bis(3--5-)-cyclic-diguanylic-acid and Cholera

The bacterial secondary messenger bis-(3',5')-cyclic-dimeric-guanosine monophosphate (c-di-GMP) has been implicated in the pathogenesis of Vibrio cholerae, due to its significant role in regulating the virulence, biofilm formation and motility of the host organism. The VC0395_0300 protein from V. cholerae, possessing a GGEEF sequence has been established as a diguanylate cyclase (DGC) capable of catalyzing the conversion of two GTP molecules to form cyclic-di-GMP. This in turn, plays a crucial role in allowing the organism to adopt a dual lifestyle, thriving both in human and aquatic systems. The difficulty in procuring sufficient amounts of homogenous soluble protein for structural assessment of the GGDEF domain in VC0395_0300 and the lack of soluble protein yield, prompted the truncation into smaller constructs (Sebox31 and Sebox32) carrying the GGDEF domain. The truncates retained their diguanylate cyclase activity comparable to the wild type, and were able to form biofilms as well. Fluorescence and circular dichroism spectroscopy measurements revealed that the basic structural elements do not show significant changes in the truncated proteins as compared to the full-length. This has also been confirmed using homology modeling and molecular docking of the wild type and truncates. This led us to conclude that the truncated constructs retain their activity in spite of the deletions in the N terminal region. This is supportive of the fact that DGC activity in GGDEF proteins is predominantly dependent on the presence of the conserved GGD(/E)EF domain and its interaction with GTP.

Topics: Amino Acid Sequence; Bacterial Proteins; Cholera; Cyclic GMP; Escherichia coli Proteins; Guanosine Triphosphate; Humans; Models, Molecular; Phosphorus-Oxygen Lyases; Vibrio cholerae

The LonA (or Lon) protease is a central post-translational regulator in diverse bacterial species. In Vibrio cholerae, LonA regulates a broad range of behaviors including cell division, biofilm formation, flagellar motility, c-di-GMP levels, the type VI secretion system (T6SS), virulence gene expression, and host colonization. Despite LonA's role in cellular processes critical for V. cholerae's aquatic and infectious life cycles, relatively few LonA substrates have been identified. LonA protease substrates were therefore identified through comparison of the proteomes of wild-type and ΔlonA strains following translational inhibition. The most significantly enriched LonA-dependent protein was TfoY, a known regulator of motility and the T6SS in V. cholerae. Experiments showed that TfoY was required for LonA-mediated repression of motility and T6SS-dependent killing. In addition, TfoY was stabilized under high c-di-GMP conditions and biochemical analysis determined direct binding of c-di-GMP to LonA results in inhibition of its protease activity. The work presented here adds to the list of LonA substrates, identifies LonA as a c-di-GMP receptor, demonstrates that c-di-GMP regulates LonA activity and TfoY protein stability, and helps elucidate the mechanisms by which LonA controls important V. cholerae behaviors.

Topics: Animals; Bacterial Proteins; Biofilms; Cholera; Cyclic GMP; Disease Models, Animal; Humans; Mice; Mutation; Protease La; Protein Processing, Post-Translational; Protein Stability; Proteolysis; Proteomics; Recombinant Proteins; Type VI Secretion Systems; Vibrio cholerae; Virulence

The bacterial enhancer-binding protein (bEBP) FlrC, controls motility and colonization of

Topics: Adenosine Triphosphatases; Bacterial Proteins; Cholera; Crystallography, X-Ray; Cyclic GMP; DNA-Binding Proteins; Flagella; Gene Expression Regulation, Bacterial; Phylogeny; Protein Structure, Tertiary; Vibrio cholerae

Vibrio cholerae of serogroups O1 and O139, the causative agent of Asiatic cholera, continues to be a major global health threat. This pathogen utilizes substratum-specific pili to attach to distinct surfaces in the aquatic environment and the human small intestine and detaches when conditions become unfavorable. Both attachment and detachment are critical to bacterial environmental survival, pathogenesis and disease transmission. However, the factors that promote detachment are less understood. In this study, we examine the role of flagellar motility and hemagglutinin/protease (HapA) in vibrio detachment from a non-degradable abiotic surface and from the suckling mouse intestine. Flagellar motility facilitated V. cholerae detachment from abiotic surfaces. HapA had no effect on the stability of biofilms formed on abiotic surfaces despite representing >50% of the proteolytic activity present in the extracellular matrix. We developed a balanced lethal plasmid system to increase the bacterial cyclic diguanylate (c-di-GMP) pool late in infection, a condition that represses motility and HapA expression. Increasing the c-di-GMP pool enhanced V. cholerae colonization of the suckling mouse intestine. The c-di-GMP effect was fully abolished in hapA isogenic mutants. These results suggest that motility facilitates detachment in a substratum-independent manner. Instead, HapA appears to function as a substratum-specific detachment factor.

