Type VI secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin

Abstract

Genes encoding type VI secretion systems (T6SS) are widely distributed in pathogenic Gram-negative bacterial species. In Vibrio cholerae, T6SS have been found to secrete three related proteins extracellularly, VgrG-1, VgrG-2, and VgrG-3. VgrG-1 can covalently cross-link actin in vitro, and this activity was used to demonstrate that V. cholerae can translocate VgrG-1 into macrophages by a T6SS-dependent mechanism. Protein structure search algorithms predict that VgrG-related proteins likely assemble into a trimeric complex that is analogous to that formed by the two trimeric proteins gp27 and gp5 that make up the baseplate "tail spike" of Escherichia coli bacteriophage T4. VgrG-1 was shown to interact with itself, VgrG-2, and VgrG-3, suggesting that such a complex does form. Because the phage tail spike protein complex acts as a membrane-penetrating structure as well as a conduit for the passage of DNA into phage-infected cells, we propose that the VgrG components of the T6SS apparatus may assemble a "cell-puncturing device" analogous to phage tail spikes to deliver effector protein domains through membranes of target host cells.

Fig. 1.

Hcp export requires the presence of VgrG-1 and VgrG-2. Plasmids allowing the inducible expression of HA-tagged VgrG-1 (pVgrG1), vsvG-tagged VgrG-2 (pVgrG2), or plasmid control (control) were introduced into wild-type V. cholerae, or isogenic mutants lacking VgrG-1 (ΔvgrG1) or VgrG-2 (ΔvgrG2). Culture supernatants from cells grown in liquid broth under inducing (+) and noninducing (−) conditions were concentrated and separated by SDS/PAGE; Hcp was visualized by Western blot analysis with Hcp antiserum. To guarantee plasmid-borne expression of HA-tagged VgrG-1 and vsvG-tagged VgrG-2, cell extracts were separated by SDS/PAGE for immunoblotting with HA and vsvG antisera, respectively.

Fig. 2.

Recombinant VgrG-1 cross-links actin in an ATP-dependent fashion. (A) Hexahistidine-tagged VgrG-1 was purified on a nickel-NTA column and separated by SDS/PAGE (see arrow). (B) Recombinant VgrG-1 was incubated at 37°C for 2 h with purified rabbit skeletal monomeric G-actin (Cytoskeleton, Denver, CO) or crude lysates from Dictyostelium and RAW 264.7. Reactions were supplemented with 2 mM Mg-ATP as indicated. Reaction mixtures were separated by SDS/PAGE, and actin was visualized by Western blot analysis with actin antiserum.

Fig. 3.

VgrG-1-mediated cross-linking of the host actin cytoskeleton. Cultured J774 cells were infected for 2 h with indicated V. cholerae strains at a multiplicity of infection (MOI) of 10. Extracts of infected cells were separated by SDS/PAGE for immunoblotting with actin antiserum.

Fig. 4.

VgrGs share structural features with the phage tail of bacteriophage T4. (A) V. cholera VgrGs are a fusion of the phage tail proteins gp27 (gray) and gp5 (hatched), omitting the OB-fold and lysozyme domains (white) of the phage gp5 protein. The gp5 domain consists of a β-helix repeat (black arrows) that forms the cell-puncturing “needle” of the phage tail. VgrG-1 and VgrG-3 contain a C-terminal actin-cross-linking domain (ACD) and peptidoglycan (PG)-binding domain, respectively. Amino acid numbers are designated for gp27, gp5, and VgrG-1. Crystal structure for the phage tail spike (gp27 and gp5) are shown for reference (24) (adapted by permission from Macmillan Publishers Ltd: Nature, 31 January 2002). (B) Graphical representation of a subset of the VgrG-family members with extended C-terminal domains. These proteins consist of a gp27-like domain (solid black), gp5-like domain (hatched) and different C-terminal extensions (bars with various patterns). On the right is shown the predicted structural similarities of the C-terminal domains identified by the PSIPRED protein structure prediction server (http://bioinf.cs.ucl.ac.uk/psipred/). Protein structures schematically shown from top to bottom have the following accession numbers: AAF94573, AAF96037, ZP_00981404, NP_248953, ZP_00832947, CAC92964, YP_652030, and YP_606302. A more extensive list of VgrG orthologs with extended C termini together with PSIPRED predicted structural similarities can be found in SI Table 1.

Fig. 5.

Immunoprecipitation of VgrG-1-myc. V. cholerae strains expressing either wild-type VgrG-1 or a VgrG-1::myc tagged protein were grown to midlogarithmic growth phase. Culture supernatants were filtered and Myc antisera conjugated to agarose beads were added to pull-down VgrG-1::myc and any interacting proteins. Resulting precipitates were separated by SDS/PAGE for immunoblotting with antisera specific for VgrG-1, VgrG-2, and VgrG-3. The bands corresponding to the size of VgrG-1, VgrG-2, and VgrG-3 were identified only in immunoprecipitations from supernatants derived from the strain expressing VgrG-1-myc.