored resistance to cell wall stress in a mutant of the SNF1 pathway. Finally, a, inducible gene deletion system revealed that SNF1 is not an essential gene.
While beneficial sponge-microbe associations have received much attention in recent years, less effort has been undertaken to investigate the interactions of sponges with potentially pathogenic microorganisms. Thus, the aim of this study was to examine two selected Caribbean disease conditions, termed “Sponge Orange Band” and “Sponge White Patch”, via ecological and molecular methods. Sponge Orange Band (SOB) disease affects the prominent Caribbean barrel sponge Xestospongia muta that is counted among the high-microbial-abundance (HMA) sponges, whereas Sponge White Patch (SWP) disease affects the abundant rope sponge Amphimedon compressa that belongs to the low-microbial-abundance (LMA) sponges. I have documented for both Caribbean sponge diseases a disease progression going along with massive tissue destruction as well as loss of the characteristic microbial signatures. Even though new bacteria were shown to colonize the bleached areas, the infection trials revealed in both cases no indication for the involvement of a microbial pathogen as an etiologic agent of disease leaving us still in the dark about the cause of Sponge Orange Band as well as Sponge White Patch disease.
Results of molecular and pathogenic studies of three different bacterial hemolysins (cytolysins) are presented. These exoproteins derive from the two gram-negative bacteria Escherichia coli and Aeromonas hydrophila and from the gram-positive pathogen Listeria monocytogenes. The hemolysin of E. coli is determined by an 8-kilobase (kb) region that includes four clustered genes (hlyC, hlyA, hlyB, and hlyD). This hemolysin determinant is part either of large transmissible plasmids or of the chromosome. The genes located chromosomally are found predominantly in E. coli strains that can cause pyelonephritis and/or other extraintestinal infections. A detailed analysis of the chromosomal hly determinants of one nephropathogenic E. coli strain revealed the existence of specific, large chromosomal insertions 75 kb and lOO kb in size that carry the hly genes but that also influence the expression of other virulence properties, i.e., adhesion and serum resistance. The direct involvement of E. coli hemolysin in virulence could be demonstrated in several model systems. The genetic determinants for hemolysin (cytolysin) formation in , A. hydrophila (aerolysin) and L. monocytogenes (listeriolysin) are less complex. Both cytolysins seem to be encoded by single genes, although two loci (aerB and aerC) that affect the expression and activity of aerolysin have been identified distal and proximal to the structural gene for aerolysin (aerA). Cytolysin-negative mutants of both bacteria were obtained by site-specific deletion and/or transposon mutagenesis. These mutants show a drastic reduction in the virulence of the respective bacteria.
Advances in stem cell research have allowed the development of 3-dimensional (3D) primary cell cultures termed organoid cultures, as they closely mimic the in vivo organization of different cell lineages. Bridging the gap between 2-dimensional (2D) monotypic cancer cell lines and whole organisms, organoids are now widely applied to model development and disease. Organoids hold immense promise for addressing novel questions in host-microbe interactions, infectious diseases and the resulting inflammatory conditions. Researchers have started to use organoids for modeling infection with pathogens, such as Helicobacter pylori or Salmonella enteritica, gut- microbiota interactions and inflammatory bowel disease. Future studies will broaden the spectrum of microbes used and continue to establish organoids as a standard model for human host-microbial interactions. Moreover, they will increasingly exploit the unique advantages of organoids, for example to address patient-specific responses to microbes.
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.
Mip protein of Legionella pneumophila exhibits peptidyl-prolyl cis-trans-isomerase (PPIase) activity
(1992)
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.
Axon degeneration and functional decline in myelin diseases are often attributed to loss of myelin but their relation is not fully understood. Perturbed myelinating glia can instigate chronic neuroinflammation and contribute to demyelination and axonal damage. Here we study mice with distinct defects in the proteolipid protein 1 gene that develop axonal damage which is driven by cytotoxic T cells targeting myelinating oligodendrocytes. We show that persistent ensheathment with perturbed myelin poses a risk for axon degeneration, neuron loss, and behavioral decline. We demonstrate that CD8\(^+\) T cell-driven axonal damage is less likely to progress towards degeneration when axons are efficiently demyelinated by activated microglia. Mechanistically, we show that cytotoxic T cell effector molecules induce cytoskeletal alterations within myelinating glia and aberrant actomyosin constriction of axons at paranodal domains. Our study identifies detrimental axon-glia-immune interactions which promote neurodegeneration and possible therapeutic targets for disorders associated with myelin defects and neuroinflammation.
Abstract
In line with the key role of methionine in protein biosynthesis initiation and many cellular processes most microorganisms have evolved mechanisms to synthesize methionine de novo. Here we demonstrate that, in the bacterial pathogen Staphylococcus aureus, a rare combination of stringent response-controlled CodY activity, T-box riboswitch and mRNA decay mechanisms regulate the synthesis and stability of methionine biosynthesis metICFE-mdh mRNA. In contrast to other Bacillales which employ S-box riboswitches to control methionine biosynthesis, the S. aureus metICFE-mdh mRNA is preceded by a 5′-untranslated met leader RNA harboring a T-box riboswitch. Interestingly, this T-box riboswitch is revealed to specifically interact with uncharged initiator formylmethionyl-tRNA \((tRNA_i^{fMet})\)while binding of elongator \(tRNA^{Met}\) proved to be weak, suggesting a putative additional function of the system in translation initiation control. met leader RNA/metICFE-mdh operon expression is under the control of the repressor CodY which binds upstream of the met leader RNA promoter. As part of the metabolic emergency circuit of the stringent response, methionine depletion activates RelA-dependent (p)ppGpp alarmone synthesis, releasing CodY from its binding site and thereby activating the met leader promoter. Our data further suggest that subsequent steps in metICFE-mdh transcription are tightly controlled by the 5′ met leader-associated T-box riboswitch which mediates premature transcription termination when methionine is present. If methionine supply is limited, and hence \((tRNA_i^{fMet})\) becomes uncharged, full-length met leader/metICFE-mdh mRNA is transcribed which is rapidly degraded by nucleases involving RNase J2. Together, the data demonstrate that staphylococci have evolved special mechanisms to prevent the accumulation of excess methionine. We hypothesize that this strict control might reflect the limited metabolic capacities of staphylococci to reuse methionine as, other than Bacillus, staphylococci lack both the methionine salvage and polyamine synthesis pathways. Thus, methionine metabolism might represent a metabolic Achilles' heel making the pathway an interesting target for future anti-staphylococcal drug development.
Author Summary
Prokaryote metabolism is key for our understanding of bacterial virulence and pathogenesis and it is also an area with huge opportunity to identify novel targets for antibiotic drugs. Here, we have addressed the so far poorly characterized regulation of methionine biosynthesis in S. aureus. We demonstrate that methionine biosynthesis control in staphylococci significantly differs from that predicted for other Bacillales. Notably, involvement of a T-box instead of an S-box riboswitch separates staphylococci from other bacteria in the order. We provide, for the first time, direct experimental proof for an interaction of a methionyl-tRNA-specific T-box with its cognate tRNA, and the identification of initiator \((tRNA_i^{fMet})\) as the specific binding partner is an unexpected finding whose exact function in Staphylococcus metabolism remains to be established. The data further suggest that in staphylococci a range of regulatory elements are integrated to form a hierarchical network that elegantly limits costly (excess) methionine biosynthesis and, at the same time, reliably ensures production of the amino acid in a highly selective manner. Our findings open a perspective to exploit methionine biosynthesis and especially its T-box-mediated control as putative target(s) for the development of future anti-staphylococcal therapeutics.
