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  • 1
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 06 March 2018, Vol.115(10), pp.E2376-E2385
    Description: The facultative human pathogen changes its transcriptional profile upon oral ingestion by the host to facilitate survival and colonization fitness. Here, we used a modified version of recombination-based in vivo expression technology to investigate gene silencing during the in vivo passage, which has been understudied. Using a murine model of cholera, we screened a transposon library composed of 10,000 randomly generated reporter fusions and identified 101 in vivo repressed () genes. Our data indicate that constitutive expression of genes reduces colonization fitness, highlighting the necessity to down-regulate these genes in vivo. For example, the gene , encoding an H/Cl transporter, could be linked to the acid tolerance response against hydrochloric acid. In a chloride-dependent manner, ClcA facilitates survival under low pH (e.g., the stomach), but its presence becomes detrimental under alkaline conditions (e.g., lower gastrointestinal tract). This pH-dependent expression is controlled by the LysR-type activator AphB, which acts in concert with AphA to initiate the virulence cascade in after oral ingestion. Thus, transcriptional networks dictating induction of virulence factors and the repression of genes overlap to regulate in vivo colonization dynamics. Overall, the results presented herein highlight the impact of spatiotemporal gene silencing in vivo. The molecular characterization of the underlying mechanisms can provide important insights into in vivo physiology and virulence network regulation.
    Keywords: Vibrio Cholerae ; Acid Tolerance Response ; Cholera ; in Vivo Expression Technology ; Murine Model ; Antiporters -- Metabolism ; Bacterial Proteins -- Metabolism ; Cholera -- Microbiology ; Gastrointestinal Tract -- Microbiology ; Vibrio Cholerae -- Metabolism
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 2
    Language: English
    In: 2012, Vol.7(8), p.e42664
    Description: Haemophilus influenzae is a Gram-negative human-restricted bacterium that can act as a commensal and a pathogen of the respiratory tract. Especially nontypeable H. influenzae (NTHi) is a major threat to public health and is responsible for several infectious diseases in humans, such as pneumonia, sinusitis, and otitis media. Additionally, NTHi strains are highly associated with exacerbations in patients suffering from chronic obstructive pulmonary disease. Currently, there is no licensed vaccine against NTHi commercially available. Thus, this study investigated the utilization of outer membrane vesicles (OMVs) as a potential vaccine candidate against NTHi infections. We analyzed the immunogenic and protective properties of OMVs derived from various NTHi strains by means of nasopharyngeal immunization and colonization studies with BALB/c mice. The results presented herein demonstrate that an intranasal immunization with NTHi OMVs results in a robust and complex humoral and mucosal immune response. Immunoprecipitation revealed the most important immunogenic proteins, such as the heme utilization protein, protective surface antigen D15, heme binding protein A, and the outer membrane proteins P1, P2, P5 and P6. The induced immune response conferred not only protection against colonization with a homologous NTHi strain, which served as an OMV donor for the immunization mixtures, but also against a heterologous NTHi strain, whose OMVs were not part of the immunization mixtures. These findings indicate that OMVs derived from NTHi strains have a high potential to act as a vaccine against NTHi infections.
    Keywords: Research Article ; Biology ; Medicine ; Genetics And Genomics ; Immunology ; Infectious Diseases ; Microbiology
    E-ISSN: 1932-6203
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  • 3
    Language: English
    In: 2012, Vol.7(10), p.e47756
    Description: Virulence factor production in Vibrio cholerae is complex, with ToxRS being an important part of the regulatory cascade. Additionally, ToxR is the transcriptional regulator for the genes encoding the major outer membrane porins OmpU and OmpT. ToxR is a transmembrane protein and contains two cysteine residues in the periplasmic domain. This study addresses the influence of the thiol-disulfide oxidoreductase system DsbAB, ToxR cysteine residues and ToxR/ToxS interaction on ToxR activity. The results show that porin production correlates with ToxR intrachain disulfide bond formation, which depends on DsbAB. In contrast, formation of ToxR intrachain or interchain disulfide bonds is dispensable for virulence factor production and in vivo colonization. This study further reveals that in the absence of ToxS, ToxR interchain disulfide bond formation is facilitated, whereat cysteinyl dependent homo- and oligomerization of ToxR is suppressed if ToxS is coexpressed. In summary, new insights into gene regulation by ToxR are presented, demonstrating a mechanism by which ToxR activity is linked to a DsbAB dependent intrachain disulfide bond formation.
