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.

Rules of Engagement: The Type VI Secretion System in Vibrio cholerae

“…The V. cholerae T6SS genes are encoded in one large operon (VCA0105-VCA0124), known as the large or major cluster, and at least three smaller operons known as auxiliary clusters 1, 2, and 3 (VCA0017-VCA0021, VC1415-VC1419, and VCA0284-VCA0286) (Figure 3) [1,43,44]. The large cluster encodes the majority of the T6SS structural components, with the exception of the essential secreted inner tube component Hcp, two of the VgrG proteins, and one PAAR-motif protein (VCA0284) [1,44].…”

“…Auxiliary clusters 1 and 2 also encode for the VgrG-1 (VC1416) and VgrG-2 (VCA0018) tip components respectively, as well as unique effector sets and their cognate immunity proteins [41,44]. The recently identified third auxiliary cluster is predicted to encode for one PAAR motif protein (VCA0284) and a unique effector-immunity pair (VCA0285-86) [22,43]. In addition to the promoters found upstream of each operon, internal promoter activity has been identified within the large cluster and auxiliary clusters 1 and 2 that lie just upstream of the immunity genes [41,45].…”

Identification of divergent type VI secretion effectors using a conserved chaperone domain

“…T6SS-dependent effectors can be experimentally identified by comparing the secretomes of WT and T6SS mutants (3,(29)(30)(31) and by screening for T6SS-encoded immunity proteins (18). Because known effectors lack a common secretion signal, bioinformatic identification of T6SS effectors is challenging.…”

Microbial Herd Protection Mediated by Antagonistic Interaction in Polymicrobial Communities

“…V. cholerae possesses four antimicrobial effectors, namely, VgrG3, which functions as a lysozyme (13,22); the lipase TseL (13,23); VasX, which has a colicin-like domain (13,24); and the hydrolase TseH (25), which seems to play only a minor role in interbacterial killing (25). A. hydrophila has one known effector, TseC, which, like VasX, carries a colicin-like domain (21).…”