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A Vibrio cholerae arcA mutant was constructed and used to examine the role of the global anaerobiosis response regulator ArcA in the expression of virulence factors in this important human pathogen. In V. cholerae, expression of the major virulence factors cholera toxin (CT) and toxin-coregulated pilus (TCP) is regulated by the transcriptional activator ToxT. toxT expression, in turn, is controlled by the transmembrane DNA binding proteins ToxR and TcpP. In the V. cholerae arcA mutant, although ToxR and TcpP were unaffected, Northern blot and reverse transcription-PCR analyses indicated that the expression of toxT was significantly decreased with concomitant reduction in the expression of CT and TCP. CT and TCP expression was completely restored in the V. cholerae arcA mutant strain by expressing a cloned toxT gene in the mutant. These results suggest that ArcA functions as a positive regulator of toxT expression under both aerobic and anaerobic conditions, although as expected, the effect was more pronounced during anaerobic growth. This was reflected in a reduction of virulence of the V. cholerae arcA mutant strain in the infant mouse cholera model.

In this study, adult volunteers were fed tcpA and mshA deletion mutants of V. cholerae O139 strain CVD 112 to determine the role of toxin-coregulated pili (TCP) and mannose-sensitive hemagglutinin (MSHA) in intestinal colonization. Eight of 10 volunteers who received CVD 112 or CVD 112 delta mshA shed the vaccine strains in their stools; the geometric mean peak excretion for both groups was 1.4 x 10(5) CFU/g of stool. In contrast, only one of nine recipients of CVD 112 delta tcpA shed vibrios in his stool (P < 0.01); during the first 24 h after inoculation, 3 x 10(2) CFU/g was recovered from this volunteer. All recipients of CVD 112 and 8 (80%) of the recipients of CVD 112 delta mshA developed at least a fourfold rise in vibriocidal titer after immunization. In contrast, only one (11%) of the nine recipients of CVD 112 delta tcpA developed a fourfold rise in vibriocidal titer (P < 0.01). We conclude that TCP are an important colonization factor of V. cholerae O139 and probably of El Tor V. cholerae O1. In contrast, MSHA does not appear to promote intestinal colonization in humans.

A previously described in-frame deletion in mshA--the gene encoding the structural subunit of the mannose-sensitive hemagglutinin pilus--has been introduced into the chromosome of three El Tor O1 strains of Vibrio cholerae. None of the deltamshA mutants showed significant attenuation or loss of colonization potential in the infant mouse cholera model. A second mutation, created by insertion of a kanamycin resistance cartridge into deltamshA, also failed to affect in vivo behavior. In contrast, strains carrying mutations in tcpA (encoding the monomer of the toxin-coregulated pilus [TCP]) were markedly attenuated and showed dramatically impaired colonization. This result was in line with those of previous studies. Protection tests performed with antibodies to TCP and to MshA showed that only the former were able to confer immunity against El Tor O1 challenge in this model. Studies with mutants constructed from two O139 strains similarly suggest that TCP but not mannose-sensitive hemagglutinin pili are critical for colonization by strains of this serogroup.

The relative contributions of toxin-coregulated pilus (TCP) and cell-associated mannose-sensitive hemagglutinin (MSHA) to the colonization ability of Vibrio cholerae O1 El Tor biotype strains and O139 Bengal strains was determined by using isogenic parental and in-frame deletion mutant pairs in the infant mouse cholera model. Both the El Tor and O139 tcpA mutant strains showed a dramatic defect in colonization as indicated by their competitive indices, whereas deletion of mshA had a negligible effect on colonization in either background.

Until recently, only Vibrio cholerae strains of the O1 serogroup have been associated with epidemic cholera. In December 1992, an outbreak of cholera gravis in Vellore, India, was attributed to a new serogroup of V. cholerae recently designated O139. Serogroup O139 cholera has since spread to 13 countries and has reached pandemic proportions. Serogroup O139 cholera evades immunity to O1 cholera and is not detected by the standard O1 antigen test. Understanding the origins of O139 cholera and determining the relatedness of O139 to O1 cholera are necessary to device strategies for detecting, reporting, and controlling this new pandemic. In order to determine the origins of this novel cholera serogroup, O139 was analyzed for virulence genes, for virulence proteins and their regulation, and for its genomic background. We found that O139 and O1 V. cholera strains of the E1 Tor biotype possess highly homologous virulence genes encoding cholera toxin and toxin-coregulated pili and that the regulation of virulence protein expression likewise was indistinguishable between O139 and O1. Pulsed-field gel electrophoresis (PFGE) revealed the restriction digest pattern of O139 strains to be closely related to that of O1 serogroup E1 Tor biotype cholera strains from the Indian subcontinent. However, PFGE showed minor differences among individual O139 cholera isolates, suggesting that O139 V. cholerae is evolving.

