We identify and characterize a gene cluster in El Tor Vibrio cholerae that encodes a cytotoxic activity for HEp-2 cells in vitro. This gene cluster contains four genes and is physically linked to the cholera toxin (CTX) element in the V. cholerae genome. We demonstrate by using insertional mutagenesis that this gene cluster is required for the cytotoxic activity. The toxin, RtxA, resembles members of the RTX (repeats in toxin) toxin family in that it contains a GD-rich repeated motif. Like other RTX toxins, its activity depends on an activator, RtxC, and an associated ABC transporter system, RtxB and RtxD. In V. cholerae strains of the classical biotype, a deletion within the gene cluster removes rtxC and eliminates cytotoxic activity. Other strains, including those of the current cholera pandemic, contain a functional gene cluster and display cytotoxic activity. Thus, the RTX gene cluster in El Tor O1 and O139 strains might have contributed significantly to their emergence. Furthermore, the RTX toxin of V. cholerae may be associated with residual adverse properties displayed by certain live, attenuated cholera vaccines.Toxigenic strains of the Gram-negative bacterium Vibrio cholerae cause a life-threatening diarrheal disease that can kill its victims within hours of the onset of symptoms. The disease is endemic in the Indian subcontinent and continues to reemerge elsewhere in Asia, Africa, and the Americas with the incidence estimated to exceed 5 million cases each year (1-3). Although over 180 serogroups of V. cholerae exist, only the O1 and O139 serovars are known to cause epidemic cholera. The O1 serogroup can be divided further into two biotypes, classical and El Tor, based on various biochemical and phenotypic differences (4). Since 1817, seven cholera pandemics have occurred, of which the classical biotype is responsible for at least the fifth (1881-1896) and the sixth . The El Tor biotype is responsible for the seventh and current pandemic (1961-present). Toxigenic strains of O139 serovar appeared in India and Bangladesh in late 1992 as the first non-O1 serovar to cause epidemic cholera (5, 6). These strains are closely related and probably derived from toxigenic El Tor O1 strains (7). The factors that allowed El Tor biotype and O139 serotype to overtake the classical strains and establish pandemics remain a mystery.Work focusing on the pathogenesis of V. cholerae led to the identification of several key virulence factors, including the cholera toxin responsible for the diarrhea and the toxincoregulated pilus essential for colonizing the human small intestine (8, 9). Several groups tried to construct live, attenuated V. cholerae vaccines by deleting the genes for cholera toxin and several other putative accessory toxins (10-16). However, many of these strains remain reactogenic in human subjects, causing diarrhea or other significant symptoms (17).The virulence factors responsible for the residual reactogenicity remain to be identified.Bacterial genomics offers an opportunity to identify undiscov...

Enteric pathogens often export toxins that elicit diarrhea as a part of the etiology of disease, including toxins that affect cytoskeletal structure. Recently, we discovered that the intestinal pathogen Vibrio cholerae elicits rounding of epithelial cells that is dependent upon a gene we designated rtxA. Here we investigate the association of rtxA with the cell-rounding effect. We ®nd that V.cholerae exports a large toxin, RTX (repeats-in-toxin) toxin, to culture supernatant¯uids and that this toxin is responsible for cell rounding. Furthermore, we ®nd that cell rounding is not due to necrosis, suggesting that RTX toxin is not a typical member of the RTX family of pore-forming toxins. Rather, RTX toxin causes depolymerization of actin stress ®bers and covalent cross-linking of cellular actin into dimers, trimers and higher multimers. This RTX toxin-speci®c cross-linking occurs in cells previously rounded with cytochalasin D, indicating that G-actin is the toxin target. Although several models explain our observations, our simultaneous detection of actin cross-linking and depolymerization points toward a novel mechanism of action for RTX toxin, distinguishing it from all other known toxins.

Agrobacterium tumefaciens can genetically transform eukaryotic cells. In many bacteria, pili are required for interbacterial DNA transfer. The formation of pili by Agrobacterium required induction of tumor-inducing (Ti) plasmid-encoded virulence genes and growth at low temperature. A genetic analysis demonstrated that virA, virG, virB1 through virB11, and virD4 are the only Ti plasmid genes necessary for pilus assembly. The loss and gain of pili in various mutants correlated with the loss and gain of transferred DNA (T-DNA) transfer functions, which is consistent with the view that Agrobacterium pili are required for transfer of DNA to plant cells in a process similar to that of conjugation.

