JB Accepts, published online ahead of print on 30 October 2009

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J. Bacteriol. doi:10.1128/JB.01260-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

A type VI secretion system effector protein VgrG1 from Aeromonas hydrophila that induces host cell toxicity by ADP-ribosylation of actin

G. Suarez, J. C. Sierra, T. E. Erova, J. Sha, A. J. Horneman, and A. K. Chopra^*

Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555; and Department of Medical and Research Technology, University of Maryland School of Medicine and Veterans Administration Medical Health Care Center, Baltimore, MD 21201

^* To whom correspondence should be addressed. Email: achopra{at}utmb.edu.

We recently delineated the importance of a type 6 secretion system (T6SS) gene cluster in the virulence of diarrheal isolate SSU of Aeromonas hydrophila and showed that VasH, a sigma 54 activator and T6SS component, was involved in the production of its associated effectors, e.g., hemolysin co-regulated protein. To identify additional T6SS effectors and/or secreted proteins, we subjected culture supernatants from deletion mutants of A. hydrophila, namely act (a T2SS-associated cytotoxic enterotoxin encoding gene) and act/vasH, to 2-dimensional gel electrophoresis and mass spectrometric analysis. Based on these approaches, we identified a member of the VgrG protein family, (VgrG1), containing 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, bacterial-host cell interaction was needed for the T6SS to induce cytotoxicity in eukaryotic cells, and we demonstrated translocation of VgrG1. Further, 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.