Topics: Animals; Bacterial Adhesion; Biofilms; Cholera; Cyclic GMP; Fimbriae, Bacterial; Flagella; Gene Expression Regulation, Bacterial; Intestinal Mucosa; Intestine, Small; Metalloendopeptidases; Mice; Movement; Polystyrenes; Vibrio cholerae

Biofilm formation is crucial to the environmental survival and transmission of Vibrio cholerae, the facultative human pathogen responsible for the disease cholera. During its infectious cycle, V. cholerae experiences fluctuations in temperature within the aquatic environment and during the transition between human host and aquatic reservoirs. In this study, we report that biofilm formation is induced at low temperatures through increased levels of the signalling molecule, cyclic diguanylate (c-di-GMP). Strains harbouring in frame deletions of all V. cholerae genes that are predicted to encode diguanylate cyclases (DGCs) or phosphodiesterases (PDEs) were screened for their involvement in low-temperature-induced biofilm formation and Vibrio polysaccharide gene expression. Of the 52 mutants tested, deletions of six DGCs and three PDEs were found to affect these phenotypes at low temperatures. Unlike wild type, a strain lacking all six DGCs did not exhibit a low-temperature-dependent increase in c-di-GMP, indicating that these DGCs are required for temperature modulation of c-di-GMP levels. We also show that temperature modulates c-di-GMP levels in a similar fashion in the Gram-negative pathogen Pseudomonas aeruginosa but not in the Gram-positive pathogen Listeria monocytogenes. This study uncovers the role of temperature in environmental regulation of biofilm formation and c-di-GMP signalling.

Topics: Biofilms; Cholera; Cyclic GMP; Escherichia coli Proteins; Humans; Listeria monocytogenes; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Pseudomonas aeruginosa; Sequence Deletion; Signal Transduction; Temperature; Vibrio cholerae

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.. Complementary 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.. 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.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Animals; Biofilms; Cholera; Cyclic GMP; Disease Models, Animal; Locomotion; Mice; Mutation; Signal Transduction; Vibrio cholerae; Virulence

Signaling through the second messenger cyclic di-GMP (c-di-GMP) is central to the life cycle of Vibrio cholerae. However, relatively little is known about the signaling mechanism, including the specific external stimuli that regulate c-di-GMP concentration. Here, we show that the phosphate responsive regulator PhoB regulates an operon, acgAB, which encodes c-di-GMP metabolic enzymes. We show that induction of acgAB by PhoB positively regulates V. cholerae motility in vitro and that PhoB regulates expression of acgAB at late stages during V. cholerae infection in the infant mouse small intestine. These data support a model whereby PhoB becomes activated at a late stage of infection in preparation for dissemination of V. cholerae to the aquatic environment and suggest that the concentration of exogenous phosphate may become limited at late stages of infection.

Topics: Animals; Bacterial Proteins; Biofilms; Cholera; Cyclic GMP; Gene Expression Regulation, Bacterial; Mice; Vibrio cholerae

In Vibrio cholerae, the second messenger cyclic di-GMP (c-di-GMP) positively regulates biofilm formation and negatively regulates virulence and is proposed to play an important role in the transition from persistence in the environment to survival in the host. Herein we describe a characterization of the infection-induced gene cdpA, which encodes both GGDEF and EAL domains, which are known to mediate diguanylate cyclase and c-di-GMP phosphodiesterase (PDE) activities, respectively. CdpA is shown to possess PDE activity, and this activity is regulated by its inactive degenerate GGDEF domain. CdpA inhibits biofilm formation but has no effect on colonization of the infant mouse small intestine. Consistent with these observations, cdpA is expressed during in vitro growth in a biofilm but is not expressed in vivo until the late stage of infection, after colonization has occurred. To test for a role of c-di-GMP in the early stages of infection, we artificially increased c-di-GMP and observed reduced colonization. This was attributed to a significant reduction in toxT transcription during infection. Cumulatively, these results support a model of the V. cholerae life cycle in which c-di-GMP must be down-regulated early after entering the small intestine and maintained at a low level to allow virulence gene expression, colonization, and motility at appropriate stages of infection.

Topics: Animals; Bacterial Proteins; Bacterial Typing Techniques; Biofilms; Cholera; Cyclic GMP; Gene Expression Regulation, Bacterial; Mice; Phosphoric Diester Hydrolases; Protein Structure, Tertiary; Vibrio cholerae

The facultative pathogen Vibrio cholerae can exist in both the human small bowel and in aquatic environments. While investigation of the infection process has revealed many factors important for pathogenesis, little is known regarding transmission of this or other water-borne pathogens. Using a temporally controlled reporter of transcription, we focus on bacterial gene expression during the late stage of infection and identify a unique class of V. cholerae genes specific to this stage. Mutational analysis revealed limited roles for these genes in infection. However, using a host-to-environment transition assay, we detected roles for six of ten genes examined for the ability of V. cholerae to persist within cholera stool and/or aquatic environments. Furthermore, passage through the intestinal tract was necessary to observe this phenotype. Thus, V. cholerae genes expressed prior to exiting the host intestinal tract are advantageous for subsequent life in aquatic environments.

Topics: Animals; Animals, Suckling; Cholera; Cyclic GMP; Diarrhea; Gene Expression Regulation, Bacterial; Genes, Bacterial; Humans; Mice; Osmolar Concentration; Reverse Transcriptase Polymerase Chain Reaction; Vibrio cholerae; Water Microbiology