The RNA Domain Vc1 Regulates Downstream Gene Expression in Response to Cyclic Diguanylate in Vibrio cholerae

In many bacterial species, including the aquatic bacterium and human pathogen Vibrio cholerae, the second messenger cyclic diguanylate (c-di-GMP) modulates processes such as biofilm formation, motility, and virulence factor production. By interacting with various effectors, c-di-GMP regulates gene expression or protein function. One type of c-di-GMP receptor is the class I riboswitch, representatives of which have been shown to bind c-di-GMP in vitro. Herein, we examined the in vitro and in vivo function of the putative class I riboswitch in Vibrio cholerae, Vc1, which lies upstream of the gene encoding GbpA, a colonization factor that contributes to attachment of V. cholerae to environmental and host surfaces containing N-acetylglucosamine moieties. We provide evidence that Vc1 RNA interacts directly with c-di-GMP in vitro, and that nucleotides conserved among this class of riboswitch are important for binding. Yet the mutation of these conserved residues individually in the V. cholerae chromosome inconsistently affects the expression of gbpA and production of the GbpA protein. By isolating the regulatory function of Vc1, we show that the Vc1 element positively regulates downstream gene expression in response to c-di-GMP. Together these data suggest that the Vc1 element responds to c-di-GMP in vivo. Positive regulation of gbpA expression by c-di-GMP via Vc1 may influence the ability of V. cholerae to associate with chitin in the aquatic environment and the host intestinal environment

Two nucleotide second messengers regulate the production of the Vibrio cholerae colonization factor GbpA

Abstract Background The nucleotide second messengers cAMP and c-di-GMP allow many bacteria, including the human intestinal pathogen Vibrio cholerae, to respond to environmental stimuli with appropriate physiological adaptations. In response to limitation of specific carbohydrates, cAMP and its receptor CRP control the transcription of genes important for nutrient acquisition and utilization; c-di-GMP controls the transition between motile and sessile lifestyles often, but not exclusively, through transcriptional mechanisms. In this study, we investigated the convergence of cAMP and c-di-GMP signaling pathways in regulating the expression of gbpA. GbpA is a colonization factor that participates in the attachment of V. cholerae to N-acetylglucosamine-containing surfaces in its native aquatic environment and the host intestinal tract. Results We show that c-di-GMP inhibits gbpA activation in a fashion independent of the known transcription factors that directly sense c-di-GMP. Interestingly, inhibition of gbpA activation by c-di-GMP only occurs during growth on non-PTS dependent nutrient sources. Consistent with this result, we show that CRP binds to the gbpA promoter in a cAMP-dependent manner in vitro and drives transcription of gbpA in vivo. The interplay between cAMP and c-di-GMP does not broadly impact the CRP-cAMP regulon, but occurs more specifically at the gbpA promoter. Conclusions These findings suggest that c-di-GMP directly interferes with the interaction of CRP-cAMP and the gbpA promoter via an unidentified regulator. The use of two distinct second messenger signaling mechanisms to regulate gbpA transcription may allow V. cholerae to finely modulate GbpA production, and therefore colonization of aquatic and host surfaces, in response to discrete environmental stimuli

A systematic analysis of the in vitro and in vivo functions of the HD-GYP domain proteins of Vibrio cholerae

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. Results 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. 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

c-di-GMP impacts the SHAPE reactivity of specific regions in Vc1 RNA.

<p>Vc1 RNA transcripts were incubated with or without 500 μM c-di-GMP and analyzed by SHAPE. SHAPE reactivity values for each nucleotide position were obtained by averaging values from five independent experiments. The inset shows in red text the location of the nucleotides with significantly altered reactivity, mapped on the predicted structure of Vc1. The boxes denote the G12, A39 and C104 residues. * <i>P</i> < 0.05 by unpaired t-test comparing SHAPE reactivity values for each nucleotide position, with or without c-di-GMP, indicating significant changes in reactivity in response to c-di-GMP.</p

Vc1 secondary structure and putative contact residues for c-di-GMP.

