Suppressor Mutations in Type II Secretion Mutants of Vibrio cholerae: Inactivation of the VesC Protease

Abstract

The type II secretion system (T2SS) is a conserved transport pathway responsible for the secretion of a range of virulence factors by many pathogens, including Vibrio cholerae Disruption of the T2SS genes in V. cholerae results in loss of secretion, changes in cell envelope function, and growth defects. While T2SS mutants are viable, high-throughput genomic analyses have listed these genes among essential genes. To investigate whether secondary mutations arise as a consequence of T2SS inactivation, we sequenced the genomes of six V. cholerae T2SS mutants with deletions or insertions in either the epsG, epsL, or epsM genes and identified secondary mutations in all mutants. Two of the six T2SS mutants contain distinct mutations in the gene encoding the T2SS-secreted protease VesC. Other mutations were found in genes coding for V. cholerae cell envelope proteins. Subsequent sequence analysis of the vesC gene in 92 additional T2SS mutant isolates identified another 19 unique mutations including insertions or deletions, sequence duplications, and single-nucleotide changes resulting in amino acid substitutions in the VesC protein. Analysis of VesC variants and the X-ray crystallographic structure of wild-type VesC suggested that all mutations lead to loss of VesC production and/or function. One possible mechanism by which V. cholerae T2SS mutagenesis can be tolerated is through selection of vesC-inactivating mutations, which may, in part, suppress cell envelope damage, establishing permissive conditions for the disruption of the T2SS. Other mutations may have been acquired in genes encoding essential cell envelope proteins to prevent proteolysis by VesC.IMPORTANCE Genome-wide transposon mutagenesis has identified the genes encoding the T2SS in Vibrio cholerae as essential for viability, but the reason for this is unclear. Mutants with deletions or insertions in these genes can be isolated, suggesting that they have acquired secondary mutations that suppress their growth defect. Through whole-genome sequencing and phenotypic analysis of T2SS mutants, we show that one means by which the growth defect can be suppressed is through mutations in the gene encoding the T2SS substrate VesC. VesC homologues are present in other Vibrio species and close relatives, and this may be why inactivation of the T2SS in species such as Vibrio vulnificus, Vibrio sp. strain 60, and Aeromonas hydrophila also results in a pleiotropic effect on their outer membrane assembly and integrity.

Keywords: Vibrio cholerae; protein structure; serine protease; suppressor; type II secretion system.

FIG 1

Vibrio cholerae T2SS mutants display reduced growth rates and lack extracellular serine protease activity. (A) Stationary-phase cultures of WT and mutant strains of V. cholerae were back-diluted to an A₆₀₀ of 0.05 and inoculated into microtiter plates in duplicate. The A₆₀₀ was measured using a Bioscreen growth curve analyzer every 15 min for 20 h. Experiments were performed in triplicate, and means ± SD are displayed. (B) Complementation of eps genes in V. cholerae T2SS mutants restores extracellular protease activity. Protease activity was measured in overnight culture supernatants using a fluorogenic peptide as described in Materials and Methods. Data shown are the means ± SD from at least three independent experiments. *, P < 0.001 versus WT and ΔepsG1pEpsG; #, P < 0.001 versus WT and ΔepsLpEpsL; §, P < 0.001 versus ΔepsMpEpsM (one-way analysis of variance [ANOVA] with Tukey’s multiple-comparison test).

FIG 2

No serine protease activity is detected following expression of VesC-Q279P and VesC-491fs in V. cholerae with a functional T2SS. (A) Protease activity was measured in log-phase culture supernatants of V. cholerae strain N16961 (WT) containing an empty vector as well as the isogenic ΔvesABC strain containing empty vector or plasmids that code for WT VesC, VesC-Q279P, VesC-491fs, or VesC-S225A. Experiments were performed in triplicate or more, with means and SD shown. ***, P < 0.0001 (one-way ANOVA with Tukey’s multiple-comparison test). (B) Culture supernatants of overnight cultures of the strains analyzed in panel A were subjected to SDS-PAGE and silver staining.

FIG 3

Crystal structure of V. cholerae VesC. (A) Structure of VesC is shown in ribbon representation with the protease domain in gold, the Ig-like domain in green, and the CBM domain in blue. The inset shows the active-site residues in stick representation. (B) A structural superposition of VesC and VesB. The protease domain of VesC is in gold, the Ig-like domain of VesC is in green, and VesB is in gray. VesB lacks a CBM domain; hence, no superposition of CBM domains is possible. (C) A structural superposition of the CBM domain of VesC (blue) and Meprin A subunit beta from Homo sapiens (PDB entry 4GWM) (red).

FIG 4

Structural environment of mutated residues in VesC. The substituted and neighboring residues are shown in stick representation. The colors correspond to those of Fig. 3. The local environment is shown for S63R (A), G159V (B), Y277H and Q279P (C), K357insL (D), and P431L and L435P (E). (F) The CBM domain of VesC in ribbon representation. The 491-frameshift alteration indicated by an arrow results in truncation of the last β-strand (cyan) that occupies a central position in one of the β-sheets of the CBM domain.

FIG 5

Overexpression of VesC interferes with growth of the T2SS ΔepsG1 mutant. WT V. cholerae TRH7000 (left column) and the ΔepsG1 mutant (middle column) containing a plasmid encoding WT vesC were grown in the absence (black line) or presence of IPTG (orange line) to induce the production of VesC. Optical density at 600 nm (OD_600nm) of the cultures was monitored over time (shown in minutes). The ΔepsG1 mutant producing the catalytically inactive VesC-S225A variant (right column) was similarly grown without and with IPTG.

FIG 6

Working model of the mechanism by which secondary mutations in vesC may suppress a potential lethal phenotype of T2SS mutants. (Left) Wild-type V. cholerae transports VesC across the outer membrane via the T2SS. Upon inactivation of the T2SS, VesC secretion is blocked and the protease accumulates in the periplasm, where it may be a contributing factor to cell envelope damage through nonspecific proteolysis and a possible lethal phenotype (right). During the process of genetic inactivation of the T2SS, we may select for mutations that inactivate VesC (A) and/or target VesC for degradation (B) to prevent proteolysis of essential components and/or avert irreparable cell envelope damage in the absence of a functional T2SS.