Metabolic conversion of CI-1040 turns a cellular MEK-inhibitor into an antibacterial compound
(2018)
Influenza virus (IV) infections cause severe respiratory illnesses that can be complicated by bacterial super-infections. Previously, we identified the cellular Raf-MEK-ERK cascade as a promising antiviral target. Inhibitors of MEK, such as CI-1040, showed potent antiviral activity. However, it remained unclear if this inhibitor and its active form, ATR-002, might sensitize host cells to either IV or secondary bacterial infections. To address these questions, we studied the anti-pathogen activity of ATR-002 in comparison to CI-1040, particularly, its impact on Staphylococcus aureus (S. aureus), which is a major cause of IV super-infections. We analysed IV and S. aureus titres in vitro during super-infection in the presence and absence of the drugs and characterized the direct impact of ATR-002 on bacterial growth and phenotypic changes. Importantly, neither CI-1040 nor ATR-002 treatment led to increased bacterial titres during super-infection, indicating that the drug does not sensitize cells for bacterial infection. In contrast, we rather observed reduced bacterial titres in presence of ATR-002. Surprisingly, ATR-002 also led to reduced bacterial growth in suspension cultures, reduced stress- and antibiotic tolerance without resistance induction. Our data identified for the first time that a particular MEK-inhibitor metabolite exhibits direct antibacterial activity, which is likely due to interference with the bacterial PknB kinase/Stp phosphatase signalling system.
Dendritic cell-based vaccination is a well established technique for preventive and therapeutic instruction of the immune system where conservative vaccine formulations fail to cure or prevent diseases, respectively. Efficiency of this technique already was demonstrated in infectious diseases as well as for cancer in animal or human studies. Well controlled manipulation and antigen-loading of immature DC is most beneficial to this technique. But, time-consuming and cost-extensive procedures for preparation of DC precursors, expansion and stimulation of DC and inpatient administration are big disadvantages regarding vaccine development for pandemic infectious diseases that occur mainly in underdeveloped countries. Therefore vaccines are needed that are pathogen-tailored and able to induce equal immune responses as their DC-based vaccine models. For vaccination against Leishmania parasites such a DC-based vaccine is feasible and its efficacy to induce protective Th1-based immune responses was already demonstrated in several animal studies. But, one of our own studies indicated supportive activity of host cells exceeding the allocation of T cells to become activated by transferred DC. IL-12, an important cytokine for the induction of Th1-related immune responses, has to be produced by host cells. Therefore, the aim of this study was to investigate the mechanism of BMDC-based vaccination with regard to simplification of the vaccine formulation. Key questions that have been addressed are: Which cells process the information that is transferred by the injected DC and what are the key components of this information? Further more, it was looked at whether altered vaccine formulations are able to induce protective immunity and whether they share equal molecular mechanisms. The current paradigm of BMDC-based vaccination proposes direct interaction of transferred BMDC with host T cells. These BMDC have to be antigen-loaded for stimulation via antigen-peptide-MHC molecule-complexes and they have to be activated for proper co-stimulation of T cells. Here, this study demonstrates that neither activation for co-stimulation nor direct interaction with adequate MHC molecules is needed for the induction of protective immunity against infection with Leishmania-parasites. Disrupted antigen-loaded BMDC are able to induce protective immunity in BALB/c mice without pre-stimulation via CpG ODN. Beyond, if BMDC were used with a different MHC-background than recipient mice then the vaccine still would be efficient in terms of reduction of footpad swelling and parasite load in draining lymph nodes. Even more, DC-specific features are no key component that leads to protective immunity as vaccination with disrupted antigen-loaded MΦ shows equal properties than before mentioned vaccine formulations. Further more, it was found that host DC play a major role in transforming the incoming signal, received from transferred antigen-loaded DC, into Th1-related stimuli and Leishmania-antigen-specific T cell activation. Suspensions of disrupted antigen-loaded DC resemble a combination of laid off soluble molecules together with exosome-like vesicles that formed after disruption of membranes. Here it was shown that separation of the membranous and soluble fractions and subsequent transfer into BALB/c mice will lead to protection of these mice against infection with L. major promastigotes only if the membranous fraction is used as vaccine. More, this vaccine formulation takes advantage of easy storage at -80°C with no need of fresh production. This clearly demonstrates that the immunity-inducing principle of disrupted DC-based vaccination lies within the membrane enclosed fraction. On a molecular level, disrupted antigen-loaded DC induce Th1-related cytokines during vaccination and as response on pathogen encounter. In vivo assays revealed IL-12 production and antigen-specific T cell proliferation among splenocytes that were stimulated with disrupted antigen-loaded DC. Splenocytes of accordingly vaccinated mice produce tremendous amounts of IFNγ after stimulation with Leishmania parasites. In summary, disrupted antigen-loaded BMDC fulfil all characteristics of DC-based vaccination against Leishmania major. But, while purification of membranes of antigen-loaded DC and subsequent transfer to BALB/c mice leads to control of the disease in the animal model, only slight levels of Th1-related cytokines are seen in the in vivo assays. Whether this points towards a loss of vaccine activity on unseen levels or unknown sites where Th1-related immunity is induced by both, complete solution and purified membranes, still has to be determined.
Marine sponge-derived Streptomyces sp SBT343 extract inhibits staphylococcal biofilm formation
(2017)
Staphylococcus epidermidis and Staphylococcus aureus are opportunistic pathogens that cause nosocomial and chronic biofilm-associated infections. Indwelling medical devices and contact lenses are ideal ecological niches for formation of staphylococcal biofilms. Bacteria within biofilms are known to display reduced susceptibilities to antimicrobials and are protected from the host immune system. High rates of acquired antibiotic resistances in staphylococci and other biofilm-forming bacteria further hamper treatment options and highlight the need for new anti-biofilm strategies. Here, we aimed to evaluate the potential of marine sponge-derived actinomycetes in inhibiting biofilm formation of several strains of S. epidermidis, S. aureus, and Pseudomonas aeruginosa. Results from in vitro biofilm-formation assays, as well as scanning electron and confocal microscopy, revealed that an organic extract derived from the marine sponge-associated bacterium Streptomyces sp. SBT343 significantly inhibited staphylococcal biofilm formation on polystyrene, glass and contact lens surfaces, without affecting bacterial growth. The extract also displayed similar antagonistic effects towards the biofilm formation of other S. epidermidis and S. aureus strains tested but had no inhibitory effects towards Pseudomonas biofilms. Interestingly the extract, at lower effective concentrations, did not exhibit cytotoxic effects on mouse fibroblast, macrophage and human corneal epithelial cell lines. Chemical analysis by High Resolution Fourier Transform Mass Spectrometry (HRMS) of the Streptomyces sp. SBT343 extract proportion revealed its chemical richness and complexity. Preliminary physico-chemical characterization of the extract highlighted the heat-stable and non-proteinaceous nature of the active component(s). The combined data suggest that the Streptomyces sp. SBT343 extract selectively inhibits staphylococcal biofilm formation without interfering with bacterial cell viability. Due to absence of cell toxicity, the extract might represent a good starting material to develop a future remedy to block staphylococcal biofilm formation on contact lenses and thereby to prevent intractable contact lens-mediated ocular infections.
