Bacterial Martyrdom: Phagocytes Disabled by Type VI Secretion after Engulfing Bacteria
Authors: Karla J.F. Satchell
Journal: Cell Host & Microbe (2009)

To colonize during disease and prevent consumption by environmental unicellular eukaryotes, bacteria often disrupt phagocytosis. In this issue, Ma et al., 2009Ma A.T. McAuley S. Pukatzki S. Mekalanos J.J. Cell Host Microbe. 2009; 5 (this issue): 234-243Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar show that Vibrio cholerae delivers the actin-crosslinking T6SS effector VgrG-1 following phagocytosis. The effector then causes irreversible cytoskeleton destruction, leading to bystander protection of the extracellular bacterial population. To colonize during disease and prevent consumption by environmental unicellular eukaryotes, bacteria often disrupt phagocytosis. In this issue, Ma et al., 2009Ma A.T. McAuley S. Pukatzki S. Mekalanos J.J. Cell Host Microbe. 2009; 5 (this issue): 234-243Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar show that Vibrio cholerae delivers the actin-crosslinking T6SS effector VgrG-1 following phagocytosis. The effector then causes irreversible cytoskeleton destruction, leading to bystander protection of the extracellular bacterial population. Vibrio cholerae is an extracellular pathogen that causes diarrhea by secretion of toxins that elicit fluid secretion from intestinal enterocytes. The ability of V. cholerae to establish persistent colonization of the small intestine has been suggested to require evasion of host phagocytic cells through the action of the actin-crosslinking toxin MARTX and other secreted factors (Olivier et al., 2007Olivier V. Salzman N.H. Satchell K.J. Infect. Immun. 2007; 75: 5043-5051Crossref PubMed Scopus (68) Google Scholar). In this issue, Ma and colleagues (Ma et al., 2009Ma A.T. McAuley S. Pukatzki S. Mekalanos J.J. Cell Host Microbe. 2009; 5 (this issue): 234-243Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar) present evidence that some strains of V. cholerae may, in addition, utilize type VI secretion to prevent phagocytosis. Type VI secretion systems (T6SS) are clusters of 12–20 genes found in many Gram-negative pathogens and include the secreted proteins Hcp and VgrG, two highly conserved components of nearly all known T6SS (Cascales, 2008Cascales E. EMBO Rep. 2008; 9: 735-741Crossref PubMed Scopus (247) Google Scholar). The functional significance of these genes was initially characterized in V. cholerae, where secretion of Hcp and VgrG proteins to supernatant fluids was shown to be important as a defense against predation by Dictyostelium discoidium (Pukatzki et al., 2006Pukatzki S. Ma A.T. Sturtevant D. Krastins B. Sarracino D. Nelson W.C. Heidelberg J.F. Mekalanos J.J. Proc. Natl. Acad. Sci. USA. 2006; 103: 1528-1533Crossref PubMed Scopus (790) Google Scholar). Hcp assembles into hexameric ring structures that may form a regulated pore for secretion of putative effectors out of the bacterium (Mougous et al., 2007Mougous J.D. Gifford C.A. Ramsdell T.L. Mekalanos J.J. Nat. Cell Biol. 2007; 9: 797-803Crossref PubMed Scopus (224) Google Scholar). Expression of Aeromonas hydrophila Hcp within eukaryotic cells is associated with induction of apoptosis, possibly related to its pore-forming activity (Suarez et al., 2008Suarez G. Sierra J.C. Sha J. Wang S. Erova T.E. Fadl A.A. Foltz S.M. Horneman A.J. Chopra A.K. Microb. Pathog. 2008; 44: 344-361Crossref PubMed Scopus (160) Google Scholar). VgrG proteins, which have homology to trimeric phage T4 tail spike proteins, are suggested to assemble into a trimer that associates with Hcp. It is proposed that VgrG proteins thus extend away from the bacterium to contact the eukaryotic cell membrane and transfer effectors directly to the target eukaryotic cell cytosol. Some of the many VgrG proteins are referred to as "evolved VgrGs," as they are fusions of VgrG to enzymes, suggesting that they are effectors directly introduced into the host cell by the attached VgrG (Pukatzki et al., 2007Pukatzki S. Ma A.T. Revel A.T. Sturtevant D. Mekalanos J.J. Proc. Natl. Acad. Sci. USA. 2007; 104: 15508-15513Crossref PubMed Scopus (514) Google Scholar). Study of T6SS has been extremely active in the past few years, but clear distinction of T6SS effector molecules has been hampered by the fact that the secreted proteins Hcp and VgrG are both potential effectors as well as necessary components of the secretion machinery. The best-characterized "evolved VgrG" is VgrG-1 secreted by V. cholerae. This VgrG protein has an attached C-terminal actin-crosslinking domain (ACD) that is 59% identical to the well-characterized ACD from the V. cholerae MARTX toxin, also known as RtxA (Sheahan et al., 2004Sheahan K.L. Cordero C.L. Satchell K.J. Proc. Natl. Acad. Sci. USA. 2004; 101: 9798-9803Crossref PubMed Scopus (113) Google Scholar). The MARTX ACD covalently links actin by introducing an isopeptide bond between lysine 50 and glutamate 270 of different G-actin monomers and sequentially adding monomeric or crosslinked actin to form long-chain oligomers (Kudryashov et al., 2008Kudryashov D.S. Durer Z.A. Ytterberg A.J. Sawaya M.R. Pashkov I. Prochazkova K. Yeates T.O. Loo R.R. Loo J.A. Satchell K.J. Reisler E. Proc. Natl. Acad. Sci. USA. 2008; 105: 18537-18542Crossref PubMed Scopus (55) Google Scholar). Actin crosslinking represents a highly efficient mechanism for cell rounding, not only because the reaction depletes