A Type VI Secretion System Effector Protein, VgrG1, from Aeromonas hydrophila That Induces Host Cell Toxicity by ADP Ribosylation of Actin

Abstract

We recently delineated the importance of a type VI secretion system (T6SS) gene cluster in the virulence of diarrheal isolate SSU of Aeromonas hydrophila and showed that VasH, a 54 activator and T6SS component, was involved in the production of its associated effectors, e.g., hemolysin-coregulated protein. To identify additional T6SS effectors and/or secreted proteins, we subjected culture supernatants from deletion mutants of A. hydrophila, namely, a ⌬act mutant (a T2SS-associated cytotoxic enterotoxin-encoding gene) and a ⌬act ⌬vasH mutant, to 2-dimensional gel electrophoresis and mass spectrometric analysis. Based on these approaches, we identified a member of the VgrG protein family, VgrG1, that contained a vegetative insecticidal protein (VIP-2) domain at its carboxyl-terminal end. Consequently, the vgrG1 gene was cloned in pBI-EGFP and pET-30a vectors to be expressed in HeLa Tet-Off cells and Escherichia coli, respectively. We assessed the ADP-ribosyltransferase (ADPRT) activity of various domains of purified recombinant VgrG1 (rVgrG1) and provided evidence that only the full-length VgrG1, as well as its carboxyl-terminal domain encoding the VIP-2 domain, showed ADPRT activity. Importantly, bacterium-host cell interaction was needed for the T6SS to induce cytotoxicity in eukaryotic cells, and we demonstrated translocation of VgrG1. Furthermore, our data indicated that expression of the genes encoding the full-length VgrG1 and its carboxyl-terminal domain in HeLa Tet-Off cells disrupted the actin cytoskeleton, which was followed by a decrease in cell viability and an increase in apoptosis. Taken together, these findings demonstrated for the first time that VgrG1 of A. hydrophila possessed actin ADPRT activity associated with its VIP-2 domain and that this domain alone was able to induce a rounded phenotype in HeLa Tet-Off cells, followed by apoptosis mediated by caspase 9 activation.

Recruitment of Dendritic Cells Is Responsible for Intestinal Epithelial Damage in the Pathogenesis of Necrotizing Enterocolitis by Cronobacter sakazakii

“…It is possible that the OmpA receptor in enterocytes could be modulated by TGF-b produced by both DCs and enterocytes. Because OmpA is present in all Cronobacter strains, it is possible that other virulence factors such as type VI secretion pathways, a newly described mechanism for protein transport across the membrane of Gram-negative bacteria that can enhance the apoptotic machinery in epithelial cells, may be expressed upon the interaction of OmpA with enterocytes (48)(49)(50). In summary, we have demonstrated a novel observation that OmpA + -C. sakazakii interaction with DCs exacerbates the production of NO in enterocytes and induces intestinal epithelial cell monolayer leakage and apoptosis.…”

In vivo actin cross-linking induced by Vibrio cholerae type VI secretion system is associated with intestinal inflammation

“…This strain required T6SS for virulence in mouse lethality assays, and immunization with purified Hcp protected against lethal infection (25). This T6SS caused contact-dependent host cell rounding and could translocate a VgrG homolog that contains an effector domain homologous to VIP-2, an insecticidal toxin with ADP ribosylation activity specific for actin (26). Catalytic activity was demonstrated in vitro, and transfection of this domain caused disruption of the actin cytoskeleton, cell rounding, and apoptosis.…”

Structural Characterization and Oligomerization of the TssL Protein, a Component Shared by Bacterial Type VI and Type IVb Secretion Systems

“…This pathway has been recently described to translocate protein substrates into both eukaryotic and prokaryotic cells (1)(2)(3)(4)(5)(6). T6SS-dependent translocation of proteins into eukaryotic cells leads to cytoskeleton rearrangements and cytotoxicity (7)(8)(9). More recently, the translocation of canonical protein effectors degrading the peptidoglycan layer into the periplasm of recipient bacterial cells has been demonstrated (10,11).…”