Identification and characterization of cyclic diguanylate signaling systems controlling rugosity in Vibrio cholerae.

Department of Microbiology and Environmental Toxicology, University of California, Santa Cruz, Santa Cruz, CA 95064, USA.
Journal of bacteriology (Impact Factor: 2.69). 10/2008; 190(22):7392-405. DOI: 10.1128/JB.00564-08
Source: PubMed

ABSTRACT Vibrio cholerae, the causative agent of the disease cholera, can generate rugose variants that have an increased capacity to form biofilms. Rugosity and biofilm formation are critical for the environmental survival and transmission of the pathogen, and these processes are controlled by cyclic diguanylate (c-di-GMP) signaling systems. c-di-GMP is produced by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs). Proteins that contain GGDEF domains act as DGCs, whereas proteins that contain EAL or HD-GYP domains act as PDEs. In the V. cholerae genome there are 62 genes that are predicted to encode proteins capable of modulating the cellular c-di-GMP concentration. We previously identified two DGCs, VpvC and CdgA, that can control the switch between smooth and rugose. To identify other c-di-GMP signaling proteins involved in rugosity, we generated in-frame deletion mutants of all genes predicted to encode proteins with GGDEF and EAL domains and then searched for mutants with altered rugosity. In this study, we identified two new genes, cdgG and cdgH, involved in rugosity control. We determined that CdgH acts as a DGC and positively regulates rugosity, whereas CdgG does not have DGC activity and negatively regulates rugosity. In addition, epistasis analysis with CdgG, CdgH, and other DGCs and PDEs controlling rugosity revealed that CdgG and CdgH act in parallel with previously identified c-di-GMP signaling proteins to control rugosity in V. cholerae. We also determined that PilZ domain-containing c-di-GMP binding proteins contribute minimally to rugosity, indicating that there are additional c-di-GMP binding proteins controlling rugosity in V. cholerae.

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    ABSTRACT: Vibrio cholerae is a Gram-negative bacterium that persists in aquatic reservoirs and causes the diarrheal disease cholera upon entry into a human host. V. cholerae employs the second messenger molecule 3', 5'-cyclic diguanylic acid (c-di-GMP) to transition between these two distinct lifestyles. C-di-GMP is synthesized by diguanylate cyclase (DGC) enzymes and hydrolyzed by phosphodiesterase (PDE) enzymes. Bacteria typically encode many different DGCs and PDEs within their genomes. Presumably, each enzyme senses and responds to cognate environmental cues by alteration of enzymatic activity. C-di-GMP represses the expression of virulence factors in V. cholerae, and it is predicted that the intracellular concentration of c-di-GMP is low during infection. Contrary to this model, we found that bile acids, a prevalent constituent of the human proximal small intestine, increase intracellular c-di-GMP in V. cholerae. We identified four c-di-GMP turnover enzymes that contribute to increased intracellular c-di-GMP in the presence of bile acids, and deletion of these enzymes eliminates the bile induction of c-di-GMP and biofilm formation. Furthermore, this bile-mediated increase in c-di-GMP is quenched by bicarbonate, the intestinal pH buffer secreted by intestinal epithelial cells. Our results lead us to propose that V. cholerae senses distinct microenvironments within the small intestine using bile and bicarbonate as chemical cues, and responds by modulating the intracellular concentration of c-di-GMP.
    Infection and immunity 05/2014; 82(7). DOI:10.1128/IAI.01664-14 · 4.16 Impact Factor
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    ABSTRACT: Background The second messenger cyclic diguanylate (c-di-GMP) plays a central role in bacterial adaptation to extracellular stimuli, controlling processes such as motility, biofilm development, cell development and, in some pathogens, virulence. The intracellular level of c-di-GMP is controlled by the complementary activities of diguanylate cyclases containing a GGDEF domain and two classes of c-di-GMP phosphodiesterases containing an EAL or HD-GYP hydrolytic domain. Compared to the GGDEF and EAL domains, the functions of HD-GYP domain family proteins are poorly characterized. The human diarrheal pathogen Vibrio cholerae encodes nine putative HD-GYP domain proteins. To determine the contributions of HD-GYP domain proteins to c-di-GMP signaling in V. cholerae, we systematically analyzed the enzymatic functionality of each protein and their involvement in processes known to be regulated by c-di-GMP: motility, biofilm development and virulence.ResultsComplementary in vitro and in vivo experiments showed that four HD-GYP domain proteins are active c-di-GMP phosphodiesterases: VC1295, VC1348, VCA0210 and VCA0681. Mutation of individual HD-GYP domain genes, as well as combinatorial mutations of multiple HD-GYP domain genes, had no effect on motility or biofilm formation of V. cholerae under the conditions tested. Furthermore, no single HD-GYP domain gene affected intestinal colonization by V. cholerae in an infant mouse model. However, inactivation of multiple HD-GYP domain genes, including the four encoding functional phosphodiesterases, significantly attenuated colonization.Conclusions These results indicate that the HD-GYP family of c-di-GMP phosphodiesterases impacts signaling by this second messenger during infection. Altogether, this work greatly furthers the understanding of this important family of c-di-GMP metabolic enzymes and demonstrates a role for HD-GYP domain proteins in the virulence of V. cholerae.
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