Type III Secretion-Dependent Modulation of Innate Immunity as One of Multiple Factors Regulated byPseudomonas aeruginosaRetS

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Abstract
Mutation ofretS(rtsM) ofPseudomonas aeruginosastrain PA103 reduces its virulence in both ocular and respiratory murine models of infection. In vitro,retSmutants exhibit loss of the ExsA-regulated type III secretion system (TTSS), reduced twitching motility, and a decrease in association with, invasion of, and survival within corneal epithelial cells. In addition, transcription of multiple other virulence genes is positively and negatively affected byretSmutation. Since our published data show that ExoU and ExoT, the two TTSS effectors encoded by strain PA103, each confer virulence in this corneal model, we hypothesized that loss of virulence ofretSmutants follows loss of type III secretion. Corneal pathology, bacterial colonization, and phagocyte infiltration were compared for wild-type PA103,retSmutants, and various TTSS mutants after infection with ∼106CFU bacteria. Results showed that either aretSor anexsA(TTSS) mutation delayed disease progression, as illustrated by reduced severity scores and colonization levels during the first 48 h postinfection. Surprisingly,retSmutant infections then became more severe than those involvingexsAmutants. By day 7, colonization levels ofretSmutants even surpassed those of wild-type bacteria (more than twofold,P = 0.028). AlthoughretSmutants caused more severe opacification of central corneas than both the wild type and theexsAmutants, neither mutant caused the peripheral ring opacity commonly associated with wild-type infection, suggesting that the TTSS was involved. Histological experiments withretSand various TTSS mutants showed that ring opacification required ExoU but not ExoT and that it consisted of dense polymorphonuclear phagocyte infiltration at the corneal periphery and the absence of any cell type in the central cornea. These data suggest that theseP. aeruginosaTTSS effectors have different effects on innate immunity and that RetS influences virulence beyond its effects on the TTSS.