General secretion pathway (eps) genes required for toxin secretion and outer membrane biogenesis in Vibrio cholerae

Abstract

The general secretion pathway (GSP) of Vibrio cholerae is required for secretion of proteins including chitinase, enterotoxin, and protease through the outer membrane. In this study, we report the cloning and sequencing of a DNA fragment from V. cholerae, containing 12 open reading frames, epsC to -N, which are similar to GSP genes of Aeromonas, Erwinia, Klebsiella, Pseudomonas, and Xanthomonas spp. In addition to the two previously described genes, epsE and epsM (M. Sandkvist, V. Morales, and M. Bagdasarian, Gene 123: 81-86, 1993; L. J. Overbye, M. Sandkvist, and M. Bagdasarian, Gene 132:101-106, 1993), it is shown here that epsC, epsF, epsG, and epsL also encode proteins essential for GSP function. Mutations in the eps genes result in aberrant outer membrane protein profiles, which indicates that the GSP, or at least some of its components, is required not only for secretion of soluble proteins but also for proper outer membrane assembly. Several of the Eps proteins have been identified by use of the T7 polymerase-promoter system in Escherichia coli. One of them, a pilin-like protein, EpsG, was analyzed also in V. cholerae and found to migrate as two bands on polyacrylamide gels, suggesting that in this organism it might be processed or otherwise modified by a prepilin peptidase. We believe that TcpJ prepilin peptidase, which processes the subunit of the toxin-coregulated pilus, TcpA, is not involved in this event. This is supported by the observations that apparent processing of EpsG occurs in a tcpJ mutant of V. cholerae and that, when coexpressed in E. coli, TcpJ cannot process EpsG although the PilD peptidase from Neisseria gonorrhoeae can.In Vibrio cholerae, the general secretion pathway (GSP) is required for extracellular secretion of several proteins including chitinase, cholera toxin (CT), and protease (51, 60). This pathway likely plays a significant role in the survival of Vibrio in different environments as well as in its pathogenicity. For instance, secreted chitinase and protease may assist in the detachment of V. cholerae from chitinous waterborne particles and from host epithelial cells, respectively, thus facilitating dissemination (14, 71). CT is the major component responsible for inducing the diarrheal disease cholera and is therefore also involved in the dissemination process (for a review, see reference 30).Escherichia coli heat-labile enterotoxin (LT) is very similar to CT in its structure, biochemical function, and ability to translocate across the outer membrane of V. cholerae (66). It is a multimeric protein which consists of a single A subunit and a pentamer of identical B-subunit polypeptides (18,64,65). Its secretion from V. cholerae proceeds in two steps, similar to the secretion of CT. The first step is translocation of the individual subunit polypeptides across the cytoplasmic membrane. This is mediated by a pathway which is presumably similar to the sec system of E. coli (63). During this step, the signal peptides of the A and B precursor polypeptides are cleaved off (8,25,52) a...

Directed polar secretion of protease from single cells of Vibrio cholerae via the type II secretion pathway

“…When GFP-EpsL and native EpsM were coexpressed from two different plasmids in the same E. coli cells, the level of GFP-EpsL fluorescence was increased (Fig. 1F), thus confirming the previous finding that showed that EpsM stabilizes EpsL and prevents its degradation (34). Furthermore, in the presence of EpsM, GFP-EpsL was detected at the cell poles in E. coli (Fig.…”

“…1A). GFP was also fused to the N terminus of the cytoplasmic membrane protein EpsM, which has previously been shown to interact with EpsL (34), to determine whether the polar location is common to other Eps proteins. Like GFP-EpsL, this fusion protein was also active and could restore extracellular secretion in a V. cholerae epsM mutant (not shown).…”

“…CT is transported in a two-step process, which first involves the translocation of the individual subunits across the cytoplasmic membrane via the Sec pathway (8). In the periplasmic compartment, the subunits assemble into the hexameric AB 5 complex, which is subsequently translocated across the outer membrane via the type II secretion pathway, encoded by the extracellular protein secretion genes (epsC-epsN) and vcpD (pilD) (9)(10)(11)(12)(13). This pathway is also responsible for extracellular secretion of other potential virulence factors that include hemagglutinin͞protease (HA͞protease), chitinase, neuraminidase, and lipase (11,14).…”

Identification of the Vibrio cholerae type 4 prepilin peptidase required for cholera toxin secretion and pilus formation

“…Often, the gene that encodes the type 4 prepilin peptidase is found at the end of the secretory locus. However, no prepilin peptidase has been identified within the eps gene cluster (Sandkvist et al, 1997). V. cholerae O1 is classified into two biotypes, classical and El Tor, based on several biochemical and phenotypic properties. One of these properties is the ability to mediate mannose-sensitive haemagglutination, an activity that is generally associated with the El Tor biotype but not with the classical biotype (Finkelstein and Mukerjee, 1963).…”

The genome sequence of Clostridium tetani , the causative agent of tetanus disease

“…Instead, a cluster of 10 genes (CTC1596-CTC1605) could be identified with similarities to subunits of type II secretion and type IV pilus biogenesis systems. Two ORFs (CTC1604, CTC1603) show strong similarities to the subunits EpsE and F of the type II secretion system in V. cholerae, which is responsible for the export of the cholera toxin (44). Three additional ORFs within the C. tetani cluster show some homology to EpsG, -H, and -I.…”