We describe a conserved family of bacterial gene products that includes the VirBl virulence factor encoded by tumor-inducing plasmids of Agrobacterium spp., proteins involved in conjugative DNA transfer of broad-hostrange bacterial plasmids, and gene products that may be involved in invasion by Shigella spp. and Salmonella enterica.Sequence analysis and structural modeling show that the proteins in this group are related to chicken egg white lysozyme and are likely to adopt a lysozyme-like structural fold. Based on their similarity to lysozyme, we predict that these proteins have glycosidase activity. Iterative data base searches with three conserved sequence motifs from this protein family detect a more distant relationship to bacterial and bacteriophage lytic transglycosylases, and goose egg white lysozyme. Two acidic residues in the VirBl protein of Agrobacterium tumefaciens form a putative catalytic dyad. Each of these residues was changed into the corresponding amide by sitedirected mutagenesis. Strains of A. tumefaciens that express mutated VirBl proteins have a significantly reduced virulence.We hypothesize that many bacterial proteins involved in export of macromolecules belong to a widespread class of hydrolases and cleave ,B-1,4-glycosidic bonds as part of their function.The soil bacterium Agrobacterium tumefaciens causes crown gall tumors in dicotyledonous plants by transferring a DNA segment (T-DNA) of its large tumor-inducing (Ti) plasmid into the host plant cell nucleus, where it becomes integrated into the plant genome and expresses oncogenes (for reviews, see refs. 1-3). At least eight plasmid-encoded vir operons and several chromosomal genes ofAgrobacterium are required for virulence (1-4). The vir genes are transcriptionally activated by the VirA/VirG two-component regulatory system, which responds to signal molecules such as specific phenolic compounds and sugars released by wounded plant cells (5). virC and virD gene products act in a concerted manner to release single-stranded transferable DNA (T-strand) from the doublestranded Ti plasmids (6-9). The VirE2 protein, a singlestranded DNA-binding protein, is believed to coat the T-strand and facilitate its transfer through the plant cell cytoplasm into the nucleus (10).There is considerable evidence that the proteins coded by the 11 genes of the virB operon are concerned with the formation of a channel for the transfer of T-DNA. These 11 genes are not required for vir gene activation or T-strand formation, but every one has been shown to be important in tumorigenesis (11). A comparative sequence analysis has revealed a high similarity between several VirB proteins and gene products required for the export of specific proteins and plasmids. These include pertussis toxin in Bordetella (12) and broad-host-range plasmids, such as plasmid pKM101, which contains the full set of VirB homologs (3, 13).How VirB proteins function in gene transfer is not well understood. The abundance of hydrophobic segments in the amino acid sequences of several ...

The contribution of accessory toxins to the acute inflammatory response to Vibrio cholerae was assessed in a murine pulmonary model. Intranasal administration of an El Tor O1 V. cholerae strain deleted of cholera toxin genes (ctxAB) caused diffuse pneumonia characterized by infiltration of PMNs, tissue damage, and hemorrhage. By contrast, the ctxAB mutant with an additional deletion in the actin-cross-linking repeats-in-toxin (RTX) toxin gene (rtxA) caused a less severe pathology and decreased serum levels of proinflammatory molecules interleukin (IL)-6 and murine macrophage inflammatory protein (MIP)-2. These data suggest that the RTX toxin contributes to the severity of acute inflammatory responses. Deletions within the genes for either hemagglutinin/protease (hapA) or hemolysin (hlyA) did not significantly affect virulence in this model. Compound deletion of ctxAB, hlyA, hapA, and rtxA created strain KFV101, which colonized the lung but induced pulmonary disease with limited inflammation and significantly reduced serum titers of IL-6 and MIP-2. 100% of mice inoculated with KFV101 survive, compared with 20% of mice inoculated with the ctxAB mutant. Thus, the reduced virulence of KFV101 makes it a prototype for multi-toxin deleted vaccine strains that could be used for protection against V. cholerae without the adverse effects of the accessory cholera toxins.