Interplay among Cyclic Diguanylate, HapR, and the General Stress Response Regulator (RpoS) in the Regulation of Vibrio cholerae Hemagglutinin/Protease

Abstract

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 ⌬hapR 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.Cholera is an acute waterborne diarrheal disease caused by Vibrio cholerae of serogroups O1 and O139. Infecting V. cholerae cells adhere to the intestinal mucosa, where they express major virulence factors such as the toxin coregulated pilus (TCP) (21) and cholera toxin (CT), which is largely responsible for the profuse rice-watery diarrhea typical of this illness (12,26). Cholera patients shed V. cholerae in their stools, which usually contain about 10 8 hyperinfective V. cholerae cells per ml (38). The V. cholerae cells can then survive and persist in fresh water and estuarine aquatic ecosystems to eventually gain entrance to a new host.The Zn-dependent metalloprotease hemagglutinin (HA)/ protease (18) has been proposed to facilitate V. cholerae detachment from the intestinal mucosa when infecting V. cholerae cells reach a high density (4,14,43,44). Inactivation of hapA, which encodes HA/protease, has been shown to enhance adherence to mucin-coated polystyrene plates (43), adherence to mucin-secreting differentiated HT29-18N2 cultured cells (4), and colonization of the suckling mouse intestine (41, 43). The mucinase activity of HA/protease (13), together with its capacity to cleave the mucin-binding adhesin GbpA at a high cell density, provides a mechanism that supports the "detachase" function attributed to this protein (25). In addition, HA/protease has been reported to perturb the paracellular barrier of cultured intestinal epithelial cells (37, 55) by acting on tight junction-associated proteins (56). In agreement with this finding, we have shown that HA/protease enhances enterotoxicity in the rabbit ileal loop model (43).The expression of flagellar motility ...

The LuxR-Type Regulator VpsT Negatively Controls the Transcription of rpoS, Encoding the General Stress Response Regulator, in Vibrio cholerae Biofilms

“…These findings suggest that biofilm development and the general stress response are inversely regulated. Consistent with this interpretation, we found that elevated levels of intracellular c-di-GMP, a condition which favors biofilm development, diminished the expression of RpoS (22). Furthermore, we showed that the negative regulation of RpoS expression by c-di-GMP was apparently counteracted by HapR (22).…”

“…At a high cell density, the quorumsensing regulator HapR inhibits biofilm formation by repressing vpsT and decreasing the c-di-GMP pool (20). In parallel, quorum sensing (HapR) positively influences the expression of rpoS (21,22). These findings suggest that biofilm development and the general stress response are inversely regulated.…”

Interaction of the Histone-Like Nucleoid Structuring Protein and the General Stress Response Regulator RpoS at Vibrio cholerae Promoters That Regulate Motility and Hemagglutinin/Protease Expression

“…V. cholerae rpoS mutants are more sensitive to starvation, high osmolarity, and oxidative stresses, are less motile than the wild type (WT), and do not express HA/protease (35,49,58). We recently reported that RpoS diminishes the cellular concentration of c-di-GMP, an inhibitor of flagellar motility (57). Consistently, microarray studies have shown that rpoS mutants express reduced levels of multiple motility and chemotaxis genes, suggesting that rpoS could act at an early stage of the motility regulatory cascade (35).…”

“…In V. cholerae, RpoS activates hapA and enhances the expression of motility genes belonging to the class II, III, and IV hierarchies (35,46,57). We examined the role of RpoS and H-NS in the expression of rpoN and flrA, which control the expression of the class II, III, and IV hierarchy motility genes (42).…”

“…Motility and HA/protease are positively regulated by the cyclic AMP (cAMP)-receptor protein (CRP), which acts by enhancing the quorum-sensing regulator HapR and the general stress response regulator RpoS (4,29,46). The expression of both phenotypes is diminished in response to an increase in the intracellular concentration of the second messenger cyclic diguanylate (c-di-GMP) (57).…”