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, Jian Sha, A. J. Horneman, Ashok Chopra

Research output: Contribution to journalArticle

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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 σ54 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 Δact mutant (a T2SS-associated cytotoxic enterotoxin-encoding gene) and a Δact ΔvasH 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.

Original languageEnglish (US)
Pages (from-to)155-168
Number of pages14
JournalJournal of Bacteriology
Volume192
Issue number1
DOIs
StatePublished - Jan 2010

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ADP Ribose Transferases
Aeromonas hydrophila
Apoptosis
Proteins
Hemolysin Proteins
Caspase 9
Enterotoxins
Eukaryotic Cells
Multigene Family
Actin Cytoskeleton
Cell Communication
Genes
Virulence
Electrophoresis
Actins
Cell Survival
Gels
Escherichia coli
Bacteria
Phenotype

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

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A type VI secretion system effector protein, VgrG1, from Aeromonas hydrophila that induces host cell toxicity by ADP ribosylation of actin. / Suarez, G.; Sierra, J. C.; Erova, T. E.; Sha, Jian; Horneman, A. J.; Chopra, Ashok.

In: Journal of Bacteriology, Vol. 192, No. 1, 01.2010, p. 155-168.

Research output: Contribution to journalArticle

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