Vibrio cholerae causes the potentiafly lethal disase cholera through the elaboration of the intestinal secretogen cholera toxin. A second toxin of V. cholerae, Zot, decreases intestinal tissue resistance by modifying interceflular tight junctions. In this report, a third toxin of V. cholerae, Ace (accessory cholera enterotoxin), is described. Ace increases short-circuit current in Ussing chambers and causes fluid secretion in ligated rabbit ileal loops. The predicted protein sequence of Ace shows striking similarit to eukaryotic iontransporting ATPases, including the product of the cystic fibrosis gene. The gene encoding Ace is located immediately upstream of the genes encoding Zot and cholera toxin. The ctx, zot, and ace genes, which are located on a dynamic sector of the chromosome, comprise a V. cholerae "virulence cassette." Development oflive attenuated Vibrio cholerae oral vaccines by recombinant technology initially targeted the genes encoding cholera toxin (ctx) for deletion. Although these initial vaccine candidates, such as JBK70 and CVD101, elicited high antibody responses and did not cause severe diarrhea in volunteers, more than half ofthe vaccinees developed mild to moderate diarrhea and many experienced abdominal cramps, anorexia, and low-grade fever (1). In search of an additional toxin which could explain this reactogenicity, we reported a second toxin, Zot (zonula occludens toxin), which decreases ileal tissue resistance by affecting intercellular tightjunctions (2). The diarrhea observed in volunteers fed Actx V. cholerae vaccine strains was hypothesized to be caused by alteration of tight junctions, with a subsequent increase in intestinal permeability.The gene encoding Zot is located immediately upstream of ctx (3) on a 4.5-kb region termed the "core region" (4). In many strains of V. cholerae, this 4.5-kb region is flanked by one or more copies of a 2.7-kb sequence called RS1; homologous recombination between RS1 elements can lead to tandem amplification of the 4.5-kb region (4). Since the zot and ctx genes comprise only 55% of the 4.5-kb core region, we investigated the potential pathogenic role ofthe remaining portion of this region. Our results show that the gene for a third toxin is included in this regionll and that this third toxin increases the short-circuit current (IS) in Ussing chambers and causes fluid secretion in ligated rabbit ileal loops. MATERIALS AND METHODSBacterial Strains. V. cholerae E7946 is an El Tor Ogawa strain previously demonstrated to produce cholera in volunteers (5). Strain CVD110 was constructed from E7946 by homologous recombination of the RS1 elements, resulting in deletion of the core region (J.M., J.E.G., A.F., and J.B.K.,The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. unpublished work). In brief, cloned ctx and zot sequences were inserted into the suicide vector pGP704 (7) and conjugated ...

Vibrio cholerae, the etiologic agent of the diarrheal disease cholera, is a Gram-negative bacterium that belongs to the ␥ subdivision of the family Proteobacteriaceae. The physical map of the genome has been reported, and the genome has been described as a single 3.2-Mb chromosome [Majumder, R., et al. (1996) J. Bacteriol. 178, 1105-1112]. By using pulsed-field gel electrophoresis of genomic DNA immobilized in agarose plugs and digested with the restriction enzymes I-CeuI, SfiI, and NotI, we have also constructed the physical map of V. cholerae. Our analysis estimates the size of the genome at 4.0 Mb, 25% larger than the physical map reported by others. Our most notable finding is, however, that the V. cholerae chromosome appears to be not the single chromosome reported but two unique and separate circular megareplicons.

We have constructed an improved recombination-based in vivo expression technology (RIVET) and used it as a screening method to identify Vibrio cholerae genes that are transcriptionally induced during infection of infant mice. The improvements include the introduction of modified substrate cassettes for resolvase that can be positively and negatively selected for, allowing selection of resolved strains from intestinal homogenates, and three different tnpR alleles that cover a range of translation initiation efficiencies, allowing identification of infection-induced genes that have low-to-moderate basal levels of transcription during growth in vitro. A transcriptional fusion library of 8,734 isolates of a V. cholerae El Tor strain that remain unresolved when the vibrios are grown in vitro was passed through infant mice, and 40 infection-induced genes were identified. Nine of these genes were inactivated by in-frame deletions, and their roles in growth in vitro and fitness during infection were measured by competition assays. Four mutant strains were attenuated >10-fold in vivo compared with the parental strain, demonstrating that infection-induced genes are enriched in genes essential for virulence.Much remains to be learned about genes that the facultative pathogen Vibrio cholerae induces during infection and how their protein products function during the complex and dynamic process in which this pathogen adapts to the human small intestine. Several new methods have been developed that are helping us to explore this process, including in vivo expression technology (IVET), signature-tagged mutagenesis, microarray technology, differential fluorescence induction, in vivo-induced antigen technology, and real-time reverse transcription-PCR, among others. A specific IVET method, recombination-based IVET (RIVET), has been used previously to identify V. cholerae genes that are induced during infection of infant mice (1, 4). RIVET is very sensitive to low or transient expression of in vivo-induced (ivi) genes during infection and is therefore capable of identifying members of this potentially interesting class of genes. However, this sensitivity is also a double-edged sword, as some ivi genes have low-to-moderate levels of expression in vitro and will therefore be lost during library construction, i.e., there will be premature excision (resolution) of the selectable cassette in such strains. In the present study, we have developed a modified RIVET that can overcome this main disadvantage and used it as a large-scale screening method to identify V. cholerae genes that are transcriptionally induced during infant mouse infection. Briefly, the new system incorporates two different resolvable cassettes that differ in the efficiency of excision, as well as three different alleles of the resolvase-encoding gene tnpR that have different efficiencies of translation initiation. These modifications extend the number of V. cholerae strains that are unresolved in vitro that can be generated in the final library. Additional modificatio...

