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- Good, James A. D., 1985-, et al.
(författare)
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Attenuating Listeria monocytogenes virulence by targeting the regulatory protein PrfA
- 2016
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Ingår i: Cell chemical biology. - : Elsevier BV. - 2451-9448 .- 2451-9456. ; 23:3, s. 404-414
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Tidskriftsartikel (refereegranskat)abstract
- The transcriptional activator PrfA, a member of the Crp/Fnr family, controls the expression of some key virulence factors necessary for infection by the human bacterial pathogen Listeria monocytogenes. Phenotypic screening identified ring-fused 2-pyridone molecules that at low micromolar concentrations attenuate L. monocytogenes infectivity by reducing the expression of virulence genes, without compromising bacterial growth. These inhibitors bind the transcriptional regulator PrfA and decrease its affinity for the consensus DNA binding site. Structural characterization of this interaction revealed that one of the ring-fused 2-pyridones, compound 1, binds within a hydrophobic pocket, located between the C- and N-terminal domains of PrfA, and interacts with residues important for PrfA activation. This indicates that these inhibitors maintain the DNA-binding helix-turn-helix motif of PrfA in a disordered state, thereby preventing a PrfA:DNA interaction. Ring-fused 2-pyridones represent a new class of chemical probes for studying virulence in L. monocytogenes.
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| 2. |
- Lindmark, Barbro, 1963-, et al.
(författare)
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Outer membrane vesicle-mediated release of cytolethal distending toxin (CDT) from Campylobacter jejuni
- 2009
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Ingår i: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 16:9, s. 220-
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Cytolethal distending toxin (CDT) is one of the well-characterized virulence factors of Campylobacter jejuni, but it is unknown how CDT becomes surface-exposed or is released from the bacterium to the surrounding environment.RESULTS: Our data suggest that CDT is secreted to the bacterial culture supernatant via outer membrane vesicles (OMVs) released from the bacteria. All three subunits (the CdtA, CdtB, and CdtC proteins) were detected by immunogold labeling and electron microscopy of OMVs. Subcellular fractionation of the bacteria indicated that, apart from the majority of CDT detected in the cytoplasmic compartment, appreciable amounts (20-50%) of the cellular pool of CDT proteins were present in the periplasmic compartment. In the bacterial culture supernatant, we found that a majority of the extracellular CDT was tightly associated with the OMVs. Isolated OMVs could exert the cell distending effects typical of CDT on a human intestinal cell line, indicating that CDT is present there in a biologically active form.CONCLUSION: Our results strongly suggest that the release of outer membrane vesicles is functioning as a route of C. jejuni to deliver all the subunits of CDT toxin (CdtA, CdtB, and CdtC) to the surrounding environment, including infected host tissue.
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| 4. |
- Vaitkevicius, Karolis, 1979-
(författare)
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Effects of Vibrio cholerae protease and pigment production on environmental survival and host interaction
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Only two out of more than 200 V. cholerae serogroups, classified on the basis of LPS structure, are associated with epidemic or pandemic cholera. These toxigenic serogroups carry phage-derived pathogenicity islands coding for the main virulence factors for establishment of cholera disease – cholera toxin (CTX) and toxin-coregulated pilus (TCP). The latter also serves as a bacterial surface receptor for the CTXΦ – the filamentous phage which carries the cholera toxin genes into otherwise harmless to human, environmental bacterium V. cholerae. In its natural aquatic habitat V. cholerae is subject to predator grazing, bacteriophage killing, temperature and pH changes, seasonality of plankton blooms and other environmental factors. Therefore understanding V. cholerae pathogenic and virulence potential requires the knowledge of its interaction not only with human host but also members of aquatic environment and environmental factors. V. cholerae is capable of killing the nematode Caenorhabditis elegans. Using a reverse genetics approach, we demonstrated that the quorum sensing regulated protease PrtV is essential for this killing. Other proteases did not seem to contribute to virulence in this model. The data from this study suggest that the PrtV could be important to V. cholerae in its natural niche for its resistance to the grazing predators. The PrtV protease belongs to an M6 family of metallopeptidases which is represented by an Immune Inhibitor A protease from the insect killing bacterium Bacillus thuringiensis. To characterize the protease in more detail, the PrtV was cloned, overexpressed in V. cholerae and purified from the culture supernatant. The enzyme was calcium stabilized and inhibited by metal ion chelators. In tests with in vitro cultured cells of the human intestinal cell line HCT8, the PrtV protein showed a cytotoxic effect leading to cell detachment and death. Using human blood plasma as a source of potential substrates, and by tests with purified candidate substrate proteins, we have identified fibrinogen (all α, β and γ chains), fibronectin and plasminogen to be degraded by the protease. Additionally, PrtV was found to alter the stability of V. cholerae cytolysin implicating its role in modulation of the reactogenicity of V. cholerae secreted factors. Pigmentation has been considered to be important in microbial pathogenesis because it has been associated with virulence in many microorganisms. Using transposon mutagenesis we identified the mutated locus of a pigment producing V. cholerae strain to encode a gene of a tyrosine catabolic pathway. The mutation in a putative homogentisate 1,2-dioxigenase gene lead to accumulation of homogentisic acid, its spontaneous oxidation and formation of a dark pigment. The pigment producing strain was altered in its ability to survive UV exposure and H2O2 stress, and was more efficient in colonizing the suckling mouse intestine compared to the wild type strain. Under the in vitro growth conditions the major virulence factor TcpA and CT expression was found to be somewhat enhanced too.
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