the cellular G-actin pool, stimulating cytoskeleton depolymerization, but also because crosslinked forms of actin actively disrupt actin polymerization by inhibiting further extension of a growing actin filament (Kudryashov et al., 2008Kudryashov D.S. Durer Z.A. Ytterberg A.J. Sawaya M.R. Pashkov I. Prochazkova K. Yeates T.O. Loo R.R. Loo J.A. Satchell K.J. Reisler E. Proc. Natl. Acad. Sci. USA. 2008; 105: 18537-18542Crossref PubMed Scopus (55) Google Scholar). Similar to the MARTX ACD, the ACD of VgrG-1 crosslinks actin when transiently expressed in HEp-2 and COS-7 cells (Sheahan et al., 2004Sheahan K.L. Cordero C.L. Satchell K.J. Proc. Natl. Acad. Sci. USA. 2004; 101: 9798-9803Crossref PubMed Scopus (113) Google Scholar), and VgrG-1 protein crosslinks purified actin in vitro (Pukatzki et al., 2007Pukatzki S. Ma A.T. Revel A.T. Sturtevant D. Mekalanos J.J. Proc. Natl. Acad. Sci. USA. 2007; 104: 15508-15513Crossref PubMed Scopus (514) Google Scholar). In the study by Ma et al. (Ma et al., 2009Ma A.T. McAuley S. Pukatzki S. Mekalanos J.J. Cell Host Microbe. 2009; 5 (this issue): 234-243Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar), mutant forms of VgrG-1 were created, in which the ACD was replaced with either an epitope tag, which allows monitoring secretion by western blotting, or with β-lactamase, which facilitates monitoring of translocation using the FRET reagent CCF2-AM. It was found that the modified VgrG-1 can still secrete Hcp and VgrG-1 to supernatant fluids, but it no longer functioned to crosslink actin, induce rounding of macrophages, or kill Dictyostelium. These results dissociate the structural function of VgrG-1 from its effector function. Since ACD directly targets monomeric actin and not filamentous actin, treatment of cells with actin polymerization inhibitors like cytochalasin D would be expected to enhance crosslinking by increasing the pool of G-actin, as was previously demonstrated for actin crosslinking by MARTX (Cordero et al., 2006Cordero C.L. Kudryashov D.S. Reisler E. Satchell K.J. J. Biol. Chem. 2006; 281: 32366-32374Crossref PubMed Scopus (70) Google Scholar). By contrast, VgrG-1-mediated actin crosslinking is completely blocked in macrophage cell lines treated with cytochalasin D. This led the authors (Ma et al., 2009Ma A.T. McAuley S. Pukatzki S. Mekalanos J.J. Cell Host Microbe. 2009; 5 (this issue): 234-243Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar) to speculate that VgrG-1 is not transferred across the eukaryotic plasma membrane but rather across a vacuolar membrane after endocytosis, a process that requires actin polymerization. Indeed, 5% of the bacteria are viable intracellularly for up to 1 hr after phagocytosis. Inhibition of acidification of the vacuole by bafilomycin A increased survival by over 200%, suggesting that the bacteria are normally eliminated by macrophages, but not before they have had a chance to deliver the VgrG-1 to the cytosol to initiate actin crosslinking. As an independent verification of a requirement for endocytosis, nonphagocytic cells that are normally resistant to actin crosslinking by VgrG-1 were shown to be susceptible after transfection with the FcγRII to facilitate opsonophagocytosis using anti-Vibrio antibodies. Thus, bacteria that are engulfed by a phagocytic cell can successfully deactivate the ability of the cell to initiate a new round of phagocytosis (Figure 1). In this way, individual V. cholerae bacteria execute a martyrdom attack, delivering an antiphagocytic toxin across the vacuolar membrane after phagocytosis that then protects the remaining extracellular bacteria. During infection, this process would defend against local phagocytic cells such as macrophages, neutrophils, and dendritic cells, while perhaps simultaneously inhibiting release of chemokines and cytokines for recruitment of reinforcements. In the environment, bacteria would be protected from unicellular eukaryotes that survive by predation on bacteria. The trigger within the vacuole that signals V. cholerae to initiate type VI effector translocation remains to be determined, as does the broader significance of the observation that delivery of VgrG-1 requires phagocytosis. Other T6SS, including that of A. hydrophila (Suarez et al., 2008Suarez G. Sierra J.C. Sha J. Wang S. Erova T.E. Fadl A.A. Foltz S.M. Horneman A.J. Chopra A.K. Microb. Pathog. 2008; 44: 344-361Crossref PubMed Scopus (160) Google Scholar), are associated with inhibition of phagocytosis. Thus, it is likely A. hydrophila and other Gram-negative bacteria use T6SS similar to V. cholerae, assuring survival of the entire population by sacrifice of a few members. Translocation of a Vibrio cholerae Type VI Secretion Effector Requires Bacterial Endocytosis by Host CellsMa et al.Cell Host & MicrobeMarch 19, 2009In BriefThe type VI secretion system (T6SS) is a virulence mechanism common to several Gram-negative pathogens. In Vibrio cholerae, VgrG-1 is required for T6SS-dependent secretion. VgrG-1 is also secreted by T6SS and displays a C-terminal actin crosslinking domain (ACD). Using a heterologous reporter enzyme in place of the ACD, we show that the effector and secretion functions of VgrG-1 are genetically dissociable with the ACD being dispensable for secretion but required for T6SS-dependent phenotypes. Furthermore, internalization of bacteria is required for ACD translocation into phagocytic target cells. Full-Text PDF Open Archive