VasH is a transcriptional regulator of the type VI secretion system functional in endemic and pandemic Vibrio cholerae

Abstract

The gram-negative bacterium Vibrio cholerae is the etiological agent of cholera, a disease characterized by the release of high volumes of watery diarrhea. Many medically important proteobacteria, including V. cholerae, carry one or multiple copies of the gene cluster that encodes the bacterial type VI secretion system (T6SS) to confer virulence or interspecies competitiveness. Structural similarity and sequence homology between components of the T6SS and the cell-puncturing device of T4 bacteriophage suggest that the T6SS functions as a molecular syringe to inject effector molecules into prokaryotic and eukaryotic target cells. Although our understanding of how the structural T6SS apparatus assembles is developing, little is known about how this system is regulated. Here, we report on the contribution of the activator of the alternative sigma factor 54, VasH, as a global regulator of the V. cholerae T6SS. Using bioinformatics and mutational analyses, we identified domains of the VasH polypeptide that are essential for its ability to initiate transcription of T6SS genes and established a universal role for VasH in endemic and pandemic V. cholerae strains.

Fig. 1.

Polymorphic VasH carries three conserved domains. (A) Schematic diagram of VasH. The protein is composed of 530 amino acids and is predicted to have three functional domains depicted here: an N-terminal regulator domain, a central domain with two Walker motifs (alignments of Walker boxes of several σ⁵⁴-dependent activators are shown), and a C-terminal DNA binding domain (HTH). Numbers represent amino acid residues. (B) Phylogenetic tree of polymorphic vasH in V. cholerae. The nucleotide sequences of vasH from 26 sequenced strains of V. cholerae were used to generate a neighbor-joining tree. The number of bootstrap data sets was set to 100; bootstrap values are listed for each branch. Serogroups of each strain are listed; N/A, information is not available.

Fig. 2.

Episomal expression of VasH_V52 induces Hcp synthesis but not secretion in N16961. (A) Expression of VasH_V52::myc establishes Hcp and VasX synthesis as shown by Western blotting. Each bacterial strain was grown in LB medium containing ampicillin in the presence or absence of arabinose. Mid-logarithmic-phase cells were separated into pellet and supernatant fractions and subjected to SDS-PAGE followed by Western blotting with anti-VasX, anti-Hcp, anti-Myc, anti-Bla, and anti-DnaK antibodies. (B) Plaque assay showing that overexpression of VasH_V52 does not restore virulence of N16961 ΔvasH. Approximately 1 × 10³ mid-logarithmic D. discoideum cells were plated onto SM/5 nutrient agar plates (with or without 0.1% arabinose) with 100 μl of bacterial strains. Pictures were taken on the third day of incubation at 22°C. (C) Expression of VasH_V52 in N16961 ΔvasH does not restore the antimicrobial property of V. cholerae. The T6SS-mediated E. coli killing property was tested with V52 ΔvasH and N16961 ΔvasH carrying plasmid pVasH_V52::myc. Each V. cholerae strain was mixed with rifampin-resistant E. coli MG1655 at a 10:1 ratio and spotted on LB agar plates in the presence or absence of 0.1% arabinose. After 4 h of incubation at 37°C, bacterial cells were recovered from the plates, and serial dilutions were spotted on LB plates supplemented with rifampin to select for surviving E. coli. The experiment was done in duplicate. α, anti.

Fig. 3.

The El Tor strain N16961 encodes a functional VasH. (A) Western blot depicting complementation of a vasH-null mutation with VasH_N16961 in V52 and N16961. V52 ΔvasH and N16961 ΔvasH strains were complemented with either VasH_V52 or VasH_N16961 and grown until mid-logarithmic phase in the presence or absence of arabinose. Both pellet and culture supernatant fractions were subjected to SDS-PAGE and Western blot analysis using the indicated antibodies to detect T6SS-dependent protein expression and secretion. (B) VasH_N16961 activates transcription of T6SS genes to wild-type level. V52 ΔvasH was transformed with pVasH_V52::myc or pVasH_N16961::myc, and cells were grown in the absence or presence of arabinose. The mRNA from each treatment was treated with DNase, reverse transcribed, and subjected to RT-qPCR to examine differences in transcription. The experiment was performed in triplicates. (C) Plaque assay showing that VasH_N16961 and VasH_V52 are equally functional. V52 ΔvasH and N16961 ΔvasH strains were complemented with pVasH_V52::myc or pVasH_N16961::myc and tested for their ability to mediate resistance toward D. discoideum grazing. A total of 100 μl of overnight culture and approximately 1 × 10³ AX3 cells were spread on SM/5 plates with or without arabinose. Plates were incubated at 22°C for 3 days before pictures were taken.

