Role of Hcp, a type 6 secretion system effector, of Aeromonas hydrophila in modulating activation of host immune cells

Abstract

Recently, we reported that the type 6 secretion system (T6SS) of Aeromonas hydrophila SSU plays an important role in bacterial virulence in a mouse model, and immunization of animals with the T6SS effector haemolysin co-regulated protein (Hcp) protected them against lethal infections with wild-type bacteria. Additionally, we showed that the mutant bacteria deleted for the vasH gene within the T6SS gene cluster did not express the hcp gene, while the vasK mutant could express and translocate Hcp, but was unable to secrete it into the extracellular milieu. Both of these A. hydrophila SSU mutants were readily phagocytosed by murine macrophages, pointing to the possible role of the secreted form of Hcp in the evasion of the host innate immunity. By using the ΔvasH mutant of A. hydrophila, our in vitro data showed that the addition of exogenous recombinant Hcp (rHcp) reduced bacterial uptake by macrophages. These results were substantiated by increased bacterial virulence when rHcp was added along with the ΔvasH mutant in a septicaemic mouse model of infection. Analysis of the cytokine profiling in the intraperitoneal lavage as well as activation of host cells after 4 h of infection with the ΔvasH mutant supplemented with rHcp indicated that this T6SS effector inhibited production of pro-inflammatory cytokines and induced immunosuppressive cytokines, such as interleukin-10 and transforming growth factor-β, which could circumvent macrophage activation and maturation. This mechanism of innate immune evasion by Hcp possibly inhibited the recruitment of cellular immune components, which allowed bacterial multiplication and dissemination in animals, thereby leading to their mortality.

Evaluation of the roles played by Hcp and VgrG type 6 secretion system effectors in Aeromonas hydrophila SSU pathogenesis

“…Cell pellets were directly lysed in SDS-PAGE loading buffer, while supernatant proteins were first precipitated with 10 % TCA and then dissolved in the loading buffer. Supernatant and pellet samples were then subjected to SDS-PAGE followed by immunoblotting with mouse polyclonal antibodies raised against Hcp, according to the procedure described by us previously (Suarez et al, 2010b). In short, following SDS-PAGE, proteins were transferred to hybond-ECL nitrocellulose membranes (GE Healthcare).…”

“…Therefore, we evaluated the ability of each of the aforementioned nine mutant strains to secrete Hcp by using a polyclonal anti-Hcp antibody that does not distinguish between Hcp-1 and Hcp-2 (Suarez et al, 2010b). As shown in Fig.…”

“…Previously, we have demonstrated that Hcp is translocated into the host cytoplasm and induced apoptosis via caspase 3 activation and that Hcp paralysed macrophages and prevented phagocytosis (Suarez et al, 2010b). The T6SS needle conduit comprises VgrG proteins, and one current model has indicated that the carboxyl terminus of VgrG punctures the host cell membrane, allowing the active domain of VgrG and Hcp to be translocated into the host cytoplasm .…”

Contribution of nuclease to the pathogenesis of Aeromonas hydrophila

“…23,24 Likewise, the T6SS effector hemolysin co-regulated protein reduces A. hydrophila uptake by macrophages and interferes with macrophage activation and maturation by inhibiting proinflammatory cytokines and inducing immunosuppressive cytokines. 25 An outer membrane endopeptidase of A. hydrophila activates big endothelin-3 and is required to induce pathognomonic skin ulcers. 26 Further, the repeat in toxin A (RtxA) of A. hydrophila possesses a functional actin cross-linking domain that induces host cell rounding and apoptosis.…”

“…The fish were maintained at [25][26] C in a recirculating freshwater system at the College of Fisheries and acclimatized for 2 weeks before experiments were performed. The fish were fed twice daily with commercial bream feed (Haid Company, China).…”