In vivo expression technology (IVET) has been widely used to study gene expression of human bacterial pathogens in animal models, but has heretofore not been used in humans to our knowledge. As part of ongoing efforts to understand Vibrio cholerae pathogenesis and develop improved V. cholerae vaccines, we have performed an IVET screen in humans for genes that are preferentially expressed by V. cholerae during infection. A library of 8,734 nontoxigenic V. cholerae strains carrying transcriptional fusions of genomic DNA to a resolvase gene was ingested by five healthy adult volunteers. Transcription of the fusion leads to resolvasedependent excision of a sacB-containing cassette and thus the selectable phenotype of sucrose resistance (Suc R ). A total of Ϸ20,000 Suc R isolates, those carrying putative in vivo-induced fusions, were recovered from volunteer stool samples. Analysis of the fusion junctions from >7,000 Suc R isolates from multiple samples from multiple volunteers identified 217 candidate genes for preferential expression during human infection. Of genes or operons induced in three or more volunteers, the majority of those tested (65%) were induced in an infant mouse model. VC0201 (fhuC), which encodes the ATPase of a ferrichrome ABC transporter, is one of the identified in vivo-induced genes and is required for virulence in the mouse model. gene expression ͉ genetics ͉ vaccinology ͉ virulence O ur understanding of the complex interactions between bacterial pathogens and humans relies heavily on the use of animal and tissue culture model systems that serve as surrogates of human infection. One useful genetic tool for discovering genes and pathways involved in virulence is in vivo expression technology (IVET), which was designed to identify genes of pathogens that are preferentially expressed during infection and has been extensively used in model systems (reviewed by refs. 1 and 2). IVET is a promoter-trapping strategy in which cells carrying a library of transcriptional fusions of genomic DNA to a reporter gene are used to infect a model host and those carrying fusions that are expressed in vivo can be identified by either a genetic screen or selection. This technique allows the identification of genes that may be expressed only under in vivo conditions [in vivo-induced genes (ivi genes)]. Such genes may be very difficult to identify during growth under laboratory conditions, but are likely to be important in the host for survival and virulence. One form of IVET, used in this work, is recombination-based IVET (3, 4) in which the reporter gene encodes a resolvase that effects a permanent genetic change, allowing a direct selection for cells that expressed the resolvase even transiently during infection.Here, we describe the use of recombination-based IVET to identify genes of Vibrio cholerae, the causative agent of the diarrheal disease cholera, that are expressed during human infection. This approach has the potential to identify important virulence genes that may not be expressed in vitro or during in...

The current pandemic of cholera is caused primarily by Vibrio cholerae O1 of the El Tor biotype. Live attenuated classical biotype V. cholerae vaccine strains prevent severe and moderate cholera due to either biotype in challenged volunteers but may provide less protection against mild cholera due to El Tor organisms. CVD 110, a new ctxA-deleted vaccine strain derived from an El Tor Ogawa parent, lacks zona occludens toxin (Zot), accessory cholera enterotoxin (Ace), and hemolysin/enterotoxin. Ten healthy adult volunteers were given 10(8) cfu of CVD 110 with buffer; 7 developed diarrhea (mean stool volume, 861 mL). Vaccine organisms were shed in stool by all vaccines and were recovered from duodenal fluid in three-quarters of vaccinees. After vaccination, the geometric mean peak reciprocal vibriocidal titer among vaccinees was 17,829. CVD 110 is a powerful immunogen but insufficiently attenuated despite the absence of known potential enterotoxins of V. cholerae. Another unrecognized toxin or colonization alone may be responsible for diarrhea after ingestion of this strain.