    Keywords: Research Article ; Biology ; Medicine ; Infectious Diseases ; Microbiology ; Biochemistry
    E-ISSN: 1932-6203
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  • 4
    In: Molecular Microbiology, September 2013, Vol.89(5), pp.816-830
    Description: Multi‐drug resistant strains of are increasingly being isolated in hospitals worldwide. Among the virulence factors identified in this bacterium there is a general ‐glycosylation system that appears to be important for biofilm formation and virulence, and the capsular polysaccharide, which is essential for resistance to complement killing. In this work, we identified a locus that is responsible for the synthesis of the ‐pentasaccharide found on the glycoproteins. Besides the enzymes required for the assembly of the glycan, additional proteins typically involved in polymerization and transport of capsule were identified within or adjacently to the locus. Mutagenesis of , the initiating glycosyltransferase prevented the synthesis of both glycoproteins and capsule, resulting in abnormal biofilm structures and attenuated virulence in mice. These results, together with the structural analysis of 17978 capsular polysaccharide via , demonstrated that the pentasaccharides that decorate the glycoproteins are also the building blocks for capsule biosynthesis. Two linked subunits, but not longer glycan chains, were detected on proteins via . The discovery of a bifurcated pathway for ‐glycosylation and capsule synthesis not only provides insight into the biology of but also identifies potential novel candidates for intervention against this emerging pathogen.
    Keywords: Polysaccharides -- Physiological Aspects ; Polysaccharides -- Analysis ; Enzymes -- Physiological Aspects ; Enzymes -- Analysis ; Glycoproteins -- Physiological Aspects ; Glycoproteins -- Analysis ; Polymerization -- Physiological Aspects ; Polymerization -- Analysis;
    ISSN: 0950-382X
    E-ISSN: 1365-2958
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  • 5
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 09 September 2014, Vol.111(36), pp.13181-6
    Description: Antibiotic therapy disrupts the human intestinal microbiota. In some patients rapid overgrowth of the enteric bacterium Klebsiella oxytoca results in antibiotic-associated hemorrhagic colitis (AAHC). We isolated and identified a toxin produced by K. oxytoca as the pyrrolobenzodiazepine tilivalline and demonstrated its causative action in the pathogenesis of colitis in an animal model. Tilivalline induced apoptosis in cultured human cells in vitro and disrupted epithelial barrier function, consistent with the mucosal damage associated with colitis observed in human AAHC and the corresponding animal model. Our findings reveal the presence of pyrrolobenzodiazepines in the intestinal microbiota and provide a mechanism for colitis caused by a resident pathobiont. The data link pyrrolobenzodiazepines to human disease and identify tilivalline as a target for diagnosis and neutralizing strategies in prevention and treatment of colitis.
    Keywords: Bacteria ; Cytotoxin ; Enteric Microbiota ; Anti-Bacterial Agents -- Adverse Effects ; Benzodiazepinones -- Toxicity ; Colitis -- Chemically Induced ; Enterotoxins -- Toxicity ; Peptides -- Toxicity
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 6
    Language: English
    In: Journal of bacteriology, April 2013, Vol.195(8), pp.1800-8
    Description: The facultative human pathogen Vibrio cholerae transits between the gastrointestinal tract of its host and aquatic reservoirs. V. cholerae adapts to different situations by the timely coordinated expression of genes during its life cycle. We recently identified a subclass of genes that are induced at late stages of infection. Initial characterization demonstrated that some of these genes facilitate the transition of V. cholerae from host to environmental conditions. Among these genes are uptake systems lacking detailed characterization or correct annotation. In this study, we comprehensively investigated the function of the VCA0682-to-VCA0687 gene cluster, which was previously identified as in vivo induced. The results presented here demonstrate that the operon encompassing open reading frames VCA0685 to VCA0687 encodes an ABC transport system for hexose-6-phosphates with Km values ranging from 0.275 to 1.273 μM for glucose-6P and fructose-6P, respectively. Expression of the operon is induced by the presence of hexose-6P controlled by the transcriptional activator VCA0682, representing a UhpA homolog. Finally, we provide evidence that the operon is essential for the utilization of hexose-6P as a C and P source. Thereby, a physiological role can be assigned to hexose-6P uptake, which correlates with increased fitness of V. cholerae after a transition from the host into phosphate-limiting environments.
