A study of the prevalence of regulatory genes controlling virulence gene expression among Vibrio choleraeeltor biovariant strains varying in their pandemic potential

The evolution of the genome of the pathogenic agent of the seventh cholera pandemia Vibrio cholerae eltor biovariant was thought to occur by acquiring not only structural genes of virulence but also regulatory systems as a result of horizontal transfer events. The polymerase chain reaction revealed the presence of the following regulatory genes that control the virulence gene expression in the chromosome of pre-pandemic and pandemic strains of cholera vibrios eltor: toxR, toxT, tcpP, tcpH, luxS, luxO, crp, vicH, pepA. The avirulent V. cholerae strain ATCC14033 isolated in 1910 (hypothetical predecessor of the cholera eltor agent) was shown to be lacking the regulatory genes toxT, tcpP, tcpHlocalized in the pathogenicity island VPI-1, and to be capable of realizing positive control over the expression of the virulence genes involved in the ToxR regulon. The virulent strains isolated from cholera patients during the local cholera outbreak in Indonesia in 1937 did not differ from the strains that caused cholera eltor pandemic in 1961. The strains had identical content of the regulatory genes tested. Only one strain of the four isolates studied contained no tcpPgene. Two key regulatory genes, toxR and toxT, were sequenced in all the isolates. The toxR nucleotide sequence of three pre-pandemic strains was shown to be indistinguishable from that of the pandemic isolates. On the other hand, the clinical strain MAK757 isolated prior to the emergence of the epidemic demonstrated an altered nucleotide sequence in its toxR gene. Experiments with the intra-intestinal challenge of suckling rabbits were indicative of similar virulence levels for the pre-pandemic and pandemic clinical strains. These results may serve as the evidence of the in vivo activity of the pre-pandemic strains of the toxT, tcpH, and tcpP positive regulatory genes that acquired in V. cholerae during the evolutionary process.     

Upregulation of human mitochondrial NADH dehydrogenase subunit 5 in intestinal epithelial cells is modulated by Vibrio cholerae pathogenesis

Cholera still remains an important global predicament especially in India and other developing countries. Vibrio cholerae, the etiologic agent of cholera, colonizes the small intestine and produces an enterotoxin that is largely responsible for the watery diarrheal symptoms of the disease. Using RNA arbitrarily primed PCR, ND5 a mitochondria encoded subunit of complex I of the mitochondrial respiratory chain was found to be upregulated in the human intestinal epithelial cell line Int407 following exposure to V. cholerae. The upregulation of ND5 was not observed when Int407 was infected with Escherichia coli strains. Incubation with heat-killed V. cholerae or cholera toxin or culture supernatant also showed no such upregulation indicating the involvement of live bacteria in the process. Infection of the monolayer with aflagellate non-motile mutant of V. cholerae O395 showed a very significant (59-fold) downregulation of ND5. In contrast, a remarkable upregulation of ND5 expression (200-fold) was observed in a hyperadherent icmF insertion mutant with reduced motility. V. cholerae cheY4 null mutant defective in adherence and motility also resulted in significantly reduced levels of ND5 expression while mutant with the cheY4 gene duplicated showing increased adherence and motility resulted in increased expression of ND5. These results clearly indicate that both motility and adherence to intestinal epithelial cells are possible triggering factors contributing to ND5 mRNA expression by V. cholerae. Interestingly infection with insertion mutant in the gene coding for ToxR, the master regulator of virulence in V. cholerae resulted in significant downregulation of ND5 expression. However, infection with ctxA or toxT insertion mutants did not show any significant changes in ND5 expression compared to wild-type. Almost no expression of ND5 was observed in case of mutation in the gene coding for OmpU, a ToxR activated protein. Thus, infection of Int407 with virulence mutant strains of V. cholerae revealed that the ND5 expression is modulated by the virulence of V. cholerae in a ToxT independent manner. Although no difference in the mitochondrial copy number could be detected between infected and uninfected cells, the modulation of the expression of other mitochondrial genes were also observed. Incidentally, upon V. cholerae infection, complex I activity was found to increase about 3-folds after 6 h. This is the first report of alteration in mitochondrial gene expression upon infection of a non-invasive enteric bacterium like V. cholerae showing its modulation with adherence, motility and virulence of the organism.