A Bistable Switch and Anatomical Site Control Vibrio cholerae Virulence Gene Expression in the Intestine

A fundamental, but unanswered question in host-pathogen interactions is the timing, localization and population distribution of virulence gene expression during infection. Here, microarray and in situ single cell expression methods were used to study Vibrio cholerae growth and virulence gene expression during infection of the rabbit ligated ileal loop model of cholera. Genes encoding the toxin-coregulated pilus (TCP) and cholera toxin (CT) were powerfully expressed early in the infectious process in bacteria adjacent to epithelial surfaces. Increased growth was found to co-localize with virulence gene expression. Significant heterogeneity in the expression of tcpA, the repeating subunit of TCP, was observed late in the infectious process. The expression of tcpA, studied in single cells in a homogeneous medium, demonstrated unimodal induction of tcpA after addition of bicarbonate, a chemical inducer of virulence gene expression. Striking bifurcation of the population occurred during entry into stationary phase: one subpopulation continued to express tcpA, whereas the expression declined in the other subpopulation. ctxA, encoding the A subunit of CT, and toxT, encoding the proximal master regulator of virulence gene expression also exhibited the bifurcation phenotype. The bifurcation phenotype was found to be reversible, epigenetic and to persist after removal of bicarbonate, features consistent with bistable switches. The bistable switch requires the positive-feedback circuit controlling ToxT expression and formation of the CRP-cAMP complex during entry into stationary phase. Key features of this bistable switch also were demonstrated in vivo, where striking heterogeneity in tcpA expression was observed in luminal fluid in later stages of the infection. When this fluid was diluted into artificial seawater, bacterial aggregates continued to express tcpA for prolonged periods of time. The bistable control of virulence gene expression points to a mechanism that could generate a subpopulation of V. cholerae that continues to produce TCP and CT in the rice water stools of cholera patients.     

Structure and secretion of CofJ,a putative colonization factor of enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) colonize the human gut, causing severe cholera‐like diarrhoea. ETEC utilize a diverse array of pili and fimbriae for host colonization, including the Type IVb pilus CFA/III. The CFA/III pilus machinery is encoded on the cof operon, which is similar in gene sequence and synteny to the tcp operon that encodes another Type IVb pilus, the Vibrio cholerae toxin co‐regulated pilus (TCP). Both pilus operons possess a syntenic gene encoding a protein of unknown function. In V. cholerae, this protein, TcpF, is a critical colonization factor secreted by the TCP apparatus. Here we show that the corresponding ETEC protein, CofJ, is a soluble protein secreted via the CFA/III apparatus. We present a 2.6 Å resolution crystal structure of CofJ, revealing a large β‐sandwich protein that bears no sequence or structural homology to TcpF. CofJ has a cluster of exposed hydrophobic side‐chains at one end and structural homology to the pore‐forming proteins perfringolysin O and α‐haemolysin. CofJ binds to lipid vesicles and epithelial cells, suggesting a role in membrane attachment during ETEC colonization.     

Comparison of the promoter proximal regions of the toxin-co-regulated tcp gene cluster in classical and E1 Tor strains of Vibrio cholerae O1

A physical map has been constructed of the 5-kb XbaI fragment encoding the promoter proximal of region the tcp gene cluster encoding the toxin-coregulated pilus (TCP) of Vibrio cholerae. This fragment contains the major regulatory regions for TCP. Comparison of the nucleotide (nt) sequences from strains of the classical and E1 Tor biotypes demonstrates that the regions are essentially identical, with several notable exceptions. The intergenic regions, between tcpI and tcpP, and between tcpH and tcpA, show significant sequence divergence which may account for the biotype-related differences in TCP, since this is the location of the major promoter sequences. The C-terminal coding regions of the major pilin subunit, TcpA, also differ. Southern hybridization analyses suggest that the tcpA nt sequence is conserved within a biotype, and Western blot analysis suggests that the two forms of TcpA are antigenically different, but related. Besides tcpA, tcpB, tcpH and tcpI, the genes encoding two additional proteins, TcpP and TcpQ, but not previously defined, were also identified. TcpH and TcpI have been previously suggested to be regulatory proteins but homology data imply that TcpI is a methyl-accepting chemotaxis protein (MCP), as recently reported [Harkey et al., Infect. Immun. 62 (1994) 2669-2678], and TcpH is predicted to be a periplasmic or exported protein. TcpP is thought to be a trans-cytoplasmic membrane (CM) protein which may have a regulatory role     

The tcp gene cluster of Vibrio cholerae

The toxin co-regulated pilus (TCP) has been identified as a critical colonization factor in both animal models and humans for Vibrio cholerae O1. The major pilin subunit, TcpA (and also TcpB), is similar to type-4 pilins but TCP probably more appropriately belongs to a sub-class which includes the bundle-forming pilus of enteropathogenic Escherichia coli. The genes for TCP biosynthesis and assembly are clustered with the exception of housekeeping functions such as TcpG (=DsbA, a periplasmic disulfide bond epimerase). The nt sequences from El Tor and classical strains show only minor differences corresponding to the major regulatory regions and in TcpA itself. These differences are thought to account for the alternate conditions required for expression of TCP by the two biotypes and the antigenic variation and lack of cross-protection. Aside from the TcpA only a few of the proteins have had their roles in TCP biogenesis defined. Regulation of TCP is controlled by the ToxR regulon via ToxT with a possible involvement of TcpP and the cAMP-CRP system. Experiments using the infant mouse cholera model have now shown that TCP is a colonization factor and protective antigen for both classical and El Tor O1 strains and in the O139 Bengal serotype and that the mannose-sensitive haemagglutinin pilus does not appear to play a comparable role.