Regulation of virulence gene expression in Vibrio cholerae by quorum sensing: HapR functions at the aphA promoter

Quorum sensing negatively influences virulence gene expression in certain toxigenic Vibrio cholerae strains. At high cell densities, the response regulator LuxO fails to reduce the expression of HapR, which, in turn, represses the expression of the virulence cascade. A critical regulatory step in the cascade is activation of tcpPH expression by AphA and AphB. We show here that HapR influences the virulence cascade by directly repressing aphA expression. In strain C6706, aphA expression was increased in a delta hapR mutant and decreased in a delta luxO mutant, indicating a negative and positive influence, respectively, of these gene products on the promoter. Overexpression of HapR also reduced aphA expression in both C6706 and Escherichia coli. DNase I footprinting showed that purified HapR binds to the aphA promoter between -85 and -58. Although it appears that quorum sensing does not influence virulence gene expression in strain O395 solely because of a frameshift in hapR, overproduced HapR did not repress expression from the O395 aphA promoter in either Vibrio or E. coli, nor did the protein bind to the promoter. Two basepair differences from C6706 are present in the O395 HapR binding site at -85 and -77. Introducing the -77 change into C6706 prevented HapR binding and repression of aphA expression. This mutation also eliminated the repression of toxin-co-regulated pilus (TCP) and cholera toxin (CT) that occurs in a delta luxO mutant, indicating that HapR function at aphA is critical for density-dependent regulation of virulence genes.     

Interplay among cyclic diguanylate, HapR, and the general stress response regulator (RpoS) in the regulation of Vibrio cholerae hemagglutinin/protease

Vibrio cholerae secretes the Zn-dependent metalloprotease hemagglutinin (HA)/protease (mucinase), which is encoded by hapA and displays a broad range of potential pathogenic activities. Expression of HA/protease has a stringent requirement for the quorum-sensing regulator HapR and the general stress response regulator RpoS. Here we report that the second messenger cyclic diguanylic acid (c-di-GMP) regulates the production of HA/protease in a negative manner. Overexpression of a diguanylate cyclase to increase the cellular c-di-GMP pool resulted in diminished expression of HA/protease and its positive regulator, HapR. The effect of c-di-GMP on HapR was independent of LuxO but was abolished by deletion of the c-di-GMP binding protein VpsT, the LuxR-type regulator VqmA, or a single-base mutation in the hapR promoter that prevents autorepression. Though expression of HapR had a positive effect on RpoS biosynthesis, direct manipulation of the c-di-GMP pool at a high cell density did not significantly impact RpoS expression in the wild-type genetic background. In contrast, increasing the c-di-GMP pool severely inhibited RpoS expression in a ΔhapR mutant that is locked in a regulatory state mimicking low cell density. Based on the above findings, we propose a model for the interplay between HapR, RpoS, and c-di-GMP in the regulation of HA/protease expression.