Abstract
Type 3 Secretion System (T3SS) is a highly conserved virulence structure that plays an essential role in the pathogenesis of many Gram-negative pathogenic bacteria, including Pseudomonas aeruginosa. Exotoxin T (ExoT) is the only T3SS effector protein that is expressed in all T3SS-expressing P. aeruginosa strains. Here we show that T3SS recognition leads to a rapid phosphorylation cascade involving Abl / PKCδ / NLRC4, which results in NLRC4 inflammasome activation, culminating in inflammatory responses that limit P. aeruginosa infection in wounds. We further show that ExoT functions as the main anti-inflammatory agent for P. aeruginosa in that it blocks the phosphorylation cascade through Abl / PKCδ / NLRC4 by targeting CrkII, which we further demonstrate to be important for Abl transactivation and NLRC4 inflammasome activation in response to T3SS and P. aeruginosa infection.
Highlights
Type 3 Secretion System (T3SS) is a highly conserved virulence structure that plays an essential role in the pathogenesis of many Gram-negative pathogenic bacteria, including Pseudomonas aeruginosa
We further show that P. aeruginosa Exotoxin T (ExoT) dampens the phosphorylation cascade through Abl/PKCδ/ NLRC4 and inhibits NLRC4 inflammasome activation by targeting CrkII, which we further demonstrate to be important for Abl transactivation and NLRC4 inflammasome activation in response to T3SS and P. aeruginosa infection
Wounds were infected with 103 wild-type or T3SS mutant strains and the impact of infection with wild-type and T3SS mutants on the production of IL-1β and IL-18 pro-inflammatory chemokines was assessed by enzyme-linked immunosorbent assay (ELISA) 24 h after infection
Summary
ExoT dampens pro-inflammatory cytokine production in wound. We used PA103—(a representative cytotoxic strain7,8,53), and PAK—(a representative invasive strain7,8), and their isogenic ExoT, ExoS, and ExoU T3SS mutant strains in a full-thickness excisional wound in C57BL/6 mice, as described previously[47,54]. Data indicated that regardless of genetic background, ExoT-deleted mutant strains were significantly impaired in their ability to colonize wound, as manifested by approximately 1–2 log-order reductions in bacterial counts in wound as compared with the wild-type strains (Fig. 2k and Supplementary Fig. 3f). The presence or absence of T3SS or ExoT did not impact the ability of P. aeruginosa to colonize wounds in caspase-1- or NLRC4 inflammasome defective mice, indicating that the primary virulence function of ExoT in wound is to dampen inflammatory responses through NLRC4 inflammasome (Fig. 2k–n, and Supplementary Fig. 3f–g). Data indicated that NLRC4−/− wounds contained significantly more bacteria than normal wounds on days 1, 3, and 6, highlighting the importance of NLRC4 in combating P. aeruginosa infection in wound (Supplementary Fig. 3i) These data indicated that T3SS-expressing P. aeruginosa recognition in wound by NLRC4 canonical inflammasome results in pro-inflammatory responses that limit P. aeruginosa infection. To evaluate the dependence of NLRC4 inflammasome activation on NLRC4 phosphorylation in response to P. aeruginosa infection, we a b 1000 IL-1β
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