Three autoinducer molecules act in concert to control virulence gene expression in Vibrio cholerae

Abstract

Bacteria use quorum sensing to monitor cell density and coordinate group behaviours. In Vibrio cholerae, the causative agent of the diarrheal disease cholera, quorum sensing is connected to virulence gene expression via the two autoinducer molecules, AI-2 and CAI-1. Both autoinducers share one signal transduction pathway to control the production of AphA, a key transcriptional activator of biofilm formation and virulence genes. In this study, we demonstrate that the recently identified autoinducer, DPO, also controls AphA production in V. cholerae. DPO, functioning through the transcription factor VqmA and the VqmR small RNA, reduces AphA levels at the post-transcriptional level and consequently inhibits virulence gene expression. VqmR-mediated repression of AphA provides an important link between the AI-2/CAI-1 and DPO-dependent quorum sensing pathways in V. cholerae. Transcriptome analyses comparing the effect of single autoinducers versus autoinducer combinations show that quorum sensing controls the expression of ∼400 genes in V. cholerae and that all three autoinducers are required for a full quorum sensing response. Together, our data provide a global view on autoinducer interplay in V. cholerae and highlight the importance of RNA-based gene control for collective functions in this major human pathogen.

Figure 1.

Quorum sensing in V. cholerae is controlled by three autoinducer molecules. The CAI-1 and AI-2 autoinducers are produced by CqsA and LuxS and detected by the membrane-bound CqsS and LuxPQ receptors, respectively. The DPO autoinducer derives from threonine catabolism, and requires the Tdh (threonine dehydrogenase) enzyme. DPO is released into the environment and binds to and activates the VqmA receptor. (A) At low autoinducer concentrations, CqsS and LuxPQ act as kinases to phosphorylate LuxU. LuxU-P transfers the phosphate to LuxO, and LuxO-P induces the expression of the Qrr1–4 sRNAs. The Qrr sRNAs act post-transcriptionally to repress hapR and activate aphA, promoting virulence gene expression and biofilm formation. AphA also activates the transcription of vpsT. (B) At high autoinducer concentrations, binding of CAI-1 and AI-2 to CqsS and LuxPQ, respectively, converts the receptors to phosphatases, which reduces LuxO-P levels and inhibits qrr1–4 expression. Under these conditions, aphA is repressed and hapR is activated. The VqmA-DPO complex induces the transcription of the VqmR sRNA. VqmR inhibits biofilm formation by repressing VpsT and virulence gene expression by inhibiting AphA. In addition, HapR and AphA antagonize each other at the transcriptional level. Active factors are highlighted in blue, inactive (repressed) factors are shown in gray.

Figure 2.

VqmR target genes and base-pairing of VqmR with the aphA 5′ UTR. (A) Secondary structure of VqmR (24). The VqmR base-pairing sequences are highlighted in red (R1), blue (R2) and green (R3). Arrows and brackets mark the truncation start sites and the internal deletion regions investigated in C, respectively. (B) E. coli harbouring plasmids carrying the five genes denoted on the x-axis each fused to gfp were co-transformed with a control plasmid (pCtr) or the indicated VqmR expressing plasmids. Transcription of vqmR and gfp was driven by constitutive promoters. Cells were cultivated in LB to OD₆₀₀ = 0.5 and GFP production was measured. GFP levels of strains carrying the control plasmid were set to 1. Error bars represent the SD of three biological replicates. (C) E. coli cells carrying the aphA::gfp reporter were tested for repression by various VqmR mutants. Cells were grown in LB to OD₆₀₀ = 0.5 and GFP production was measured. Error bars indicate the SD of three biological replicates. (D) Predicted base-pairing of the VqmR R3 sequence (green) with the 5′ UTR of aphA. The arrows indicate the single nucleotide mutations tested in E and the start codon is underlined. (E) Repression of AphA::GFP and AphA*::GFP (G-3C) by VqmR and VqmR* (C133G). Cells were grown in LB to OD₆₀₀ = 0.5 and GFP levels were measured using Western Blot. RNAP served as the loading control.

Figure 3.

DPO inhibits AphA production. (A) V. cholerae wild-type and vqmR mutants carrying the indicated plasmids were cultivated in M9 minimal media supplemented with casamino acids (0.4% final conc.). At the indicated growth phases, total RNA and protein samples were collected. AphA::3XFLAG production was monitored on Western Blots and RNAP served as the loading control. VqmR and aphA::3XFLAG mRNA levels were probed on Northern Blots using 5S rRNA as loading control. (B) Total RNA and protein samples were collected from V. cholerae wild-type, ΔvqmA, ΔvqmR and Δtdh strains at low cell density (OD₆₀₀ = 0.2). Cells were cultivated in M9 minimal media and one set of cultures was supplemented with DPO (100 μM final conc.). AphA::3XFLAG production was determined using Western Blot. Northern Blot was used to probe the expression of aphA-3XFLAG and VqmR. RNAP and 5S rRNA served as loading controls for the Western and Northern Blot analyses, respectively. (C) V. cholerae Δtdh cells were cultivated in M9 medium supplemented with the indicated DPO concentrations (x-axis) and total RNA and protein samples were harvested at low cell densities (OD₆₀₀ = 0.2). AphA production was analyzed on Western Blots (left y-axis), sRNA levels (VqmR and Qrr4) were determined on Northern Blots (right y-axis). Error bars represent the SD of five (AphA) and three (sRNAs) biological replicates, respectively.

Figure 4.

DPO and VqmR inhibit virulence gene expression. (A) The virulence cascade of V. cholerae. (B) V. cholerae wild-type and ΔvqmR strains carrying the indicated plasmids were cultivated under AKI conditions. Cellular and secreted protein (SP) fractions were harvested 2h and 16 h after switching from static to aerating conditions, respectively and tested for AphA-3XFLAG and CtxAB production on Western Blots. RNAP and a coomassie-stained SDS gel (bottom) confirmed equal loading of the two protein fractions. (C) V. cholerae Δtdh or Δtdh, vqmR cells carrying plasmids with the indicated transcriptional reporters were cultivated under AKI conditions in the presence or absence of DPO (100 μM final conc.) and fluorescence was measured 2 h after switching to aerating conditions. mKate2 levels of the Δtdh cells cultivated without DPO were set to 1. Error bars represent the SD of three biological replicates.

Figure 5.

AI-2, CAI-1 and DPO act in concert to repress AphA production. (A) The V. cholerae ΔluxS, cqsA, tdh mutant was cultivated in M9 minimal media containing the indicated autoinducers (5 μM final conc. each) to OD₆₀₀ = 0.2. AphA::3XFLAG, Qrr4 and VqmR levels were monitored on Western Blots and Northern Blots, respectively. RNAP (Western Blot) and 5S rRNA (Northern Blot) served as loading controls. (B) Quantification of (A). AphA levels in the mock-treated sample was set to 1. Error bars represent the SD of three biological triplicates.

Figure 6.

Genome-wide transcriptome changes in response to the AI-2, DPO and CAI-1 autoinducers. Heatmap displaying 420 genes differentially expressed (≥2-fold) in response to at least one of the autoinducers. V. cholerae ΔluxS, cqsA, tdh cells were cultivated in M9 minimal media containing single or combinations of the autoinducers (5 μM final conc. each). Selected gene clusters showing significant regulation are highlighted on the right. Fold changes of the normalized expression values were calculated relative to the normalized expression values of the mock treated replicates.