Screening of TnphoA mutants of Vibrio cholerae O139 for identification of antigens involved in colonisation.

A new serogroup of Vibrio cholerae non-O1, designated as O139, has emerged causing cholera-like disease among adults. Laboratory and field studies clearly show that there is no cross-protection between O1 and O139 pathogenic strains. Since colonisation of the intestine is a most important step in the pathogenesis of cholera caused by O1 strains and colonising antigens are known to be protective, investigation of the colonising antigens of O139 strain was initiated. By TnphoA mutagenesis, mutants were generated with insertions in the genome encoding membrane spanning or secretory proteins. Screening of the mutants for adherence to rabbit intestinal surface and colonisation in 5-day-old mice resulted in the identification of mutant clones, which were less adhesive than was the wild-type parent strain and which could not efficiently colonise the gut. Such non-colonising strains were attenuated in virulence. Analysis of the proteins by SDS-PAGE revealed that the non-colonising mutants did not express a 40-kDa outer-membrane protein.

Evaluation of different subcellular fractions of Vibrio cholerae O139 in protection to challenge in experimental cholera.

Various cellular fractions of Vibrio cholerae O139 were prepared and evaluated in the rabbit ileal loop model of experimental cholera for identification of the protective antigen(s) relevant for vaccine development. Lipopolysaccharides (LPS) and capsular polysaccharides (CPS) of O139 strains and its cell surface, membrane and cytosolic fractions were assayed for antibacterial immunity, whereas the cholera toxin was examined for antitoxic immunity. The lipopolysaccharides, membrane fraction and cholera toxin induced moderate protection, however there was a significant synergistic effect when cholera toxin was combined with membrane proteins or lipopolysaccharides. The O139 strains strongly resembled O1 strains in the profile of proteins and immunological cross reactivity, yet there was no cross protection. The results warrant further investigation of the pathogenesis of O139 strains and identify the critical somatic antigens relevant to protection.

Comparative efficacy of biodegradable liposomes and microspheres as carriers for delivery of Vibrio cholerae antigens in the intestine.

The effect of the encapsulated antigens of Vibrio cholerae and their route of administration in induction of immune response was studied in experimental cholera. The antigenic proteins of V. cholerae El Tor strain KB207 were obtained by fractionation of cell-free lysate by high-performance liquid chromatography. The antigenic proteins were pooled and encapsulated in biodegradable liposomes and poly(D,L) lactic co-glycolic acid microspheres. Rabbits were immunized with free as well as encapsulated antigens by different routes. Liposome-encapsulated antigens delivered intraintestinally offered maximum protection. Orally or intraintestinally delivered antigens in microspheres failed to elicit a significant immune response, although as a carrier microspheres were comparable to liposomes when judged by the subcutaneous route. The results suggested that liposomes and microspheres could be used as carriers of protective antigens of V. cholerae for effective immunization.

A 53 kDa protein of Vibrio cholerae classical strain 0395 involved in intestinal colonization.

Mutants of Vibrio cholerae 01 strain 0395 (classical) mutated in genes encoding secretory or cell surface proteins were induced by TnphoA mutagenesis and were selected as blue colonies on L-agar plates containing 5-bromo-4-chloro-3-indolyl phosphate. Southern analysis of the total DNA from blue colonies showed that all mutants had TnphoA insertion in genomic DNA. These mutants were analysed for adherence, colonization and protein profile. Adherence to freshly isolated rabbit intestinal discs was affected in some mutants. The less adhesive mutants were examined for colonization of the intestine of infant mice. One mutant, designated T-87, was extremely poor at colonization and less diarrhaegenic than the parent strain. Analysis of T-87 by SDS-PAGE revealed that two proteins of 53 and 38 kDa were lacking. The 38 kDa protein was identified as OmpU. The 53 kDa protein was extracellular and cells treated with anti-53-kDa antibodies could not colonize the gut of infant mice. The expression of the 53 and 38 kDa proteins in T-87 was dependent of the growth medium. The data suggest that T-87 is mutated in a regulatory gene which regulates the expression of proteins involved in intestinal colonization.

recA mutations reduce adherence and colonization by classical and El Tor strains of Vibrio cholerae.

Two recA mutants of Vibrio cholerae (classical and El Tor biotypes) were constructed by disruption of the wild-type recA gene with mutated recA sequences of V. cholerae cloned in the suicide vector pGP704. Mutants defective in the recA gene were compared with their respective RecA+ parent strains with regard to their adherence to isolated rabbit intestine and colonization of intestine of infant mice. The recA mutation in V. cholerae was found to diminish adherence and markedly affected colonization.

Identification of the flagellar antigens of Vibrio cholerae El Tor and their role in protection.

Antiserum to the surface antigens of the wild-type flagellate strain KB207 of Vibrio cholerae El Tor was absorbed with isogenic aflagellate mutant CD12. Antibodies remaining in the absorbed serum exhibited specificity to KB207 but not to CD12 and inhibited motility of KB207. Proteins from cell-free lysates of KB207 and CD12 were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. CD12 differed from KB207 in the absence of two proteins of 40 and 38 kDa. These proteins were detected in KB207 when electrophoretically separated proteins were immunoblotted with unabsorbed and absorbed sera. These two proteins were isolated by immunoaffinity chromatography using purified antibodies from absorbed serum. Although antigenic, the 40 and 38 kDa proteins did not induce protection against cholera in the rabbit ileal loop model.

