Molecular characterization of a functional type VI secretion system from a clinical isolate of Aeromonas hydrophila

Abstract

Our laboratory recently molecularly characterized the type II secretion system (T2SS)-associated cytotoxic enterotoxin (Act) and the T3SS-secreted AexU effector from a diarrheal isolate SSU of Aeromonas hydrophila. The role of these toxin proteins in the pathogenesis of A. hydrophila infections was subsequently delineated in in vitro and in vivo models. In this study, we characterized the new type VI secretion system (T6SS) from isolate SSU of A. hydrophila and demonstrated its role in bacterial virulence. Study of the role of T6SS in bacterial virulence is in its infancy, and there are, accordingly, only limited, recent reports directed toward a better understanding its role in bacterial pathogenesis. We have provided evidence that the virulence-associated secretion (vas) genes vasH (Sigma 54-dependent transcriptional regulator) and vasK (encoding protein of unknown function) are essential for expression of the genes encoding the T6SS and/or they constituted important components of the T6SS. Deletion of the vasH gene prevented expression of the potential translocon hemolysin coregulated protein (Hcp) encoding gene from bacteria, while the vasK gene deletion prevented secretion but not translocation of Hcp into host cells. The secretion of Hcp was independent of the T3SS and the flagellar system. We demonstrated that secreted Hcp could bind to the murine RAW 264.7 macrophages from outside, in addition to its ability to be translocated into host cells. Further, the vasH and vasK mutants were less toxic to murine macrophages and human epithelial HeLa cells, and these mutants were more efficiently phagocytosed by macrophages. We also provided evidence that the expression of the hcp gene in the HeLa cell resulted in apoptosis of the host cells. Finally, the vasH and vasK mutants of A. hydrophila were less virulent in a septicemic mouse model of infection, and animals immunized with recombinant Hcp were protected from subsequent challenge with the wild-type (WT) bacterium. In addition, mice infected with the WT A. hydrophila had circulating antibodies to Hcp, indicating an important role of T6SS in the pathogenesis of A. hydrophila infections. Taken together, we have characterized the T6SS from Aeromonas for the first time and provided new features of this secretion system not yet known for other pathogens.

Phytobacterial Type VI Secretion System - Gene Distribution, Phylogeny, Structure and Biological Functions

“…Hcp secretion was independent of the T3SS and the flagellar system and the secreted protein was capable of binding to the murine macrophages from the outside, in addition to being translocated into mammalian model host cells; heterologous expression of this protein in HeLa cells increased the rate of apoptosis mediated by caspase 3 activation (Suarez et al, 2008). These findings are consistent with Hcp being secreted/translocated by T6SS, along with other yet unidentified effectors.…”

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

“…A. hydrophila clinical isolate SSU encodes a VgrG C-terminal effector domain. 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).…”

Structural similarity of tailed phages and pathogenic bacterial secretion systems

“…However, less is known about the organization, function, and mechanism of the T6SS system first discovered by Mekalanos and coworkers in 2006 (3). After their initial report, a number of bioinformatics-based comparative analyses of the T6SS gene clusters between bacterial strains have been made to assess possible function (4)(5)(6)(7)(8)(9)(10). In this issue of PNAS, both Leiman et al (11) and Pell et al (12) use structure-based analysis to demonstrate the existence of a structure/function relationship between the molecular components of T6SS and the tail proteins of bacteriophages T4 (11) and (12).…”