A full understanding of the contribution of small RNAs (sRNAs) to bacterial virulence demands knowledge of their target suites under infection-relevant conditions. Here, we take an integrative approach to capturing targets of the Hfq-associated sRNA PinT, a known post-transcriptional timer of the two major virulence programs of Salmonella enterica. Using MS2 affinity purification and RNA sequencing (MAPS), we identify PinT ligands in bacteria under in vitro conditions mimicking specific stages of the infection cycle and in bacteria growing inside macrophages. This reveals PinT-mediated translational inhibition of the secreted effector kinase SteC, which had gone unnoticed in previous target searches. Using genetic, biochemical, and microscopic assays, we provide evidence for PinT-mediated repression of steC mRNA, eventually delaying actin rearrangements in infected host cells. Our findings support the role of PinT as a central post-transcriptional regulator in Salmonella virulence and illustrate the need for complementary methods to reveal the full target suites of sRNAs.
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.
Background: Treatment options for NAFLD are still limited. Bariatric surgery, such as Roux-en-Y gastric bypass (RYGB), has been shown to improve metabolic and histologic markers of NAFLD. Glucagon-like-peptide-1 (GLP-1) analogues lead to improvements in phase 2 clinical trials. We directly compared the effects of RYGB with a treatment using liraglutide and/or peptide tyrosine tyrosine 3-36 (PYY\(_{3-36}\)) in a rat model for early NAFLD. Methods: Obese male Wistar rats (high-fat diet (HFD)-induced) were randomized into the following treatment groups: RYGB, sham-operation (sham), liraglutide (0.4 mg/kg/day), PYY\(_{3-36}\) (0.1 mg/kg/day), liraglutide+PYY\(_{3-36}\), and saline. After an observation period of 4 weeks, liver samples were histologically evaluated, ELISAs and RNA sequencing + RT-qPCRs were performed. Results: RYGB and liraglutide+PYY\(_{3-36}\) induced a similar body weight loss and, compared to sham/saline, marked histological improvements with significantly less steatosis. However, only RYGB induced significant metabolic improvements (e.g., adiponectin/leptin ratio 18.8 ± 11.8 vs. 2.4 ± 1.2 in liraglutide+PYY\(_{3-36}\)- or 1.4 ± 0.9 in sham-treated rats). Furthermore, RNA sequencing revealed a high number of differentially regulated genes in RYGB treated animals only. Conclusions: The combination therapy of liraglutide+PYY\(_{3-36}\) partly mimics the positive effects of RYGB on weight reduction and on hepatic steatosis, while its effects on metabolic function lack behind RYGB.
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.
The hemolytic, uropathogenic Escherichia coli 536 (06:K15:H31) contains two inserts in its chromosome (insert I and insert II), both of which carried hly genes, were rather unstable, and were deleted spontaneously with a frequen~y of 10-3 to 10-4• These inserts were not found in the chromosome of two nonhemolytic E. coli strains, whereas the chromosomal ~equences adjacent to these inserts appeared tobe again homologous in the uropathogenic and two other E. coü strains. Insert I was 75 kilobases in size and was ftanked at both ends by 16 base pairs (bp) (TTCGACTCCTGTGATC) which were arranged in direct orientation. For insert I it was demonstrated that deletion occurred by recombination between the two 16-bp ftanking sequences, since mutants lacking this insert still carried a single copy of the 16-bp sequence in the chromosome. 8oth inserts contained a functional hemolysin determinant. However, the loss of the inserts not only atfected the hemolytic phenotype bot led to a considerable reduction in serum resistance and the loss of mannose-resistant hemagglutination, caused by the presence of S-type funbriae (sja). lt is shown that the Sfa-negative phenotype is due to a block in transcription of the sfa genes. Mutants of strain 536 which lacked both inserts were entirely avirulent when tested in several animal model systems.
During a study of the nutritional requirements of clinical isolates of Escherichia coli, we found that 21 (7.0%) of 301 strains required nicotinamide to grow in minimal medium. The nicotinamide- requiring strains were present in 16 (15.8%) of 101 cultures of urine from young women with acute cystitis, in 5 (5.0%) of 100 stool specimens from healthy adults, and in none of 100 blood samples from adult patients with bacteremia. Most of the strains belonged to serogroup OI8:KI:H7, were hemolytic, possessed type I fimbriae, and exhibited similar patterns of antibiotic susceptibility. Two of the urinary isolates expressed S fimbriae, and all 16 urinary isolates contained the s/aS homologue gene on their chromosomes. One of the stool isolates contained the s/aS gene. The urinary isolates closely resembled a large clone of E. coli that is reportedly associated with neonatal meningitis and sepsis. It may be possible to detect this and related clones by their requirement for nicotinamide and to screen strains for S fimbriae by relatively inexpensive hemagglutination methods, including the use of avian PI antigens to detect mannose- resistant, non-P-fimbriated E. coli; the agglutination of bovine erythrocytes; and the use of bovine mucin to detect sialyl galactosides in S fimbriae.
The \(\alpha\)-Sialyl-\(\beta\) 2-3-Galactosyl-specific adhesin (S adhesin) was isolated from cells of a recombinant Escherichia coli K-12 strain expressing the S-flmbrial adhesin complex. A crude cell extract was partiaUy dissociated into fimbriae and an adhesin-enriched fraction by heating to 7O°C. From the latter, adhesin was purified to apparent homogeneity (by fast protein liquid chromatography, immunoblot, and NaDodSO\(_4\)/PAGE) by differential ammonium sulfate precipitation, dissociation in 8 M guanidine hydrochloride, and high-resolution anion-exchange chromatography in 8 M urea. The purified adhesin formed an aggregate of M\(_r\)\(\approx\)10\(^6\) that was made up of one type of 12-kDa polypeptide (fimbrillin is 16.5 kDa). It had pI value of 4.7 (fimbriae has a pI value of 6). Adhesin and fimbrillin had different amino add compositions. The purified adhesins agglutinated human and bovine erythrocytes with the same speclfkity as the whole bacteria; purified fimbriae were not adhesive. Monoclonal anti-adhesin and anti-fimbriae antibodies were obtained. Monoclonal antiadhesin, but none of the anti-fimbriae, antibodies inhibited the agglutination of erythrocytes. The anti-adhesive antibodies were used in immuno-gold electron microscopy to localize adhesin exclusively on the fimbriae, with a possible preference to their tips.
The search for new antibiotics against multidrug-resistant (MDR), Gram-negative bacteria is crucial with respect to filling the antibiotics development pipeline, which is subject to a critical shortage of novel molecules. Screening of natural products is a promising approach for identifying antimicrobial compounds hosting a higher degree of novelty. Here, we report the isolation and characterization of four galloylglucoses active against different MDR strains of Escherichia coli and Klebsiella pneumoniae. A crude acetone extract was prepared from Paeonia officinalis Linnaeus leaves, and bioautography-guided isolation of active compounds from the extract was performed by liquid–liquid extraction, as well as open column, flash, and preparative chromatographic methods. Isolated active compounds were characterized and elucidated by a combination of spectroscopic and spectrometric techniques. In vitro antimicrobial susceptibility testing was carried out on E. coli and K. pneumoniae using 2 reference strains and 13 strains hosting a wide range of MDR phenotypes. Furthermore, in vivo antibacterial activities were assessed using Galleria mellonella larvae, and compounds 1,2,3,4,6-penta-O-galloyl-β-d-glucose, 3-O-digalloyl-1,2,4,6-tetra-O-galloyl-β-d-glucose, 6-O-digalloyl-1,2,3,4-tetra-O-galloyl-β-d-glucose, and 3,6-bis-O-digalloyl-1,2,4-tri-O-galloyl-β-d-glucose were isolated and characterized. They showed minimum inhibitory concentration (MIC) values in the range of 2–256 µg/mL across tested bacterial strains. These findings have added to the number of known galloylglucoses from P. officinalis and highlight their potential against MDR Gram-negative bacteria.
