Pathoadaptive Conditional Regulation of the Type VI Secretion System in Vibrio cholerae O1 Strains

Abstract

The most recently discovered secretion pathway in Gram-negative bacteria, the type VI secretion system (T6SS), is present in many species and is considered important for the survival of non-O1 non-O139 Vibrio cholerae in aquatic environments. Until now, it was not known whether there is a functionally active T6SS in wild-type V. cholerae O1 strains, the cause of cholera disease in humans. Here, we demonstrate the presence of a functionally active T6SS in wild-type V. cholerae O1 strains, as evidenced by the secretion of the T6SS substrate Hcp, which required several gene products encoded within the putative vas gene cluster. Our analyses showed that the T6SS of wild-type V. cholerae O1 strain A1552 was functionally activated when the bacteria were grown under high-osmolarity conditions. The T6SS was also active when the bacteria were grown under low temperature (23°C), suggesting that the system may be important for the survival of the bacterium in the environment. A test of the interbacterial virulence of V. cholerae strain A1552 against an Escherichia coli K-12 strain showed that it was strongly enhanced under high osmolarity and that it depended on the hcp genes. Interestingly, we found that the newly recognized osmoregulatory protein OscR plays a role in the regulation of T6SS gene expression and secretion of Hcp from V. cholerae O1 strains.T he severe diarrheal disease cholera is caused by Vibrio cholerae bacteria of serogroups O1 and O139 that carry the ctxAB and tcp genes, encoding cholera toxin and toxin-coregulated pili, respectively (22). These bacteria have been the cause of seven pandemics since 1817 (12,38,42). Non-O1, non-O139 V. cholerae (NOVC) constitutes a large group of V. cholerae bacteria found as environmental isolates. Typically, they lack the ctx and tcp genes and do not cause cholera but have recently been emerging as potential extraintestinal pathogens. NOVC isolates possess a protein secretion system, the type VI secretion system (T6SS), which appears to play an important role in the bacterium's environmental survival by promoting killing of predator organisms, like amoebas (37).The T6SS is present in several Gram-negative bacterial species ranging from environmental to pathogenic bacteria and is involved in a variety of cellular processes (3-5, 10, 13, 43). It can secrete certain effector proteins into the extracellular milieu and/or translocate them into the eukaryotic host cell cytoplasm (31,32,36,37). Intriguingly, a role for T6SS in interbacterial competition was recently described in Pseudomonas aeruginosa, Burkholderia thailandensis, and a NOVC isolate (19,24,44). Although little is known about the contribution of the T6SS to virulence in general, expression of T6SS loci is precisely modulated to adapt T6SS production to the specific needs of and conditions encountered by individual bacteria (3, 4). Two T6SS-associated proteins, hemolysin-coregulated protein (Hcp) and valine-glycine repeat protein G (VgrG), have recently been characterized in NOVC isolates and in P. aeruginosa (1...

Response of Vibrio cholerae to Low-Temperature Shifts: CspV Regulation of Type VI Secretion, Biofilm Formation, and Association with Zooplankton

“…3A and B). It was previously reported that Hcp is increased in cells grown at 23°C compared to levels at 37°C (48), further corroborating our results. A molecular mechanism by which hcp transcription is decreased at 15°C is yet to be determined.…”

The LonA Protease Regulates Biofilm Formation, Motility, Virulence, and the Type VI Secretion System in Vibrio cholerae

“…The genes coding for the T6SS apparatus are located in one main cluster (VCA0105 to VCA0124) and two auxiliary clusters (VCA0017 to VCA0022 and VC1415 to VC1421) (21,25). The genes of the T6SS are negatively regulated by the global regulator TsrA, the quorumsensing (QS) regulator LuxO, and the osmolarity-responsive transcriptional regulator OscR, and they are positively regulated by HapR, cyclic AMP receptor protein (CRP), response regulator VxrB, and VasH (26)(27)(28)(29)(30).…”

Screening for Inhibition of Vibrio cholerae VipA-VipB Interaction Identifies Small-Molecule Compounds Active against Type VI Secretion

“…To this end, the secretion of the very-well-characterized T6SS effector, Hcp, was analyzed. A normal VipA-VipB interaction is an absolute requirement for Hcp secretion since VipA variants that are unable to bind to VipB efficiently do not support secretion of Hcp (26,34). KS100, KS200, and the analogues were serially diluted to final concentrations of 50 M, 100 M, or 200 M and incubated with V. cholerae V52 for 4 h. Extracts from the cells and culture supernatants were examined by Western blotting, and a dose-dependent inhibition of Hcp secretion was observed ( Fig.…”

“…The VipA-VipB interaction is an absolute prerequisite for the function of the T6S, and, thus, it would be postulated that the inhibition of the interaction will lead to the abrogation of Hcp secretion or phospholipase activity, both shown to be important T6SS effector mechanisms (28,34). In fact, numerous studies have demonstrated not only a close link between T6SS and bacterial secretion, necessary for virulence, but also the structural roles of VipA, VipB, and Hcp in the needle-like structure required for secretion (2, 4, 13, 14, 27-29, 34, 39, 53, 60).…”

“…Thus, the coordinated functions of VipA, VipB, and ClpV are needed for active secretion. In most T6SSs, hemolysin-coregulated protein (Hcp) and valine-glycine repeat protein G (VgrG) are exported by the secretion machinery, but most likely numerous other proteins are secreted as well (30)(31)(32)(33)(34)(35)(36). The well-characterized Hcp protein is not only secreted but also suggested to be a chaperone that binds to cognate T6SS effector molecules, and it forms a hexameric pore that is required for secretion of diverse effectors encompassing several enzymatic classes (37)(38)(39).…”