    Keywords: Carbon -- Metabolism ; Gene Expression Regulation, Bacterial -- Physiology ; Hexosephosphates -- Metabolism ; Monosaccharide Transport Proteins -- Metabolism ; Phosphates -- Metabolism ; Vibrio Cholerae -- Metabolism
    ISSN: 00219193
    E-ISSN: 1098-5530
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  • 7
    Language: English
    In: 2014, Vol.9(7), p.e102664
    Description: Transcutaneous immunization (TCI) approaches utilize skin associated lymphatic tissues to elicit specific immune responses. In this context, the imidazoquinoline derivative imiquimod formulated in Aldara applied onto intact skin together with a cytotoxic T lymphocyte (CTL) epitope induces potent CTL responses. However, the feasibility and efficacy of the commercial imiquimod formulation Aldara is limited by its physicochemical properties as well as its immunogenicity. ; To overcome these obstacles, we developed an imiquimod-containing emulsion gel (IMI-Gel) and characterized it in comparison to Aldara for rheological properties and mouse skin permeation in a Franz diffusion cell system. Imiquimod was readily released from Aldara, while IMI-Gel showed markedly decreased drug release. Nevertheless, comparing vaccination potency of Aldara or IMI-Gel-based TCI in C57BL/6 mice against the model cytotoxic T-lymphocyte epitope SIINFEKL, we found that IMI-Gel was equally effective in terms of the frequency of peptide-specific T-cells and cytolytic activity. Importantly, transcutaneous delivery of IMI-Gel for vaccination was clearly superior to the subcutaneous or oral route of administration. Finally, IMI-Gel based TCI was at least equally effective compared to Aldara-based TCI in rejection of established SIINFEKL-expressing E.G7 tumors in a therapeutic setup indicated by enhanced tumor rejection and survival. ; In summary, we developed a novel imiquimod formulation with feasible pharmaceutical properties and immunological efficacy that fosters the rational design of a next generation transcutaneous vaccination platform suitable for the treatment of cancer or persistent virus infections.
    Keywords: Research Article ; Biology And Life Sciences ; Medicine And Health Sciences ; Physical Sciences ; Research And Analysis Methods
    E-ISSN: 1932-6203
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  • 8
    Language: English
    In: Infection and Immunity, 2010, Vol. 78(10), p.4402
    Description: Vibrio cholerae is the causative agent of cholera, a severe diarrheal disease that remains endemic in many parts of the world and can cause outbreaks wherever sanitation and clean water systems break down. Prevention of disease could be achieved through improved sanitation and clean water provision supported by vaccination. V. cholerae serogroup O1 is the major cause of cholera; O1 serotypes Inaba and Ogawa have similar disease burdens, while O139 is the only non-O1 serogroup to cause epidemics. We showed previously that immunization of adult female mice with purified V. cholerae outer membrane vesicles (OMVs) elicits an antibody response that protect neonates from oral V. cholerae challenge and that suckling from an immunized dam accounts for the majority of protection from V. cholerae colonization. Here we report that lipopolysaccharide (LPS) is the major OMV protective antigen. Mucosal immunization with OMVs from Inaba or Ogawa provides significant cross-serotype protection from V. cholerae colonization, although serotype-specific antigens are dominant. OMVs from O1 or O139 do not provide cross-serogroup protection, but by immunization with a mixture of O1 and O139 OMVs, cross-serogroup protection was achieved. Neonatal protection is not associated with significant bacterial death but may involve inhibition of motility, as antibodies from OMV-immunized mice inhibit V. cholerae motility in vitro, with trends that parallel in vivo protection. Motility assays also reveal that a higher antibody titer is required to immobilize O139 compared to O1, a phenotype that is O139 capsule dependent.