Isolation and characterization of coliphage Omega18A specific for Escherichia coli O18ac strains
(1987)
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.
Background:
Ethiopia, a high tuberculosis (TB) burden country, reports one of the highest incidence rates of extra-pulmonary TB dominated by cervical lymphadenitis (TBLN). Infection with Mycobacterium bovis has previously been excluded as the main reason for the high rate of extra-pulmonary TB in Ethiopia.
Methods:
Here we examined demographic and clinical characteristics of 953 pulmonary (PTB) and 1198 TBLN patients visiting 11 health facilities in distinct geographic areas of Ethiopia. Clinical characteristics were also correlated with genotypes of the causative agent, Mycobacterium tuberculosis.
Results:
No major patient or bacterial strain factor could be identified as being responsible for the high rate of TBLN, and there was no association with HIV infection. However, analysis of the demographic data of involved patients showed that having regular and direct contact with live animals was more associated with TBLN than with PTB, although no M. bovis was isolated from patients with TBLN. Among PTB patients, those infected with Lineage 4 reported "contact with other TB patient" more often than patients infected with Lineage 3 did (OR = 1.6, CI 95% 1.0-2.7; p = 0.064). High fever, in contrast to low and moderate fever, was significantly associated with Lineage 4 (OR = 2.3; p = 0.024). On the other hand, TBLN cases infected with Lineage 4 tended to get milder symptoms overall for the constitutional symptoms than those infected with Lineage 3.
Conclusions:
The study suggests a complex role for multiple interacting factors in the epidemiology of extra-pulmonary TB in Ethiopia, including factors that can only be derived from population-based studies, which may prove to be significant for TB control in Ethiopia.
The anaerobe Fusobacterium nucleatum (F. nucleatum) is an important member of the oral microbiome but can also colonize different tissues of the human body. In particular, its association with multiple human cancers has drawn much attention.
This association has prompted growing interest into the interaction of F. nucleatum with cancer, with studies focusing primarily on the host cells. At the same time, F. nucleatum itself remains poorly understood, which includes its transcriptomic architecture but also gene regulation such as global stress responses that typically enable survival of bacteria in new environments. An important aspect of such regulatory networks is the post-transcriptional regulation, which is entirely unknown in F. nucleatum. This paucity extents to any knowledge on small regulatory RNAs (sRNAs), despite their important role as post-transcriptional regulators of the bacterial physiology.
Investigating the above stated aspects is further complicated by the fact that F. nucleatum is phylogenetically distant from all other bacteria, displays very limited genetic tractability and lacks genetic tools for dissecting gene function.
This leaves many open questions on basic gene regulation in F. nucleatum, such as if the bacterium combines transcriptional and post-transcriptional regulation in its adaptation to a changing environment.
To begin answering this question, this works elucidated the transcriptomic landscape of F. nucleatum by performing differential RNA-seq (dRNA-seq). Conducted for five representative strains of all F. nucleatum subspecies and the closely related F. periodonticum, the analysis globally uncovered transcriptional start sites (TSS), 5'untranslated regions (UTRs) and improved the existing annotation. Importantly, the dRNA-seq analysis also identified a conserved suite of sRNAs specific to Fusobacterium.
The development of five genetic tools enabled further investigations of gene functions in F. nucleatum. These include vectors that enable the expression of different fluorescent proteins, inducible gene expression and scarless gene deletion in addition to transcriptional and translational reporter systems.
These tools enabled the dissection of a Sigma E response and uncovered several commonalities with its counterpart in the phylogenetically distant Proteobacteria. The similarities include the upregulation of genes involved in membrane homeostasis but also a Simga E-dependent regulatory sRNA. Surprisingly, oxygen was found to activated Sigma E in F. nucleatum contrasting the typical role of the factor in envelope stress.
The non-coding Sigma E-dependent sRNA, named FoxI, was shown to repress the translation of several envelope proteins which represented yet another parallel to the envelope stress response in Proteobacteria.
Overall, this work sheds light on the RNA landscape of the cancer-associated bacterium leading to the discovery of a conserved global stress response consisting of a coding and a non-coding arm. The development of new genetic tools not only aided the latter discovery but also provides the means for further dissecting the molecular and infection biology of this enigmatic bacterium.
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: Intrazelluläres Überleben
Staphylococcus aureus is a major human pathogen, which can invade and survive in non-professional and professional phagocytes. Uptake by host cells is thought to contribute to pathogenicity and persistence of the bacterium. Upon internalization by epithelial cells, cytotoxic S. aureus strains can escape from the phagosome, replicate in the cytosol and induce host cell death. Here, we identified a staphylococcal cysteine protease to induce cell death after translocation of intracellular S. aureus into the host cell cytoplasm. We demonstrated that loss of staphopain A function leads to delayed onset of host cell death and prolonged intracellular replication of S. aureus in epithelial cells. Overexpression of staphopain A in a non-cytotoxic strain facilitated intracellular killing of the host cell even in the absence of detectable intracellular replication. Moreover, staphopain A contributed to efficient colonization of the lung in a mouse pneumonia model. In phagocytic cells, where intracellular S. aureus is exclusively localized in the phagosome, staphopain A did not contribute to cytotoxicity. Our study suggests that staphopain A is utilized by S. aureus to exit the epithelial host cell and thus contributes to tissue destruction and dissemination of infection.
Author summary Staphylococcus aureus is an antibiotic-resistant pathogen that emerges in hospital and community settings and can cause a variety of diseases ranging from skin abscesses to lung inflammation and blood poisoning. The bacterium can asymptomatically colonize the upper respiratory tract and skin of humans and take advantage of opportune conditions, like immunodeficiency or breached barriers, to cause infection. Although S. aureus was not regarded as intracellular bacterium, it can be internalized by human cells and subsequently exit the host cells by induction of cell death, which is considered to cause tissue destruction and spread of infection. The bacterial virulence factors and underlying molecular mechanisms involved in the intracellular lifestyle of S. aureus remain largely unknown. We identified a bacterial cysteine protease to contribute to host cell death of epithelial cells mediated by intracellular S. aureus. Staphopain A induced killing of the host cell after translocation of the pathogen into the cell cytosol, while bacterial proliferation was not required. Further, the protease enhanced survival of the pathogen during lung infection. These findings reveal a novel, intracellular role for the bacterial protease staphopain A.
