Outer membrane vesicle-mediated release of cytolethal distending toxin (CDT) from Campylobacter jejuni

<p>Abstract</p> <p>Background</p> <p>Background: Cytolethal distending toxin (CDT) is one of the well-characterized virulence factors of <it>Campylobacter jejuni</it>, but it is unknown how CDT becomes surface-exposed or is released from the bacterium to the surrounding environment.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>Our results strongly suggest that the release of outer membrane vesicles is functioning as a route of <it>C. jejuni </it>to deliver all the subunits of CDT toxin (CdtA, CdtB, and CdtC) to the surrounding environment, including infected host tissue.</p

Quorum Sensing Regulation of the Two hcp Alleles in Vibrio cholerae O1 Strains

BACKGROUND: The type VI secretion system (T6SS) has emerged as a protein secretion system important to several gram-negative bacterial species. One of the common components of the system is Hcp, initially described as a hemolysin co-regulated protein in a serotype O17 strain of Vibrio cholerae. Homologs to V. cholerae hcp genes have been found in all characterized type VI secretion systems and they are present also in the serotype O1 strains of V. cholerae that are the cause of cholera diseases but seemed to have non-functional T6SS. METHODOLOGY/PRINCIPAL FINDINGS: The serotype O1 V. cholerae strain A1552 was shown to express detectable levels of Hcp as determined by immunoblot analyses using polyclonal anti-Hcp antiserum. We found that the expression of Hcp was growth phase dependent. The levels of Hcp in quorum sensing deficient mutants of V. cholerae were compared with the levels in wild type V. cholerae O1 strain A1552. The expression of Hcp was positively and negatively regulated by the quorum sensing regulators HapR and LuxO, respectively. In addition, we observed that expression of Hcp was dependent on the cAMP-CRP global transcriptional regulatory complex and required the RpoN sigma factor. CONCLUSION/SIGNIFICANCE: Our results show that serotype O1 strains of V. cholerae do express Hcp which is regarded as one of the important T6SS components and is one of the secreted substrates in non-O1 non-O139 V. cholerae isolates. We found that expression of Hcp was strictly regulated by the quorum sensing system in the V. cholerae O1 strain. In addition, the expression of Hcp required the alternative sigma factor RpoN and the cAMP-CRP global regulatory complex. Interestingly, the environmental isolates of V. cholerae O1 strains that showed higher levels of the HapR quorum sensing regulator in comparison with our laboratory standard serotype O1 strain A1552 where also expressing higher levels of Hcp

Modulators of Vibrio cholerae predator interaction and virulence

Vibrio cholerae, the causal agent of cholera typically encodes two critical virulence factors: cholera toxin (CT), which is primarily responsible for the diarrhoeal purge, and toxin-co-regulated pilus (TCP), an essential colonisation factor. Nontoxigenic strains expressing TCP can efficiently acquire the CT gene through lysogenic conversion with CTXΦ, a filamentous phage that encodes CT and uses TCP as a receptor.  V. cholerae is a Gram-negative bacterium and a natural inhabitant of estuarine and coastal waters throughout both temperate and tropical regions of the world. In the aquatic environment, V. cholerae encounters several environmental stresses, such as change in salinity, UV stress, nutrient limitation, temperature fluctuations, viral infections and protozoan predation. To fully understand the pathogenic and virulence potential of V. cholerae, knowledge is required of its interactions with, not only human, but also environmental factors. By using the nematode Caenorhabditis elegans as host model, we were able to identify a previously uncharacterised protein, the extracellular protease PrtV. PrtV was shown to be required for the killing of. elegans and also necessary for survival from grazing by the ciliate Tetrahymena pyriformis and the flagellate Cafeteria roenbergensis. The PrtV protein, which belongs to a M6 family of metallopeptidases was cloned and purified for further characterisations. The purified PrtV was cytotoxic against the human intestinal cell line HCT8. By using human blood plasma, fibrinogen, fibronectin and plasminogen were identified as candidate substrates for the PrtV protease. Outer membrane vesicles (OMVs) are released to the surroundings by most Gram-negative bacteria through “bulging and pinching” of the outer membrane.  OMVs have been shown to contain many virulence factors important in pathogenesis. Therefore, we investigated the association of PrtV with OMVs. PrtV was not associated with OMVs from the wild type O1 strain. In contrast, in an LPS mutant lacking two sugar chains in the core oligosaccharide PrtV was found to be associated with the OMVs. The OMV-associated PrtV was shown to be proteolytically and cytotoxically active. V. cholerae strains are grouped into &gt;200 serogroups. Only the O1 and O139 serogroups have been associated with pandemic cholera, a severe diarrhoeal disease.  All other serogroups are collectively referred to as non-O1 non-O139 V. cholerae. Non-O1 non-O139 V. cholerae can cause gastroenteritis and extraintestinal infections, but unlike O1 and O139 strains of V. cholerae, little is known about the virulence gene content and their potential to become human pathogens. We analysed clinical and environmental non-O1 non-O139 isolates for their putative virulence traits. None of them carry the genes encoding CT or the TCP, but other putative virulence factors were present in these isolates. The incidence of serum resistance was found to vary considerably and was independent of encapsulation. Three strains were strongly serum-resistant, and these same strains could also kill C. elegans

Analysis of Hcp stability in <i>V. cholerae</i> O1 wild type strain A1552.

<p>(A) The growth curve and time of sampling for Hcp analyses of <i>V. cholerae</i> wild type strain A1552 with and without chloramphenicol (Cm) treatment. The bacterial cells were grown to OD 2.0 and 25 µg/ml Cm was added. Arrows indicate the time points of sampling for immunoblot analysis after the addition of Cm. (B) Immunoblot analysis of the stability of Hcp in <i>V. cholerae</i> wild type strain A1552. The samples were taken at different time points after the addition of Cm (for the test sample). For the control experiment, the samples were taken at different time points during normal growth of bacteria. The vertical arrows show the time points when the samples were taken.</p

Bacterial strains and plasmids.

<p>Bacterial strains and plasmids.</p

Levels of Hcp and HapR in different <i>V. cholerae</i> O1 isolates.

<p>(A) Immunoblot analysis of the expression of the Hcp in different <i>V. cholerae</i> O1 isolates was performed using anti-Hcp antiserum. (B) Detection of the HapR protein in same samples used for the detection of the Hcp. Samples were taken at OD 2.0. (-); negative controls for Hcp (Δ<i>hcp</i>1,2) and HapR (Δ<i>hapR</i>)</p

Primers used in this study.

<p>Primers used in this study.</p

The effect of Δ<i>hapR</i> and Δ<i>luxO</i> mutations on Hcp levels in <i>V. cholerae</i> strain A1552.

<p>The whole cell lysate samples were taken at OD 1.0 and OD 2.0 and immunoblot analysis was performed using anti-Hcp polyclonal antiserum.</p

Hcp levels in Δ<i>crp</i> and Δ<i>cya</i> mutants of <i>V. cholerae</i> strain 1552.

<p>Immunoblot analysis of Hcp levels in whole cell lysates of <i>V. cholerae</i> O1 wild type strain A1552, Δ<i>crp</i>, Δ<i>cya</i>, Δ<i>crp</i>/p<i>crp</i>, and Δ<i>crp</i>/vector control strains. The whole cell lysates were taken at OD 1.0 and OD 2.0 and immunoblot was done using anti-Hcp antiserum.</p