Date Published: December 5, 2013
Publisher: Public Library of Science
Author(s): Sarah T. Miyata, Daniel Unterweger, Sydney P. Rudko, Stefan Pukatzki, Joseph D. Mougous.
The Vibrio cholerae type VI secretion system (T6SS) assembles as a molecular syringe that injects toxic protein effectors into both eukaryotic and prokaryotic cells. We previously reported that the V. cholerae O37 serogroup strain V52 maintains a constitutively active T6SS to kill other Gram-negative bacteria while being immune to attack by kin bacteria. The pandemic O1 El Tor V. cholerae strain C6706 is T6SS-silent under laboratory conditions as it does not produce T6SS structural components and effectors, and fails to kill Escherichia coli prey. Yet, C6706 exhibits full resistance when approached by T6SS-active V52. These findings suggested that an active T6SS is not required for immunity against T6SS-mediated virulence. Here, we describe a dual expression profile of the T6SS immunity protein-encoding genes tsiV1, tsiV2, and tsiV3 that provides pandemic V. cholerae strains with T6SS immunity and allows T6SS-silent strains to maintain immunity against attacks by T6SS-active bacterial neighbors. The dual expression profile allows transcription of the three genes encoding immunity proteins independently of other T6SS proteins encoded within the same operon. One of these immunity proteins, TsiV2, protects against the T6SS effector VasX which is encoded immediately upstream of tsiV2. VasX is a secreted, lipid-binding protein that we previously characterized with respect to T6SS-mediated virulence towards the social amoeba Dictyostelium discoideum. Our data suggest the presence of an internal promoter in the open reading frame of vasX that drives expression of the downstream gene tsiV2. Furthermore, VasX is shown to act in conjunction with VasW, an accessory protein to VasX, to compromise the inner membrane of prokaryotic target cells. The dual regulatory profile of the T6SS immunity protein-encoding genes tsiV1, tsiV2, and tsiV3 permits V. cholerae to tightly control T6SS gene expression while maintaining immunity to T6SS activity.
Vibrio cholerae is the etiological agent of the diarrheal disease cholera. This pathogen utilizes a wide array of virulence factors during host infection including the well-characterized cholera toxin (CT) and toxin-coregulated pilus (TCP). In addition, V. cholerae possesses numerous other virulence factors including the type VI secretion system (T6SS), a recently described mechanism used by numerous Gram-negative bacteria to export effectors across their cell envelopes. In contrast to TCP and CT, whose presence is restricted to a subset of virulent V. cholerae strains, the V. cholerae T6SS is highly conserved and is present in strains of all serogroups sequenced to date. Structurally, the T6SS resembles an inverted bacteriophage; it assembles in the V. cholerae cytoplasm and docks onto a baseplate complex situated in the bacterial envelope. Two alleles on the small and large chromosome encode hemolysin-coregulated protein (Hcp) , which polymerizes  and forms the inner tube of the T6SS injectosome and acts as a chaperone for T6SS effectors . Contraction of the outer sheath (formed by VipA and VipB) around the formed Hcp nanotube leads to the ejection of the Hcp tube decorated with a VgrG trimer consisting of three different VgrG proteins: VgrG-1, VgrG-2, and VgrG-3 –. VgrG-1 and VgrG-3 carry enzymatic C-terminal extensions that crosslink actin or degrade the peptidoglycan layer, respectively, upon translocation into target cells , , –. ATP hydrolysis by the inner membrane protein VasK provides the energy for Hcp secretion . A recent report suggests that proteins from the PAAR (proline-alanine-alanine-arginine) repeat superfamily form a sharp conical extension on the VgrG cap. This extension is able to load additional T6SS effectors besides the VgrGs, which are then delivered simultaneously into target cells in a single contraction-driven translocation event .
TA systems are commonly employed by bacteria to ward off bacterial competitors , , , , –. In some bacteria, the presence of an antitoxin or immunity protein prevents self-intoxication and killing by toxins produced by sister cells. Similarly, it appears that T6SS immunity proteins protect against an oncoming attack by neighboring T6SS-active bacteria. By screening a C6706 T6SS transposon library for susceptibility to killing by V. cholerae V52 (Figure S1), we identified three T6SS immunity protein-encoding genes, namely tsiV1, tsiV2, and tsiV3 (Figure 1A). These data agree with recent findings of Dong et al. who also identified tsiV1, tsiV2, and tsiV3 as V. cholerae T6SS immunity protein-encoding genes using a Tn-seq approach .