The massive remodeling of the heart tissue, as observed in response to pressure overload or myocardial infarction, is considered to play a causative role in the development of heart failure. Alterations in the heart architecture clearly affect the mechanical properties of the heart muscle, but they are rooted in changes at the cellular level including modulation of gene expression. Together with integrins, the transmembrane receptors linking the extracellular environment to the cytoskeleton, extracellular matrix (ECM) proteins and matricellular proteins are key components of the remodeling process in the heart. Therefore, this thesis was aimed at analysing the role of integrins in the regulation of gene expression and heart muscle performance during cardiac wound repair induced by pressure overload or myocardial infarction (MI). To investigate the contribution of integrin Beta 1, we characterised the response of mice with a conditional, cardiac-specific deletion of the integrin Beta 1 gene in an experimental model of pressure overload by aortic banding (AB). In particular, we measured physiological alterations and gene expression events in the stressed heart in the presence or absence of integrin Beta 1. Interestingly, mice containing a knock-out allele and the ventricular myocyte-specific conditional allele of the integrin Beta 1 gene were born and grew up to adulthood. Though these animals still exhibited minor amounts of integrin Beta1 in the heart (expressed by non-myocytes), these mice displayed abnormal cardiac function and were highly sensitive to AB. Whereas a compensatory hypertrophic response to pressure overload was observed in wildtype mice, the integrin Beta 1-deficient mice were not able to undergo heart tissue remodeling. Furthermore, ECM gene expression was altered and, in particular, the increased expression of the matricellular protein SPARC after AB was abolished in integrin Beta 1–deficient mice. Interestingly, we also found a transient upregulation of SPARC mRNA during heart remodeling after MI using cDNA macroarrays. Indeed, increased SPARC protein levels were observed starting at day 2 (2.55±0.21fold, p<0.01), day 7 (3.72±0.28 fold, p<0.01) and 1 month (1.9±0.16 fold, p<0.01) after MI, which could be abolished by using an integrin alpha v inhibitor in vivo. Immunofluorescence analysis of heart tissue demonstrated that the increased SPARC expression was confined to the infarcted area and occurred together with the influx of fibroblasts into the heart. In vitro, either TGF-Beta 1 or PDGF-BB stimulated SPARC expression by fibroblasts. Inhibition of integrin alpha v did not interfere with TGF-Beta1 or PDGF induced SPARC secretion as determined by ELISA assays or Western blot. However, secretion of TGF-Beta1 and PDGF-BB by cardiomyocytes was induced by vitronectin, a ligand of integrin alpha v, and this response was blocked by the integrin alpga v inhibitor. Functionally, SPARC modulated the migratory response of fibroblasts towards ECM proteins suggesting that the local deposition of SPARC following MI contributes to scar formation. Taken together, our combined in vivo and in vitro data demonstrate that several integrin subunits play critical roles during tissue remodeling in the injured heart. Integrin-dependent gene expression events such as the upregulation of SPARC following MI are critical to orchestrate the healing response. These processes appear to involve complex cross-talk between different cell types such as cardiomyocytes and fibroblasts to allow for locally confined scar formation. The elucidation of the sophisticated interplay between integrins, matricellular proteins such as SPARC, and growth factors will undoubtedly provide us with a better and clinically useful understanding of the molecular mechanisms governing heart remodeling.
The prevention of restenosis after percutaneous coronary intervention is a major task for researchers and clinicians in cardiovascular pharmacology. Nearly 1.5 million PTCA are performed every year worldwide and, due to the implantation of stents, most of the cases can be treated successfully. 60% of those patients develop restenosis within 6 months. SMC migration and ECM deposition are known to be responsible for neointima formation. Among many processes, integrin initiated signalling events play a central role in SMC migration. Many integrins recognize a specific RGD sequence which is present in several ECM proteins and cell surface immunoglobulin super family molecules. Until now, there are various integrin antagonists such as antibodies, cyclic peptides, peptidomimetics, and non-peptides have been shown to interfere with such pathological situations indicating the importance of integrin initiated signalling pathways in SMC migration. Therefore, in this study SMC migration induced by ECM proteins was inhibited either using pharmacological inhibitor or by overexpressing the endogenous inhibitor of FAK by AAV vector system. In the first part of the thesis, the effect of integrin-ligand stimulation on hCASMCs was studied. The tyrosine phosphorylation of many cellular proteins was observed from serum starved hCASMCs replated on VN but not on PL coated plates. The major tyrosine phosphorylated protein was identified as FAK by immunoprecipitation and also phosphorylation was found at Tyr 397, the autophosphorylation site of FAK. Further, VN induced the dose dependent migration of hCASMCs in haptotaxis assay. The integrin v inhibitor was used to block those ECM stimulated integrin signalling pathways and cell migration. It inhibited the ECM stimulated tyrosine phosphorylation in a dose dependent manner. Interestingly, specific potent antagonism of integrin v abrogated both ECM induced haptotaxis and growth factor induced chemotaxis. The inhibition of migration is consistent with the replating assay results that show interference with integrin induced signalling pathways particularly the FAK tyrosine phosphorylation. The integrin v inhibitor also is able to interfere with hCASMC invasion through matrigel by reducing MMP-2 secretion. Importantly, integrin v inhibitor did not induce the apoptosis in hCASMCs. FAK is a key player in many cellular events and its involvement in cell migration was extensively studied in various cell types. The present study explored the function of FAK in hCASMC migration by overexpression of FRNK, the C-terminal domain of FAK. Overexpression of FRNK inhibited the in vitro SMC migration as well as the neointima formation in a porcine restenosis model in vivo. The last part of this thesis focused on the identification of putative binding partners for the N-terminal domain of FAK by bacterial two-hybrid screen. One of the interesting binding partners was a putative protein of 17.9 kDa. Its human homolog is AGS4, which acts as a GTPase activator. The preliminary results revealed that it is able to interact with N-FAK domain and its expression is high in haematopoietic cells. Taken together the above results suggest that integrin v and FAK are promising targets for inhibition of SMC migration. Disruption of FAK-mediated signalling pathways by a pharmacological inhibitor or by overexpression of FRNK, which acts as dominant-negative regulator, resulted in decreased migration of SMCs and thus can lead to reduction of neointima formation.
Integrative "Omics"-Approach Discovers Dynamic and Regulatory Features of Bacterial Stress Responses
(2013)
Bacteria constantly face stress conditions and therefore mount specific responses to ensure adaptation and survival. Stress responses were believed to be predominantly regulated at the transcriptional level. In the phototrophic bacterium Rhodobacter sphaeroides the response to singlet oxygen is initiated by alternative sigma factors. Further adaptive mechanisms include post-transcriptional and post-translational events, which have to be considered to gain a deeper understanding of how sophisticated regulation networks operate. To address this issue, we integrated three layers of regulation: (1) total mRNA levels at different time-points revealed dynamics of the transcriptome, (2) mRNAs in polysome fractions reported on translational regulation (translatome), and (3) SILAC-based mass spectrometry was used to quantify protein abundances (proteome). The singlet oxygen stress response exhibited highly dynamic features regarding short-term effects and late adaptation, which could in part be assigned to the sigma factors RpoE and RpoH2 generating distinct expression kinetics of corresponding regulons. The occurrence of polar expression patterns of genes within stress-inducible operons pointed to an alternative of dynamic fine-tuning upon stress. In addition to transcriptional activation, we observed significant induction of genes at the post-transcriptional level (translatome), which identified new putative regulators and assigned genes of quorum sensing to the singlet oxygen stress response. Intriguingly, the SILAC approach explored the stress-dependent decline of photosynthetic proteins, but also identified 19 new open reading frames, which were partly validated by RNA-seq. We propose that comparative approaches as presented here will help to create multi-layered expression maps on the system level ("expressome"). Finally, intense mass spectrometry combined with RNA-seq might be the future tool of choice to re-annotate genomes in various organisms and will help to understand how they adapt to alternating conditions.
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.
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.
