ToxR Antagonizes H-NS Regulation of Horizontally Acquired Genes to Drive Host Colonization

Abstract

The virulence regulator ToxR initiates and coordinates gene expression needed by Vibrio cholerae to colonize the small intestine and cause disease. Despite its prominence in V. cholerae virulence, our understanding of the direct ToxR regulon is limited to four genes: toxT, ompT, ompU and ctxA. Here, we determine ToxR's genome-wide DNA-binding profile and demonstrate that ToxR is a global regulator of both progenitor genome-encoded genes and horizontally acquired islands that encode V. cholerae's major virulence factors and define pandemic lineages. We show that ToxR shares more than a third of its regulon with the histone-like nucleoid structuring protein H-NS, and antagonizes H-NS binding at shared binding locations. Importantly, we demonstrate that this regulatory interaction is the critical function of ToxR in V. cholerae colonization and biofilm formation. In the absence of H-NS, ToxR is no longer required for V. cholerae to colonize the infant mouse intestine or for robust biofilm formation. We further illustrate a dramatic difference in regulatory scope between ToxR and other prominent virulence regulators, despite similar predicted requirements for DNA binding. Our results suggest that factors in addition to primary DNA structure influence the ability of ToxR to recognize its target promoters.

Fig 1. ToxR positively and negatively regulates genes important for biofilm formation.

(A) ToxR ChIP fold enrichment of the promoter regions of ryhB, vpsL, VC1599, and leuO. Enrichment of a non-ToxR regulated icd promoter region is shown as a negative control. ToxR enrichment of the ryhB, vpsL, and VC1599 promoter regions is statistically significant relative to the icd promoter. ****p < 0.0001; **p < 0.01; *p < 0.05, unpaired two-tailed Student’s t test. (B) Northern blot for RyhB. Equal amounts of total RNA were loaded. The 5S blot is shown for a loading control. All Northern blots were performed in biological triplicate. RyhB expression increased 3.3 ± 0.1 fold in the ΔtoxRS mutant compared to the wild type strain. Mean with standard error of the mean (SEM) reported, p < 0.001, unpaired two-tailed Student’s t test. A representative image is shown. All samples for this image were processed on the same gel. (C) qRT-PCR analysis of vpsL and VC1599 gene expression. The expression level of these genes in the ΔtoxRS+ptoxRS strain is shown, normalized to expression levels in the ΔtoxRS+vector control strain, which was set at 1. Expression of vpsL increased, while expression of VC1599 decreased in the ΔtoxRS+ptoxRS strain compared to the control. **p < 0.005, unpaired two-tailed Student’s t test. (D) Quantification of biofilm formation in rich broth at 30°C. ΔtoxRS, ΔVC1599, ΔryhB::kan ^R and ΔvpsL mutant strains show a defect in biofilm formation compared to the wild-type strain. ****p < 0.0001; *p < 0.05, unpaired two-tailed Student’s t test. (E) Representative images of biofilm formation. For panels A, C and D, mean with standard error of the mean (SEM) is shown.

Fig 2. ToxR binds to promoter regions on all four major Vibrio pathogenicity islands.

ToxR ChIP enrichment of the promoter regions of selected genes located on horizontally acquired islands (A) VPI-1, (B) VSP-1, (C) VSP-2, and (D) VPI-2. Enrichment of a non-ToxR-dependent promoter region of icd is shown as the negative control. ToxR enrichment of indicated promoter regions is statistically significant compared to the control. ****p < 0.0001; **p < 0.01; *p < 0.05, unpaired two-tailed Student’s t test. Mean with standard error of the mean (SEM) is shown.

Fig 3. ToxR negatively regulates VSP-1 encoded genes affecting host colonization.

(A) qRT-PCR analysis of VC0176, VC0178, and VC0493 gene expression. The expression level of these genes in the ΔtoxRS+ptoxRS strain is shown, normalized to expression levels in the ΔtoxRS+vector control strain, which was set at 1. VC0176, VC0178, and VC0493 gene expression is decreased in ΔtoxRS+ptoxRS compared to the control. ****p < 0.0001, unpaired two-tailed Student’s t test. (B) Competition assays of ΔVC0176 mutant vs. wild type in vitro and in vivo in the infant mouse model. Each point represents an individual mouse result. ΔVC0176 has a colonization defect vs. wild type in vivo in the infant mouse model compared to in vitro. ****p < 0.0001, unpaired two-tailed Student’s t test.

Fig 4. ToxR antagonizes H-NS function to control host colonization and biofilm formation.

(A) ChIP enrichment of H-NS on the indicated promoter regions in the presence (ptoxRS) or absence (control) of toxRS ectopic expression. H-NS enrichment on the indicated promoter regions is decreased in the presence of toxRS ectopic expression (ptoxRS) compared to in the absence of toxRS ectopic expression (control). *p < 0.05, unpaired two-tailed Student’s t test. (B) Competition assays of indicated V. cholerae mutants vs. wild type strain in the infant mouse intestinal colonization model. The fold change difference between the indicated strains is shown alongside the statistical significance. Statistical significance was determined by One-Way ANOVA analysis followed by a Tukey’s multiple comparison post-test, ***p < 0.001. (C) Quantification of biofilm formation in rich broth at 30°C, All biofilm measurements were normalized to the wild-type strain which was set at 1. ΔtoxRS mutant shows a defect in biofilm formation compared to the wild-type strain, while the Δhns mutant and ΔtoxRSΔhns double mutant have increased biofilm formation compared to the wild-type strain. ****p < 0.0001, unpaired two-tailed Student’s t test. Mean with standard error of the mean (SEM) is shown.

Fig 5. TcpP directly regulates toxT, ompT, and VCA0536.

(A) TcpP ChIP enrichment of the promoter regions of toxT, ompT, and VCA0536. Enrichment of a non-TcpP-dependent promoter icd is shown as a control. TcpP enrichment of the promoter regions of toxT, ompT, and VCA0536 is statistically significant compared to the control. **p < 0.01; *p < 0.05, unpaired two-tailed Student’s t test. (B) qRT-PCR analysis of ompT and VCA0536 gene expression. The expression level of these genes in the ΔtcpPH+ptcpPH strain is shown, normalized to expression levels in the ΔtcpPH+vector control strain, which was set at 1. ompT expression is decreased in ΔtcpPH+ptcpPH compared to the control strain, while VCA0536 expression is increased in ΔtcpPH+ptcpPH compared to the control strain. ****p < 0.0001; **p < 0.005, unpaired two-tailed Student’s t test. Mean with standard error of the mean (SEM) is shown.

Fig 6. Predicted ToxR binding motif from ChIP-seq data.

Sequences within ToxR ChIP-Seq peaks were used as input to identify a potential ToxR binding motif using GLAM2 software. This motif predicted from our ChIP-Seq data contains the previously established ToxR consensus sequence (TNAAA-N₅-TNAAA) within it, which corresponds to the sequence underlined in blue. Larger letters indicate stronger base preferences at those sites.

Fig 7. The direct ToxR regulon and its relationship to H-NS regulation.

Genes clustered by general function or by location on acquired elements under ToxR and H-NS regulation are shown. Horizontally acquired elements are underlined. Arrows indicate positive regulation and perpendicular lines indicate negative regulation. Clusters containing genes bound by H-NS are shaded.