Secretome analysis of Vibrio cholerae type VI secretion system reveals a new effector-immunity pair

Abstract

The type VI secretion system (T6SS) is a dynamic macromolecular organelle that many Gram-negative bacteria use to inhibit or kill other prokaryotic or eukaryotic cells. The toxic effectors of T6SS are delivered to the prey cells in a contact-dependent manner. In Vibrio cholerae, the etiologic agent of cholera, T6SS is active during intestinal infection. Here, we describe the use of comparative proteomics coupled with bioinformatics to identify a new T6SS effector-immunity pair. This analysis was able to identify all previously identified secreted substrates of T6SS except PAAR (proline, alanine, alanine, arginine) motif-containing proteins. Additionally, this approach led to the identification of a new secreted protein encoded by VCA0285 (TseH) that carries a predicted hydrolase domain. We confirmed that TseH is toxic when expressed in the periplasm of Escherichia coli and V. cholerae cells. The toxicity observed in V. cholerae was suppressed by coexpression of the protein encoded by VCA0286 (TsiH), indicating that this protein is the cognate immunity protein of TseH. Furthermore, exogenous addition of purified recombinant TseH to permeabilized E. coli cells caused cell lysis. Bioinformatics analysis of the TseH protein sequence suggest that it is a member of a new family of cell wall-degrading enzymes that include proteins belonging to the YD repeat and Rhs superfamilies and that orthologs of TseH are likely expressed by species belonging to phyla as diverse as Bacteroidetes and Proteobacteria.

Importance: The Gram-negative bacterium Vibrio cholerae causes cholera, a severe and often lethal diarrheal disease. The 2010-2012 epidemic in Haiti and new explosive epidemics in Africa show that cholera remains a significant global public health problem. The type VI secretion system (T6SS) is a dynamic organelle expressed by many Gram-negative bacteria, which use it to inject toxic effector proteins into eukaryotic and bacterial prey cells. In this study, we applied a comparative proteomics approach to the V. cholerae T6SS secretome to identify new substrates of this secretion apparatus. We show that the product of the gene VCA0285 is likely a new peptidoglycan hydrolase that is secreted by T6SS and that its cognate immunity protein is encoded by the gene that is immediately downstream (VCA0286). Bioinformatics analysis shows that VCA0285 carries four conserved motifs that likely define a large family of hydrolases with antibacterial activity. The identification of new antibacterial T6SS effectors provides useful information for the development of novel antibiotics and therapeutic agents.

FIG 1

Comparative secretome analysis identifies a novel V. cholerae T6SS effector candidate. Comparison of individual proteins in the parental strain (ΔFlgG) to those in mutant 1 (ΔFlgG ΔClpV) (A) and mutant 2 (ΔFlgG ΔClpV Δgp25) (B) using quantitative proteomics. A MatLab application was used to compare the average spectral counts (total peptide number) of each protein to analyze the abundance in different samples. The abundances of proteins around the range from −2 to 2 are approximately the same. Previously identified T6SS substrates that were not identified in the mutant secretomes are indicated by red dots. TseH, the new effector identified in this study, is shown by a green dot. Proteins below −6 were not identified in the mutants’ secretomes but only in the parental strain’s secretomes.

FIG 2

Amino acid sequence of the novel T6SS effector TseH and bioinformatics analysis. (A) The peptides of TseH identified by mass spectrometry are in red font. The green highlighting defines the four conserved motifs in 62 BLASTp homologs of TseH. Red highlighting shows the YD repeat regions of the protein. GHAG region is the conserved catalytic motif among different cell wall amidases. (B) HHpred analysis reveals an amidase (amidase_6) or peptidase (peptidase_C97) domain for the TseH protein. (C) Conserved motif of YD repeats generated from the Pfam database. (D) Clustal Omega alignment of TseH and RhsP1. Boxes indicate the most conserved regions between two proteins. The arrow points to the conserved YDY residues. The asterisks indicate identical residues, double dots indicate strong similarity and the single dots indicate weaker similarity. Red indicates small, blue indicates acidic, magenta indicates basic, green indicates Hydroxyl + sulfhydryl + amine and grey indicates unusual amino acids.

FIG 3

Genomic arrangements and conserved motifs of TseH orthologs. (A) Genomic arrangement of TseH ortholog proteins from different species. Colors indicate candidate effector (red), neighboring PAAR proteins (grey), candidate immunity protein (orange), Rhs proteins (blue), and gp25 (purple). (B) WebLogo sequence alignment of TseH orthologs identified with BLASTp. The four conserved motifs are shown by boxes. The accession numbers of protein sequences used to prepare this WebLogo diagram are listed in Table S2 in the supplemental material.

FIG 4

TseH is secreted in a T6SS-dependent manner, and TsiH is the cognate immunity protein. (A) Western blot analysis of the supernatants and the whole-cell pellets of V. cholerae V52 wild type (WT) and the ΔVipA strain carrying the pBAD18 vector encoding VCA0285 with a 3×V5 epitope tag. Protein secretion was tested by using tag-specific antibody. The RNA polymerase subunit RpoB was used to control the level of cell lysis. (B) TseH is toxic in the periplasm of E. coli, and the toxicity is neutralized by coexpression of the cognate immunity protein TsiH. Gene expression was induced by arabinose and repressed by glucose in the medium.

FIG 5

TseH is toxic to V. cholerae. (A) Light microscopic imaging of V. cholerae expressing TseH in the periplasm of the ΔtseH ΔtsiH double mutant. Yellow arrows highlight cells with a spherical shape that are seen frequently when TseH is expressed in the periplasm. Left, cells before induction; right, cells after 5 s of induction. (B) Effect of TseH on T6SS-mediated killing. Left, E. coli survival when E. coli cells were mixed with the wild type, the ΔvasK mutant, and the ΔtseH ΔtsiH mutant of V. cholerae V52; right, the prey is the ΔtseH ΔtsiH mutant. (C) Addition of exogenous TseH to E. coli leads to cell lysis. Results are the means of three independent experiments. Error bars represent the standard deviations derived from all data points.