Identification of a 33 kDa antigen associated with an adhesive and colonizing strain of Vibrio cholerae El Tor and its role in protection.

Proteins from the cell-free lysates of the wild-type strain KB207 of Vibrio cholerae El Tor and the isogenic non-adhesive mutant CD11 were analysed by native and denaturing polyacrylamide gel electrophoresis. A protein of 33 kDa present in KB207 was absent from CD11. Antiserum to the surface antigens of KB207 was absorbed with CD11. Antibodies remaining in the serum after absorption reacted to KB207 but not to CD11 as judged by slide agglutination, double gel diffusion and dot blot ELISA. Antibodies in the absorbed serum inhibited adherence of KB207 to rabbit intestinal mucosa and colonization in an infant mice model. The 33 kDa protein was isolated from KB207 by immunoaffinity chromatography. Antibodies present in the absorbed serum were used as ligand. The 33 kDa antigen was immunogenic and conferred protection in the rabbit ileal loop model. Combined administration of 33 kDa protein and B-subunit of cholera toxin offered full protection.

Chromosomal transfer and in vivo cloning of genes in Vibrio cholerae using RP4::mini-Mu.

The RP4::mini-Mu replicon has been used to transfer chromosomal genes by conjugation and to clone in vivo metabolic, toxin and flagellar genes of Vibrio cholerae. RP4::mini-Mu was introduced into several strains of V. cholerae and these strains were mated with V. cholerae or Escherichia coli K-12 recipients. R'-episomes carrying the respective genes were maintained in recA recipients and were detected by complementation of auxotrophic, nontoxinogenic and aflagellate mutations in V. cholerae.

Regulation of toxin biosynthesis by plasmids in Vibrio cholerae.

Vibrio cholerae strain 569B Inaba harbouring P plasmid produced less toxin than the parent strain. To examine the effect of plasmid loss on toxin production, temperature-sensitive (ts) mutants of P, unable to replicate at 42 degrees C, were isolated. One ts plasmid was unstable at 42 degrees C and its loss yielded a cured strain that resumed a normal level of toxin biosynthesis characteristic of the plasmid-free parent strain. Toxin production was again suppressed in the cured strain after reacquisition of P plasmid. This suggested a role for plasmid-borne genes in the regulation of toxin biosynthesis. A mutant of strain 569B Inaba that produced mutant toxin was isolated by transfer of P and V plasmids. The mutant toxin was similar to choleragenoid because it did not give rise to symptoms of cholera but induced antitoxin immunity in rabbits.

Events in the pathogenesis of experimental cholera: role of bacterial adherence and multiplication.

Pathogenic, and laboratory-derived non-adherent, non-motile, streptomycin-dependent and attenuated strains of Vibrio cholerae, were injected into the ileal loops of adult rabbits. The pattern of bacterial adherence and multiplication was studied. It was shown that all the strains multiplied to the same extent in the intestine; multiplication per se had no role in pathogenesis except when the infecting dose was low. Vibrio strains differed in their capacity to adhere to the intestine. A good correlation was found between adherence and pathogenesis. While adhesive strains were pathogenic, the poorly adhesive strains proved to be poor pathogens. There was no trace of toxin the ileal loops inoculated with poorly adhesive strains and very little in the diarrhoeal fluid produced by pathogenic strains. Adhesive strains adhered poorly to the intestine of immunised animals. It is suggested that adherence is concerned in the release of toxin and thus plays an important role in the pathogenesis of cholera.

Attenuation of virulence by P and V plasmids in Vibrio cholerae: strains suitable for oral immunization.

In a virulent strain of Vibrio cholerae, KB9, P and V plasmids were introduced by bacterial conjugation. Characterization of PV isolates and systematic screening of them in animal models of cholera revealed that a large number of PV isolates were non-pathogenic, owing to the loss of ability to synthesize toxin. Results obtained with two such strains, designated as KB9:PV and CD24, are described. The strains with plasmids were stable during in vitro cultivation or during two successive passages in rabbit intestine. Protection conferred by PV strains was determined in mouse protection tests and in the rabbit ileal loop model. The plasmid strains were immunogenic. In view of the results, it is proposed that PV-bearing attenuated strains should be tried in oral immunization.

Attenuated recombinant strains of Vibrio cholerae for oral immunization.

Two attenuated strains of Vibrio cholerae, CD1 and CD3, have been isolated that have remained stable since 1976. These strains are motile, adhere to and multiply in rabbit intestine, and colonize the gut of infant mice for 6-7 days. Both strains are antigenic and provide protection to challenge in the mouse protection test and in the rabbit ileal loop model. Because of their ability to adhere to and colonize the gut, and since they are antigenic, strains CD1 and CD3 have the potentiality of oral vaccines.