Coordinated regulation of accessory genetic elements produces cyclic di-nucleotides for V. cholerae virulence

Abstract

The function of the Vibrio 7(th) pandemic island-1 (VSP-1) in cholera pathogenesis has remained obscure. Utilizing chromatin immunoprecipitation sequencing and RNA sequencing to map the regulon of the master virulence regulator ToxT, we identify a TCP island-encoded small RNA that reduces the expression of a previously unrecognized VSP-1-encoded transcription factor termed VspR. VspR modulates the expression of several VSP-1 genes including one that encodes a novel class of di-nucleotide cyclase (DncV), which preferentially synthesizes a previously undescribed hybrid cyclic AMP-GMP molecule. We show that DncV is required for efficient intestinal colonization and downregulates V. cholerae chemotaxis, a phenotype previously associated with hyperinfectivity. This pathway couples the actions of previously disparate genomic islands, defines VSP-1 as a pathogenicity island in V. cholerae, and implicates its occurrence in 7(th) pandemic strains as a benefit for host adaptation through the production of a regulatory cyclic di-nucleotide.

Figure 1

(A) Schematic outlining ToxT regulon. Major regulatory targets grouped by functional class are shown. Solid arrows denote direct ToxT regulation identified by integrated ChIP-seq and RNA-seq analysis. Dashed arrows denote indirect ToxT regulation identified by RNA-seq alone. Green indicates positive gene expression, red indicates negative gene expression. Specific pathways are indicated beside their functional group and colored to indicate the direction of their regulation. (B) Schematic of the 6^th ToxT ChIP-peak site in the Tcp island between VC0845 and VC0846. The heat map shows enrichment (red) of sequenced reads at ToxT ChIP-peak. Below, alignment of RNA-seq reads shows the predicted sRNA outlined by the black box. RNA-seq reads colored red align to the reverse strand of the genome while reads colored green align to the forward strand. The predicted sequence of the sRNA is shown at the top in upper case font from 5′ to 3′. The nucleotides in bold highlight a rho independent hairpin terminator predicted by the ARNold program server. (C) ToxT ChIP enrichment of the promoter regions of ctxA, tcpA and 6^th ToxT ChIP peak were determined by qPCR relative to sample DNA input. Enrichment of a non ToxT-dependent promoter of VC1141 is shown as a control. Significance was determined by t-test relative to the VC1141 promoter; *** p < 0.001; **** p < 0.0001 (D) Northern blot of RNA from indicated strains probed for the presence of the sRNA (TarB) as predicted by RNAseq alignment (top panel). The size markers of a ssRNA ladder are indicated. The same samples were probed for 5S rRNA as a loading control (bottom panel). (E) qPCR analysis of the effect of TarB on VC0177 (VspR) expression levels in V. cholerae wild type strains C6706 and E7946 following expression of ToxT from a plasmid. Expression for each sample was determined relative to 16S rRNA and at least 3 independent replicates tested. Fold changes are normalized relative to expression in the wild type strain. Significance was determined by t-test relative to the wild type strain; ** p < 0.01; **** p < 0.0001. See also Table S1.

Figure 2

(A) Expression analysis of VSP-1 genes in WT and VC0177::Tn C6706 strains. The expression of each gene was determined relative to total 16S rRNA. 8 replicates were sampled for each gene. Significance was determined by t-test; * p < 0.05; ** p < 0.01; **** p < 0.0001. (B) VC0177 (VspR) ChIP enrichment of the 5′ UTR regions of VC0176, VC0178, VC0179 and VC0180 was determined by qPCR relative to sample DNA input. Enrichment of the promoter region of non VSP-1 gene VC1141 is shown as a control. Significance was determined by t-test relative to control enrichment; * p < 0.05; ** p < 0.01 (C) In vivo competition experiments measuring the ability of mutant strains to colonize the infant mouse intestine compared to the parental strain. Significance was determined by t-test relative to colonization ratio of parental strains wild type C6706 vs. C6706 ΔlacZ; **** p < 0.0001. See also Figure S1.

Figure 3

Di-nucleotide cyclase activity of VC0179 (DncV). (A) Purified wild type or mutated VC0179 was incubated with 1 mM ATP and 10 µCi α-P³² ATP for the indicated time (t in min) and the products (P1) separated by denaturing PAGE. (B) The product from the reaction between VC0179 and ATP (P1) was incubated with calf intestinal phosphatase (CIP) or snake venom phosphodieasterase (SVPD) for 30 min and separated by denaturing PAGE. (C) Purified wild type VC0179 was incubated with 1 mM GTP and 10 µCi α-P³² GTP for the indicated time (t in min) and the products (P2) separated by denaturing PAGE. (D) VC0179 was incubated with ATP or GTP as described above or with 1mM GTP and 10 µCi α-P³² ATP and the products (P1–3) separated by denaturing PAGE. (E) Purified wild type VC0179 was incubated with 1 mM GTP and 10 µCi α-P³² ATP for the indicated time (t in min) and the products (P3) separated by denaturing PAGE. P1, c-di-AMP; P2, c-di-GMP; P3, c-AMP-GMP. (F) Commercial standards of ATP, GTP, c-di-AMP and c-di-GMP were fractionated by RP-HPLC. The peaks are labeled by their respective compound. VC0179 incubated with 2mM ATP (G), 2mM GTP (H), 1mM ATP + 1mM GTP (I), or 1mM of each of the 5 NTPs (J) for 30 min at 37 °C was fractionated by RPHPLC. 50 nM of VC0179 (DncV) wild type or mutant enzyme was used per reaction. See also Figure S2, S3, S4, S5; Table S2, S3.

Figure 4

(A) LCMS chromatogram trace corresponding to the ion extraction of c-AMP-GMP (theoretical mass [M-H] = 673.0927 Da; ion extraction: 673.0922 – 673.0947 Da) in cell lysate from V. cholerae ΔdncV strain expressing plasmid borne WT or D131A/D133A mutant DncV. High resolution mass found under the peak eluting at approximately 4.75 min in chromatogram A corresponding to c-AMP-GMP from V. cholerae ΔdncV strain expressing WT DncV is shown on the inset. This peak eluted with the same retention time as in vitro generated c-AMP-GMP. (B) Functional categories of differentially expressed genes in response to expression of DncV. The number of genes induced or repressed in response to DncV expression is presented. Green indicates genes in the respective category are induced > 2 fold, red indicated genes in respective category are repressed > 2 fold (FDR corrected p-value < 0.01). (C) Examination of chemotactic behavior of V. cholerae wild type, ΔdncV mutant, and ΔdncV mutant expressing wild type or D131A/D131A mutant DncV from an arabinose inducible plasmid, with and without induction by arabinose. See also Figure S6, S7; Table S4; Movie S1, S2.

Figure 5

Model for ToxT-dependent TarB-mediated control of VSP-1. (1) Host signals induce ToxT activity resulting in transcription of TarB from the Tcp Island. (2) Stabilized by Hfq, TarB downregulates expression of transcriptional repressor VC0177 (VspR) resulting in de-repression of VSP-1 genes including VC0179 (DncV). (3) DncV activity increases cellular concentration of c-AMP-GMP (and possibly c-di-AMP and c-di-GMP) that affects chemotactic behavior and other metabolic aspects of 7^th pandemic V. cholerae.