<p>(A) The +1 site of transcription, which is 244 base pairs upstream of the experimentally determined translational start site (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148478#pone.0148478.s001" target="_blank">S1 Fig</a>), was identified using 5’ RACE. Vc2 and Vc1 were aligned using ClustalW2. The alignment begins with the +1 transcriptional start site for Vc1 and the nucleotide at position +9 according to the sequence annotation for Vc2 (PDB 3IRW [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148478#pone.0148478.ref011" target="_blank">11</a>]). Asterisks represent nucleotides that are conserved between Vc1 and Vc2. Nucleotides bolded and underlined are contact residues for c-di-GMP in Vc2 and are predicted contact residues for c-di-GMP in Vc1. The regions predicted to encode P2 and P3 are labeled. (B) The predicted structure of Vc1 based on an alignment with Vc2 and the consensus GEMM aptamer structure. The predicted tetraloop and tetraloop receptor are noted.</p

Vc1 influences downstream gene expression.

<p>(A) β-galactosidase activity of <i>V</i>. <i>cholerae</i> strains with plasmid-borne <i>lacZ</i> translational fusions to wild type Vc1 or mutant derivatives. Transcription initiation is controlled by the <i>lac</i> promoter. Three independent experiments were done, and the means and standard deviations are shown. *** <i>P</i> < 0.001, * <i>P</i> < 0.05 by unpaired t-test. (B) Western blot analysis of the GbpA levels in the supernatants of cultures of wild type <i>V</i>. <i>cholerae</i>, an isogenic Δ<i>gbpA</i> strain, or strains with mutations in Vc1 (P1, G12T, A39T, or C104G). The image shown is representative of three independent experiments. (C) Densitometry of the western blots was done by comparing the intensity of the GbpA band to that of a cross-reactive band in the same lane (indicated by an asterisk), and then normalizing to the wild type values. Shown are the means and standard deviations for three independent experiments. *** <i>P</i> < 0.001 by unpaired t-test.</p

Lowering intracellular c-di-GMP reduces Vc1-dependent gene expression.

<p>β-galactosidase activity of <i>V</i>. <i>cholerae</i> strains with chromosomal, translational fusions of <i>E</i>. <i>coli lacZ</i> to the <i>gbpA</i> 5’ UTR with wild type Vc1, Vc1^G12T, or Vc1^A39T, with transcription initiation under the control of the constitutive P_{<i>lacUV5</i>} promoter: PlacUV5-Vc1UTR-<i>lacZ</i>, PlacUV5-Vc1UTR^G12T-<i>lacZ</i>, PlacUV5-Vc1UTR^A39T-<i>lacZ</i>, respectively [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148478#pone.0148478.ref038" target="_blank">38</a>]. Each reporter strain carried pPDE and was grown without arabinose (wild-type c-di-GMP level; white bars) or with 0.2% arabinose (reduced c-di-GMP; black bars). (B) Dose response analysis using the P_{<i>lacUV5</i>}-Vc1UTR-<i>lacZ</i> reporter strain, with vector or pPDE, grown in rich medium with a range of arabinose concentrations. Increasing PDE production corresponds with decreasing intracellular c-di-GMP. (A and B) Measurements of β-galactosidase activity were done with at least three independent biological samples, and the means and standard error are shown. * <i>P</i> < 0.05, *** <i>P</i> < 0.001, n.s. = not significant by one-way ANOVA and Bonferroni’s multiple comparison test.</p

Vc1 directly and specifically interacts with c-di-GMP.

<p>(A) Binding of c-di-GMP³² to <i>in vitro</i> transcribed Vc1 RNA and mutant derivatives was measured by equilibrium dialysis as described in the Materials and Methods. Vc2 and Vc2^G20U RNA were included as controls, and no-RNA samples were used as a negative control. (B) The specificity of binding to c-di-GMP to Vc1 and Vc2 RNA was assessed by equilibrium dialysis, allowing c-di-GMP³² to bind and reach equilibrium as the first measurement (black bars), then competing with unlabeled, excess c-di-GMP or GTP and determining the percentage of c-di-GMP³² retained in the RNA chambers (grey bars). **<i>P</i> < 0.01, **<i>P</i> < 0.001 by unpaired t-test.</p