A Type VI Secretion System Effector Protein, VgrG1, from Aeromonas hydrophila That Induces Host Cell Toxicity by ADP Ribosylation of Actin
- 1 January 2010
- journal article
- Published by American Society for Microbiology in Journal of Bacteriology
- Vol. 192 (1), 155-168
- https://doi.org/10.1128/jb.01260-09
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 σ 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 Δ act mutant (a T2SS-associated cytotoxic enterotoxin-encoding gene) and a Δ act Δ 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.Keywords
This publication has 49 references indexed in Scilit:
- Translocation of a Vibrio cholerae Type VI Secretion Effector Requires Bacterial Endocytosis by Host CellsCell Host & Microbe, 2009
- The phage λ major tail protein structure reveals a common evolution for long-tailed phages and the type VI bacterial secretion systemProceedings of the National Academy of Sciences of the United States of America, 2009
- Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary originProceedings of the National Academy of Sciences of the United States of America, 2009
- Use of mchI Encoding Immunity to the Antimicrobial Peptide Microcin H47 as a Plasmid Selection Marker in Attenuated Bacterial Live VectorsInfection and Immunity, 2008
- The type VI secretion toolkitEMBO Reports, 2008
- Structural basis of actin recognition and arginine ADP-ribosylation by Clostridium perfringens ι-toxinProceedings of the National Academy of Sciences of the United States of America, 2008
- Molecular characterization of a functional type VI secretion system from a clinical isolate of Aeromonas hydrophilaMicrobial Pathogenesis, 2008
- Type VI secretion system translocates a phage tail spike-like protein into target cells where it cross-links actinProceedings of the National Academy of Sciences of the United States of America, 2007
- CDD: a conserved domain database for interactive domain family analysisNucleic Acids Research, 2007
- A Virulence Locus of Pseudomonas aeruginosa Encodes a Protein Secretion ApparatusScience, 2006