    Keywords: Immunity, Maternally-Acquired ; Cholera -- Prevention & Control ; Cholera Vaccines -- Immunology ; Immunoglobulins -- Immunology ; Secretory Vesicles -- Immunology ; Vibrio Cholerae -- Immunology;
    ISSN: 0019-9567
    ISSN: 00199567
    E-ISSN: 10985522
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  • 9
    Language: English
    In: Medical Microbiology and Immunology, 2012, Vol.201(4), pp.463-473
    Description: Activation of CD8 + cytotoxic T cells is crucial for the adaptive immune response against viral infections and the control of malignant transformed cells. Together with activation of costimulatory molecules like CD3 and CD28, CD8 + T cells need activation of their unique T cell receptor via recognition of foreign peptide epitopes in combination with major histocompatibility complexes class I on the cell surface of professional antigen-presenting cells. Presentation of pathogen-associated proteins is the result of a complex proteolytic process. It starts with the breakdown of proteins by a cytosolic endopeptidase, the proteasome, and is continued by subsequent N-terminal trimming events in the cytosol and/or the endoplasmic reticulum. Analysis of the proteolytic aminopeptidase activity in the former cellular compartment showed that the cytosol harbors a multitude of aminopeptidases that have singular specificities, but on the other hand also show redundancy in the trimming of N-terminal residues. The observed pattern of the overall trimming in the cytosol is reflected by the activity of the four identified aminopeptidases, and the administration of protease inhibitors made it possible to assign specificity of cleaving of proteinogenic amino acids to one or more identified aminopeptidase. The only exception was the cleavage of aspartic acid, which is performed by one yet unidentified enzyme.
    Keywords: Aminopeptidase ; Cytosol ; PSA ; BH ; Aminopeptidase B ; LTAH
    ISSN: 0300-8584
    E-ISSN: 1432-1831
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  • 10
    Language: English
    In: Infection and immunity, July 2013, Vol.81(7), pp.2379-93
    Description: The causative agent of the life-threatening gastrointestinal infectious disease cholera is the Gram-negative, facultative human pathogen Vibrio cholerae. We recently started to investigate the potential of outer membrane vesicles (OMVs) derived from V. cholerae as an alternative approach for a vaccine candidate against cholera and successfully demonstrated the induction of a long-lasting, high-titer, protective immune response upon immunization with OMVs using the mouse model. In this study, we present immunization data using lipopolysaccharide (LPS)-modified OMVs derived from V. cholerae, which allowed us to improve and identify the major protective antigen of the vaccine candidate. Our results indicate that reduction of endotoxicity can be achieved without diminishing the immunogenic potential of the vaccine candidate by genetic modification of lipid A. Although the protective potential of anti-LPS antibodies has been suggested many times, this is the first comprehensive study that uses defined LPS mutants to characterize the LPS-directed immune response of a cholera vaccine candidate in more detail. Our results pinpoint the O antigen to be the essential immunogenic structure and provide a protective mechanism based on inhibition of motility, which prevents a successful colonization. In a detailed analysis using defined antisera, we can demonstrate that only anti-O antigen antibodies, but not antibodies directed against the major flagellar subunit FlaA or the most abundant outer membrane protein, OmpU, are capable of effectively blocking the motility by binding to the sheathed flagellum and provide protection in a passive immunization assay.
    Keywords: Cholera -- Prevention & Control ; Cholera Vaccines -- Immunology ; Lipid A -- Immunology ; O Antigens -- Immunology ; Vibrio Cholerae -- Immunology
    E-ISSN: 1098-5522
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