| Pseudomonas aeruginosa is a gram-negative pathogen responsible for opportunistic infections that result in devastating inflammatory responses. One important virulence factor of P. aeruginosa is exoenzyme S (ExoS), which prior to this study, was known to contribute to the inflammatory response, although by an unidentified mechanism. This work has determined that ExoS directly activated both monocytes and T cells. Monocyte activation was not dependent on the presence of T cells and resulted in the production of numerous biologically active pro-inflammatory cytokines and chemokines. T cell activation was in part dependent on monocyte secretion of tumor necrosis factor-alpha TNF-alpha and interleukin-6 (IL-6), although direct T cell stimulation by ExoS did result in T cell activation and also T cell death.; A more detailed analysis of the interactions between monocytes and ExoS led to the observation that ExoS bound a specific receptor on the surface of monocytic cells. ExoS activated both Toll-like receptor-2 (TLR2) and TLR4/MD-2 dependent pathways. The N-terminal domain of ExoS induced TLR4/MD-2-dependent activation, while the C-terminal domain was responsible for TLR2-dependent activity. ExoS activated several intracellular signaling pathways, including the NF-kappaB extracellular signal-regulated kinase 1/2 (Erk 1/2) and Src pathways, which were all involved in ExoS-induced TNF-alpha production.; Src kinases are concentrated within cholesterol-containing detergent resistant membrane (DRM) microdomains, which act as signaling platforms/amplifiers on the surface of cells. ExoS bound a subset of DRM, and induced the phosphorylation of multiple proteins within DRM including Src kinases. Disruption of DRM by cholesterol extraction prevented NF-kappaB and Erk 1/2 activation, as well as TNF-alpha production by ExoS. Disruption of DRM did not prevent ExoS binding, but did release the Src kinase Lyn from DRM and into the detergent-soluble fraction, a site in which Lyn is not active. Additionally, activation of monocytic cells by other TLR agonist such as peptidoglycan, lipopolysaccharide and lipoteichoic acid was also dependent on DRM, suggesting a common requirement for DRM in TLR-mediated signaling. |