The virulence of the uropathogenic E. coli strain 536 (06: K 1 5: H31) which produces the S-fimbrial adhesin (Sfa•), is serum-resistant (Sre+) and hemolytic (Hiy+) and its derivatives were assessed in five different animal models. Cloned hemolysin (h/y) determinants from the Chromosomes of 06,018 and 075 E. colistrains and from the plasmid pHiy152 were introduced into the spontaneaus Sfa-, Sre-, Hly- mutant 536-21 and its Sfa+, Sre+, Hly- variant 536-31. As already demonstrated for the 536-21 strains {lnfect. Immun. 42: 57-63) the 018-hly determinant but not the plasmid-encoded hly determinant of pHiy 1 52 transformed into 536-31 contribute to lethality in a mouse peritonitis modal. Similar results were obtained with both Hlyhost strains and their Hly+ transformants in a chicken embryo test and in a mouse nephropathogenicity assay in which the renal bacterial counts were measured 1 5 min to 8 hours after i.v. infection. S-fimbriae and serum resistance had only a marginal influence in these three in vivo systems. ln centrast all three factors, S-fimbriae, serum resistance and hemolysin, were necessary for full virulence in a respiratory mouse infection assay. ln a subcutaneously-induced sepsis model in the mouse restoration of S-fimbriae and serum resistance and separately chromosomally-encoded hemolysis increased virulence to a Ievel comparable to that of the parental 536 strain.
Meningococcal meningitis is a severe central nervous system infection that occurs when Neisseria meningitidis (Nm) penetrates brain endothelial cells (BECs) of the meningeal blood-cerebrospinal fluid barrier. As a human-specific pathogen, in vivo models are greatly limited and pose a significant challenge. In vitro cell models have been developed, however, most lack critical BEC phenotypes limiting their usefulness. Human BECs generated from induced pluripotent stem cells (iPSCs) retain BEC properties and offer the prospect of modeling the human-specific Nm interaction with BECs. Here, we exploit iPSC-BECs as a novel cellular model to study Nm host-pathogen interactions, and provide an overview of host responses to Nm infection. Using iPSC-BECs, we first confirmed that multiple Nm strains and mutants follow similar phenotypes to previously described models. The recruitment of the recently published pilus adhesin receptor CD147 underneath meningococcal microcolonies could be verified in iPSC-BECs. Nm was also observed to significantly increase the expression of pro-inflammatory and neutrophil-specific chemokines IL6, CXCL1, CXCL2, CXCL8, and CCL20, and the secretion of IFN-γ and RANTES. For the first time, we directly observe that Nm disrupts the three tight junction proteins ZO-1, Occludin, and Claudin-5, which become frayed and/or discontinuous in BECs upon Nm challenge. In accordance with tight junction loss, a sharp loss in trans-endothelial electrical resistance, and an increase in sodium fluorescein permeability and in bacterial transmigration, was observed. Finally, we established RNA-Seq of sorted, infected iPSC-BECs, providing expression data of Nm-responsive host genes. Altogether, this model provides novel insights into Nm pathogenesis, including an impact of Nm on barrier properties and tight junction complexes, and suggests that the paracellular route may contribute to Nm traversal of BECs.
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.
FinO-domain proteins represent an emerging family of RNA-binding proteins (RBPs) with diverse roles in bacterial post-transcriptional control and physiology. They exhibit an intriguing targeting spectrum, ranging from an assumed single RNA pair (FinP/traJ) for the plasmid-encoded FinO protein, to transcriptome-wide activity as documented for chromosomally encoded ProQ proteins. Thus, the shared FinO domain might bear an unusual plasticity enabling it to act either selectively or promiscuously on the same cellular RNA pool. One caveat to this model is that the full suite of in vivo targets of the assumedly highly selective FinO protein is unknown. Here, we have extensively profiled cellular transcripts associated with the virulence plasmid-encoded FinO in Salmonella enterica. While our analysis confirms the FinP sRNA of plasmid pSLT as the primary FinO target, we identify a second major ligand: the RepX sRNA of the unrelated antibiotic resistance plasmid pRSF1010. FinP and RepX are strikingly similar in length and structure, but not in primary sequence, and so may provide clues to understanding the high selectivity of FinO-RNA interactions. Moreover, we observe that the FinO RBP encoded on the Salmonella virulence plasmid controls the replication of a cohabitating antibiotic resistance plasmid, suggesting cross-regulation of plasmids on the RNA level.
Bulk RNA sequencing technologies have provided invaluable insights into host and bacterial gene expression and associated regulatory networks. Nevertheless, the majority of these approaches report average expression across cell populations, hiding the true underlying expression patterns that are often heterogeneous in nature. Due to technical advances, single-cell transcriptomics in bacteria has recently become reality, allowing exploration of these heterogeneous populations, which are often the result of environmental changes and stressors. In this work, we have improved our previously published bacterial single-cell RNA sequencing (scRNA-seq) protocol that is based on multiple annealing and deoxycytidine (dC) tailing-based quantitative scRNA-seq (MATQ-seq), achieving a higher throughput through the integration of automation. We also selected a more efficient reverse transcriptase, which led to reduced cell loss and higher workflow robustness. Moreover, we successfully implemented a Cas9-based rRNA depletion protocol into the MATQ-seq workflow. Applying our improved protocol on a large set of single Salmonella cells sampled over different growth conditions revealed improved gene coverage and a higher gene detection limit compared to our original protocol and allowed us to detect the expression of small regulatory RNAs, such as GcvB or CsrB at a single-cell level. In addition, we confirmed previously described phenotypic heterogeneity in Salmonella in regard to expression of pathogenicity-associated genes. Overall, the low percentage of cell loss and high gene detection limit makes the improved MATQ-seq protocol particularly well suited for studies with limited input material, such as analysis of small bacterial populations in host niches or intracellular bacteria.
IMPORTANCE: Gene expression heterogeneity among isogenic bacteria is linked to clinically relevant scenarios, like biofilm formation and antibiotic tolerance. The recent development of bacterial single-cell RNA sequencing (scRNA-seq) enables the study of cell-to-cell variability in bacterial populations and the mechanisms underlying these phenomena. Here, we report a scRNA-seq workflow based on MATQ-seq with increased robustness, reduced cell loss, and improved transcript capture rate and gene coverage. Use of a more efficient reverse transcriptase and the integration of an rRNA depletion step, which can be adapted to other bacterial single-cell workflows, was instrumental for these improvements. Applying the protocol to the foodborne pathogen Salmonella, we confirmed transcriptional heterogeneity across and within different growth phases and demonstrated that our workflow captures small regulatory RNAs at a single-cell level. Due to low cell loss and high transcript capture rates, this protocol is uniquely suited for experimental settings in which the starting material is limited, such as infected tissues.