Fig. 4.

VasH requires Walker motifs. (A) Western blot analysis comparing Hcp and VasX expression by V52 ΔvasH strains complemented by wild-type and Walker motif mutants of VasH_V52. Bacterial cultures were grown to mid-logarithmic phase of growth in the presence or absence of 0.1% arabinose, and pellet fractions were subjected to SDS-PAGE followed by Western blot analysis using anti-VasX, anti-DnaK (loading control), anti-Myc, and anti-Hcp antibodies. (B) Chromatographs confirming alanine substitutions in the Walker motifs (h indicates hydrophobic residues, x indicates any residue). The codon (AAA) encoding lysine (K) was changed to GCA to encode alanine (A) in the Walker A motif. Aspartic acid (D), encoded by GAT in the Walker B motif, was changed to alanine (GCT). (C) Plaque assay demonstrating that Walker mutants of VasH_V52 are unable to restore virulence toward D. discoideum. Overnight bacterial culture volumes of 100 μl were mixed with 1 × 10³ amoebae on SM/5 nutrient agar plate with or without 0.1% arabinose and incubated for 3 days at 22°C. This figure represents three independent experiments. (D) Walker mutants are unable to initiate transcription of the T6SS genes. Total mRNA was extracted from mid-logarithmically growing cells. Equal amounts of RNA were treated with DNase, reverse transcribed into cDNA, and analyzed with RT-qPCR to determine the transcriptional profile of the T6SS genes: VCA0017 (hcp-2), VCA0018 (vgrG-2), and VCA0020 (vasX). Fold change in transcription is plotted on a log₁₀ scale. Each experiment was carried out in triplicates. WT, wild type.

Fig. 5.

VasH requires its C terminus. (A) Western blot depicting Hcp and VasX pellet fractions of V52 ΔvasH carrying truncated VasH (VasH_V52-ΔN and VasH_V52-ΔHTH). Pellet fractions from mid-logarithmic bacterial cultures were subjected to SDS-PAGE, followed by Western blotting with anti-VasX, anti-DnaK, anti-Myc, and anti-Hcp antibodies. (B) Plaque assay using V52 ΔvasH/pVasH_V52-ΔN and pVasH_V52-ΔHTH. A total of 100 μl of overnight bacterial culture was spread onto SM/5 nutrient agar plates (with or without 0.1% arabinose) together with approximately 1 × 10³ D. discoideum cells. Images were taken after 3 days of incubation at 22°C. (C) RT-qPCR data showing the ability of VasH_V52-ΔN and VasH_V52-ΔHTH to initiate T6SS gene transcription. V52 ΔvasH/pVasH_V52-ΔN and V52 ΔvasH/pVasH_V52-ΔHTH were grown to mid-logarithmic phase in the presence or absence of arabinose. Total mRNA extracted from each sample was treated with DNase, followed by reverse transcription and RT-qPCR analysis against VCA0017 (hcp-2), VCA0018 (vgrG-2), and VCA0020 (vasX). Fold increases of T6SS genes are plotted in log₁₀ scale on the y axis. Each experiment was done in triplicates.

Fig. 6.

Deletion of the N-terminal regulatory domain of VasH affects Hcp levels. Bacterial cultures were grown to late logarithmic stage in the presence of decreasing amounts of arabinose. Pellet samples were subjected to SDS-PAGE, and Western blotting was performed using anti-DnaK (loading control), anti-Myc (VasH), and anti-Hcp antibodies. MW, molecular weight in thousands.

Fig. 7.

Defined VasH mutants have a dominant negative effect. (A) Western blot depicting the effect of Walker motif mutants on Hcp synthesis. Wild-type V52 strains carrying nonfunctional VasH (VasH_V52-K227A, VasH_V52-D292A, and VasH_V52-ΔHTH) in pBAD24 were grown to mid-logarithmic phase with or without 0.1% arabinose and separated into pellet and supernatant fractions. Both fractions were subjected to SDS-PAGE and Western blotting with the antibodies shown. (B) Episomally expressed VasH Walker mutants compete with the endogenous VasH (VCA0117). Approximately 1 × 10³ amoebae were plated onto SM/5 nutrient agar plates with 100 μl of overnight bacterial culture and incubated for 3 days at 22°C. Results are representative of three independent experiments.