Vibrio cholerae O1, which exists as two biotypes, classical and El Tor, expresses fimbrial antigens called toxin-coregulated pili (TCP) and mannose-sensitive hemagglutinin (MSHA) pili, respectively. We have raised rabbit antisera and monoclonal antibodies against these fimbrial antigens and prepared Fab fragments which possess specific antibodies directed against the respective fimbrial antigens from these antisera. The protective effect of these antibody preparations was studied in the infant mouse cholera model. Antibodies against TCP were able to protect baby mice against challenge with V. cholerae O1 of the classical but not of the El Tor biotype. Similar but reverse biotype differences in protection against challenge with classical and El Tor vibrios were observed when antibodies against MSHA pili were used. The protective effect of V. cholerae O1 antilipopolysaccharide (anti-LPS) antibodies, both alone and in combination with antifimbrial antibodies, was also evaluated. We showed that antibodies to the LPS component also prevented infections with V. cholerae O1. Moreover, our results indicate that antibodies against TCP or MSHA pili and against LPS cooperate at least additively, and possible even synergistically, in protecting baby mice against challenge with group O1 vibrios. These results indicate that TCP and MSHA pili as well as LPS play an important role in the pathogenesis of experimental cholera. We could also demonstrate that antibacterial immunity preventing colonization is biotype specific. Our results might be used for the generation of new oral cholera vaccines including both TCP and MSHA fimbrial antigens.

Secretory immunoglobulin A (IgA) antibodies directed against cholera toxin (CT) are thought to be important in resistance to oral challenge with virulent Vibrio cholerae, although alternative mechanisms for protection of intestinal epithelia against CT-induced fluid secretion have been proposed. The ability of anti-CT IgA to block the effects of CT on human enterocytes has not been directly tested because of the lack of a well-defined in vitro intestinal epithelial cell system to directly measure toxin action and the limited availability of purified anti-CT IgA antibodies. We have generated hybridomas that produce monoclonal IgA and IgG antibodies directed against CT by fusion of Peyer's patch cells with mouse myeloma cells after oral-systemic immunization of mice with CT and CT B-subunit protein. All of the anti-CT antibodies recognized the B subunit. Three clones (designated anti-CTB IgA-1, IgA-2, and IgA-3) which produced IgA antibodies in dimeric and polymeric forms were selected. Checkerboard immunoblotting demonstrated that IgA-1 recognized an epitope distinct from that recognized by IgA-2 and IgA-3 and that none of the antibodies were directed against the binding site of GM1, the intestinal cell membrane toxin receptor. The protective capacity of these IgAs was tested in vitro with human T84 colon carcinoma cells grown on permeable supports as confluent monolayers of polarized enterocytes. When each anti-CTB IgA was mixed with 10 nM CT and applied to the apical surfaces of T84 cell monolayers, all three IgAs blocked CT-induced Cl- secretion in a dose-dependent manner and completely inhibited binding of rhodamine-labelled CT to apical cell membranes. Thus, monoclonal anti-CTB IgA antibodies are sufficient to protect human enterocytes in vitro against CT binding and action.

The role of motility in the pathogenesis of cholera was evaluated in ligated ileal loops of adult rabbits. Four strains of Vibrio cholerae (including both Inaba and Ogawa serotypes of both classical and El Tor biotypes) were compared with their aflagellated, but fully toxigenic and prototrophic, isogenic derivatives as to their ability to produce fluid accumulation in the rabbit gut. The nonmotile mutants required an at least 100-fold-higher dose than their respective wild-type strains to produce comparable fluid accumulation responses. The decreased ability of nonmotile strains to produce a fluid response was not due to their failure to multiply in vivo, since they increased in numbers in the rabbit ileum at the same rate as the wild-type strains, but probably was related to their inability to associate with the intestinal mucosa. After 3 h of incubation, 45 to 53% of motile, [35S]-labeled cells adsorbed to the intestinal wall, whereas only 3 to 15% (depending upon the strain) of the nonmotile bacteria were associated.

This study was undertaken to determine the sequence of events in the microenvironment of the intestinal tract that culminate in the symptoms of cholera and to attempt to define more clearly the mechanisms involved in antibacterial immunity. The extent to which mucus occurs in the normal intestine of rabbits and the appearance of the intestinal villi in unfixed frozen sections was demonstrated. The villi and intervillous spaces were found to be normally covered by a layer of mucoid material that formed a mucous zone between the intestinal contents and the tips of the villi. The distribution of cholera organisms in normal and immunized animals was demonstrated by the staining of frozen-tissue sections with specific fluorescent antibody. Study of tissue sections from normal animals showed that the onset of fluid accumulation was concomitant with the establishment of large masses of organisms in the intervillous spaces and crypts of the intestine after the successful penetration of this mucous zone. Tissue sections from animals actively or passively immunized against a cell wall antigen of Vibrio cholerae showed clumping of vibrios in the lumen and restricted distribution in the lumen and luminal border of the mucous zone. Antibody was not lytic in vivo.

The enterotoxicity of an El Tor-like hemolysin purified from non-O1 Vibrio cholerae was investigated. Fluid accumulation was induced by injection of purified hemolysin into the ligated intestinal loops in adult rabbits (De test), intraintestinal administration in infant rabbits (Dutta test), and oral inoculation in suckling mice. The accumulated fluid was invariably mucous and bloody, and a histological change in the mucosa was observed. These results suggest that the hemolysin is an enterotoxic factor that is responsible for non-O1 V. cholerae gastroenteritis.