As multidrug-resistant bacteria represent a concerning burden, experts insist on the need for a dramatic rethinking on antibiotic use and development in order to avoid a post-antibiotic era. New and rapidly developable strategies for antimicrobial substances, in particular substances highly potent against multidrug-resistant bacteria, are urgently required. Some of the treatment options currently available for multidrug-resistant bacteria are considerably limited by side effects and unfavorable pharmacokinetics. The glycopeptide vancomycin is considered an antibiotic of last resort. Its use is challenged by bacterial strains exhibiting various types of resistance. Therefore, in this study, highly active polycationic peptide-vancomycin conjugates with varying linker characteristics or the addition of PEG moieties were synthesized to optimize pharmacokinetics while retaining or even increasing antimicrobial activity in comparison to vancomycin. The antimicrobial activity of the novel conjugates was determined by microdilution assays on susceptible and vancomycin-resistant bacterial strains. VAN1 and VAN2, the most promising linker-modified derivatives, were further characterized in vivo with molecular imaging and biodistribution studies in rodents, showing that the linker moiety influences both antimicrobial activity and pharmacokinetics. Encouragingly, VAN2 was able to undercut the resistance breakpoint in microdilution assays on vanB and vanC vancomycin-resistant enterococci. Out of all PEGylated derivatives, VAN:PEG1 and VAN:PEG3 were able to overcome vanC resistance. Biodistribution studies of the novel derivatives revealed significant changes in pharmacokinetics when compared with vancomycin. In conclusion, linker modification of vancomycin-polycationic peptide conjugates represents a promising strategy for the modulation of pharmacokinetic behavior while providing potent antimicrobial activity.
Mouse H-Y-specific and I-Ab restricted T-cell clones have been established and compared for their helper effects in the differentiation ofboth T and B Iymphocytes. The results demonstrate that three individual T -cell clones and one subclone could help in the antigen-driven induction of cytotoxic Iymphocytes (CTL) from their precursor cells (CTL-P), and were able to activate B cells to develop into antibody-secreting cells (PFC) in the presence of SRBC, provided the cloned T cells were restimulated by H-Y antigen on antigen-presenting cells. In addition, antigen or lectin could induce the same H -Y -specific T -cell clones to secrete factor(s) expressing helper activities similar to that ofthe cloned T cells. Furthermore, it is shown that the T cell-derived soluble mediator(s) was distinct from T-cell growth factor (TCGF) and from immune interferon (lFN-y). The data reveal a new type ofT cell with helper potential for the activation ofCTL-P and B Iymphocytes, and suggest the existence of distinct T helper cells which can provide help for both cytotoxic and antibody responses by virtue of different Iymphokine activities.
H-Y-specific and H-2Db-restricted, Lyt-1 "2+ T-cell clones (CTLL) with graded specific cytotoxic activities on male C57BL/6 (B6) target cells (1E3, +++; 2C5, ++; 2A5, +, 3E6, ±) were tested for their capacity to inhibit the generation of H-Y-specific cytotoxic T lymphocytes (CTL) in vitro. Addition of irradiated lymphocytes of CTLL 1E3 and CTLL 3E6 but not those of CTLL 2A5 or CTLL 2C5 abolished the generation of CTL from in vivo primed H-Y-specific precursor cells (CTLP) when added to fresh mixed-lymphocyte cultures (MLC). Exogenous sources of T-cell growth factors (TCGF) did not overcome suppression. Rather the presence of TCGF resulted in a further enhancement of suppressive activities in CTLL 1E3 and 3E6 and the induction of similar activities in cells from CTLL 2A5 and 2C5, which by themselves were not inhibitory. Moreover when added to similar MLC on Day 1 instead of Day 0, only irradiated cells of CTLL 3E6 but not those of the other three CTLL were suppressive. Induction of suppressive activities in H-Y-specific CTLL was independent of the appropriate male stimulator cells since it was also observed in MLC induced by irrelevant antigens (H-2, trinitrophenol). Furthermore at low cell numbers, irradiated lymphocytes from any of the CTLL consistently enhanced CTL activities generated from H-Y-specific CTLP. This augmenting activity, which was not TCGF, could be transferred by soluble mediators present in antigen-sensitized CTLL cultures. Thus, these data indicate (i) that cytotoxic effector cells can function as suppressor cells in the generation of CTL, (ii) that the cytotoxic activity of cloned CTL does not correlate with their capacity to suppress CTL responses, (iii) that the inhibition of CTL responses by CTLL is not due to simple consumption of T-cell growth factors produced in MLC, and (iv) that different CTL clones may interfere with the generation of CTL at different stages of their maturation. Moreover, the experiments suggest an antigen-independent enhancement of suppression by the interaction of CTL with lymphokines. Together with the augmenting activity evoked by cloned CTL the data provide strong evidence for the expression of multiple immunological functions by one particular subset of T cells and suggest that cytotoxic effector cells can differentially regulate the maturation and/or clonal expression of their precursor cells.
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!.
Background
Oncolytic virotherapy of tumors is an up-coming, promising therapeutic modality of cancer therapy. Unfortunately, non-invasive techniques to evaluate the inflammatory host response to treatment are rare. Here, we evaluate \(^{19}\)F magnetic resonance imaging (MRI) which enables the non-invasive visualization of inflammatory processes in pathological conditions by the use of perfluorocarbon nanoemulsions (PFC) for monitoring of oncolytic virotherapy.
Methodology/Principal Findings
The Vaccinia virus strain GLV-1h68 was used as an oncolytic agent for the treatment of different tumor models. Systemic application of PFC emulsions followed by \(^1H\)/\(^{19}\)F MRI of mock-infected and GLV-1h68-infected tumor-bearing mice revealed a significant accumulation of the \(^{19}\)F signal in the tumor rim of virus-treated mice. Histological examination of tumors confirmed a similar spatial distribution of the \(^{19}\)F signal hot spots and \(CD68^+\)-macrophages. Thereby, the \(CD68^+\)-macrophages encapsulate the GFP-positive viral infection foci. In multiple tumor models, we specifically visualized early inflammatory cell recruitment in Vaccinia virus colonized tumors. Furthermore, we documented that the \(^{19}\)F signal correlated with the extent of viral spreading within tumors.
Conclusions/Significance
These results suggest \(^{19}\)F MRI as a non-invasive methodology to document the tumor-associated host immune response as well as the extent of intratumoral viral replication. Thus, \(^{19}\)F MRI represents a new platform to non-invasively investigate the role of the host immune response for therapeutic outcome of oncolytic virotherapy and individual patient response.
Converging evidence suggests a role of serotonin (5-hydroxytryptamine, 5-HT) and tryptophan hydroxylase 2 (TPH2), the rate-limiting enzyme of 5-HT synthesis in the brain, in modulating long-term, neurobiological effects of early-life adversity. Here, we aimed at further elucidating the molecular mechanisms underlying this interaction, and its consequences for socio-emotional behaviors, with a focus on anxiety and social interaction. In this study, adult, male Tph2 null mutant (Tph2\(^{-/-}\)) and heterozygous (Tph2\(^{+/-}\)) mice, and their wildtype littermates (Tph2\(^{+/+}\)) were exposed to neonatal, maternal separation (MS) and screened for behavioral changes, followed by genome-wide RNA expression and DNA methylation profiling. In Tph2\(^{-/-}\) mice, brain 5-HT deficiency profoundly affected socio-emotional behaviors, i.e., decreased avoidance of the aversive open arms in the elevated plus-maze (EPM) as well as decreased prosocial and increased rule breaking behavior in the resident-intruder test when compared to their wildtype littermates. Tph2\(^{+/-}\) mice showed an ambiguous profile with context-dependent, behavioral responses. In the EPM they showed similar avoidance of the open arm but decreased prosocial and increased rule breaking behavior in the resident-intruder test when compared to their wildtype littermates. Notably, MS effects on behavior were subtle and depended on the Tph2 genotype, in particular increasing the observed avoidance of EPM open arms in wildtype and Tph2\(^{+/-}\) mice when compared to their Tph2\(^{-/-}\) littermates. On the genomic level, the interaction of Tph2 genotype with MS differentially affected the expression of numerous genes, of which a subset showed an overlap with DNA methylation profiles at corresponding loci. Remarkably, changes in methylation nearby and expression of the gene encoding cholecystokinin, which were inversely correlated to each other, were associated with variations in anxiety-related phenotypes. In conclusion, next to various behavioral alterations, we identified gene expression and DNA methylation profiles to be associated with TPH2 inactivation and its interaction with MS, suggesting a gene-by-environment interaction-dependent, modulatory function of brain 5-HT availability.
