Conversely, mutating fleQ in PAO1 reduced internalization by >10-fold, also with or without ExoS. Correcting the fleQ mutation in effector-null PA103 promoted internalization by >10-fold with or without ExoS. Intracellular bacteria showed T3SS activation that continued in replicating daughter cells. Except for effector-null PA103, all strains were internalized while encoding ExoS. aeruginosa with and without ExoS transformation, antibiotic exclusion assays, and imaging using a T3SS-GFP reporter. aeruginosa and corresponding effector-null isogenic T3SS mutants, effector-null mutants of cytotoxic P. Here, we addressed mechanisms for this dichotomy using invasive (ExoS-expressing) P. aeruginosa and its occupation of bleb niches in epithelial cells. Paradoxically, ExoS, a type three secretion system (T3SS) effector, has antiphagocytic activities but is required for intracellular survival of P. Pseudomonas aeruginosa is internalized into multiple types of epithelial cell in vitro and in vivo and yet is often regarded as an exclusively extracellular pathogen. These experiments suggest that loss of flagellar motility may primarily benefit PA103 by attenuating pathogen recognition and clearance during acute infection. Restoration of flagellar assembly did, however, amplify IL-1 signals measured during murine pulmonary infection and was associated with increased bacterial clearance. Although a negative correlation between flagellar assembly and Type 3 secretion system (T3SS) expression has been reported previously, we did not observe downregulation of T3SS expression or function in Fla+ PA103. We also investigated the consequences of restoring flagellar assembly on PA103 virulence. Reversal of this seemingly conservative amino acid substitution is sufficient to restore swimming motility to PA103, despite the presence of mutations in other flagellar genes of PA103. This mutation, which lies just outside the Walker B box of FleQ, abrogates the ability of FleQ to positively regulate flagellar gene expression. We determined that PA103's aflagellate phenotype is the result of a single amino acid change (G240V) in the master flagellar regulator, FleQ. Nonetheless, aflagellate clinical strains are often isolated from acutely and chronically infected patients and include the virulent laboratory strain PA103. Flagellar-based motility plays a critical role in Pseudomonas aeruginosa pathogenesis, influencing both the establishment of bacterial infection and the host's response to the pathogen.
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