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.
The Vpr protein from type 1 and type 2 Human Immunodeficiency Viruses (HIV-1 and HIV-2) is thought to inactivate several host proteins through the hijacking of the DCAF1 adaptor of the Cul4A ubiquitin ligase. Here, we identified two transcriptional regulators, ZIP and sZIP, as Vpr-binding proteins degraded in the presence of Vpr. ZIP and sZIP have been shown to act through the recruitment of the NuRD chromatin remodeling complex. Strikingly, chromatin is the only cellular fraction where Vpr is present together with Cul4A ubiquitin ligase subunits. Components of the NuRD complex and exogenous ZIP and sZIP were also associated with this fraction. Several lines of evidence indicate that Vpr induces ZIP and sZIP degradation by hijacking DCAF1: (i) Vpr induced a drastic decrease of exogenously expressed ZIP and sZIP in a dose-dependent manner, (ii) this decrease relied on the proteasome activity, (iii) ZIP or sZIP degradation was impaired in the presence of a DCAF1-binding deficient Vpr mutant or when DCAF1 expression was silenced. Vpr-mediated ZIP and sZIP degradation did not correlate with the growth-related Vpr activities, namely G2 arrest and G2 arrest-independent cytotoxicity. Nonetheless, infection with HIV-1 viruses expressing Vpr led to the degradation of the two proteins. Altogether our results highlight the existence of two host transcription factors inactivated by Vpr. The role of Vpr-mediated ZIP and sZIP degradation in the HIV-1 replication cycle remains to be deciphered.
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.
Campylobacter jejuni is currently the leading cause of bacterial gastroenteritis in humans. Comparison of multiple Campylobacter strains revealed a high genetic and phenotypic diversity. However, little is known about differences in transcriptome organization, gene expression, and small RNA (sRNA) repertoires. Here we present the first comparative primary transcriptome analysis based on the differential RNA–seq (dRNA–seq) of four C. jejuni isolates. Our approach includes a novel, generic method for the automated annotation of transcriptional start sites (TSS), which allowed us to provide genome-wide promoter maps in the analyzed strains. These global TSS maps are refined through the integration of a SuperGenome approach that allows for a comparative TSS annotation by mapping RNA–seq data of multiple strains into a common coordinate system derived from a whole-genome alignment. Considering the steadily increasing amount of RNA–seq studies, our automated TSS annotation will not only facilitate transcriptome annotation for a wider range of pro- and eukaryotes but can also be adapted for the analysis among different growth or stress conditions. Our comparative dRNA–seq analysis revealed conservation of most TSS, but also single-nucleotide-polymorphisms (SNP) in promoter regions, which lead to strain-specific transcriptional output. Furthermore, we identified strain-specific sRNA repertoires that could contribute to differential gene regulation among strains. In addition, we identified a novel minimal CRISPR-system in Campylobacter of the type-II CRISPR subtype, which relies on the host factor RNase III and a trans-encoded sRNA for maturation of crRNAs. This minimal system of Campylobacter, which seems active in only some strains, employs a unique maturation pathway, since the crRNAs are transcribed from individual promoters in the upstream repeats and thereby minimize the requirements for the maturation machinery. Overall, our study provides new insights into strain-specific transcriptome organization and sRNAs, and reveals genes that could modulate phenotypic variation among strains despite high conservation at the DNA level.
Extraintestinal pathogenic and intestinal pathogenic (diarrheagenic) Escherichia coli differ phylogenetically and by virulence profiles. Classic theory teaches simple linear descent in this species, where non-pathogens acquire virulence traits and emerge as pathogens. However, diarrheagenic Shiga toxin-producing E.coli (STEC) O2:H6 not only possess and express virulence factors associated with diarrheagenic and uropathogenic E.coli but also cause diarrhea and urinary tract infections. These organisms are phylogenetically positioned between members of an intestinal pathogenic group (STEC) and extraintestinal pathogenic E.coli. STEC O2:H6 is, therefore, a 'heteropathogen,' and the first such hybrid virulent E.coli identified. The phylogeny of these E.coli and the repertoire of virulence traits they possess compel consideration of an alternate view of pathogen emergence, whereby one pathogroup of E.coli undergoes phased metamorphosis into another. By understanding the evolutionary mechanisms of bacterial pathogens, rational strategies for counteracting their detrimental effects on humans can be developed.
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.
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.
The harlequin ladybird beetle Harmonia axyridis has been introduced in many countries as a biological control agent, but has become an invasive species threatening the biodiversity of native ladybirds. Its invasive success has been attributed to its vigorous resistance against diverse pathogens. This study demonstrates that harmonine ((17R,9Z)-1,17-diaminooctadec-9-ene), which is present in H. axyridis haemolymph, displays broad-spectrum antimicrobial activity that includes human pathogens. Antibacterial activity is most pronounced against fast-growing mycobacteria and Mycobacterium tuberculosis, and the growth of both chloroquine-sensitive and -resistant Plasmodium falciparum strains is inhibited. Harmonine displays gametocytocidal activity, and inhibits the exflagellation of microgametocytes and zygote formation. In an Anopheles stephensi mosquito feeding model, harmonine displays transmission-blocking activity.
Background: Despite availability of efficient treatment regimens for early stage colorectal cancer, treatment regimens for late stage colorectal cancer are generally not effective and thus need improvement. Oncolytic virotherapy using replication-competent vaccinia virus (VACV) strains is a promising new strategy for therapy of a variety of human cancers.
Methods: Oncolytic efficacy of replication-competent vaccinia virus GLV-1h68 was analyzed in both, cell cultures and subcutaneous xenograft tumor models.
Results: In this study we demonstrated for the first time that the replication-competent recombinant VACV GLV-1h68 efficiently infected, replicated in, and subsequently lysed various human colorectal cancer lines (Colo 205, HCT-15, HCT-116, HT-29, and SW-620) derived from patients at all four stages of disease. Additionally, in tumor xenograft models in athymic nude mice, a single injection of intravenously administered GLV-1h68 significantly inhibited tumor growth of two different human colorectal cell line tumors (Duke’s type A-stage HCT-116 and Duke’s type C-stage SW-620), significantly improving survival compared to untreated mice. Expression of the viral marker gene ruc-gfp allowed for real-time analysis of the virus infection in cell cultures and in mice. GLV-1h68 treatment was well-tolerated in all animals and viral replication was confined to the tumor. GLV-1h68 treatment elicited a significant up-regulation of murine immune-related antigens like IFN-γ, IP-10, MCP-1, MCP-3, MCP-5, RANTES and TNF-γ and a greater infiltration of macrophages and NK cells in tumors as compared to untreated controls.
Conclusion: The anti-tumor activity observed against colorectal cancer cells in these studies was a result of direct viral oncolysis by GLV-1h68 and inflammation-mediated innate immune responses. The therapeutic effects occurred in tumors regardless of the stage of disease from which the cells were derived. Thus, the recombinant vaccinia virus GLV-1h68 has the potential to treat colorectal cancers independently of the stage of progression.