Type III secretion-dependent modulation of innate immunity as one of multiple factors regulated by Pseudomonas aeruginosa RetS - PubMed (original) (raw)

Comparative Study

Type III secretion-dependent modulation of innate immunity as one of multiple factors regulated by Pseudomonas aeruginosa RetS

Irandokht Zolfaghar et al. Infect Immun. 2006 Jul.

Abstract

Mutation of retS (rtsM) of Pseudomonas aeruginosa strain PA103 reduces its virulence in both ocular and respiratory murine models of infection. In vitro, retS mutants 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 by retS mutation. 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 of retS mutants follows loss of type III secretion. Corneal pathology, bacterial colonization, and phagocyte infiltration were compared for wild-type PA103, retS mutants, and various TTSS mutants after infection with approximately 10(6) CFU bacteria. Results showed that either a retS or an exsA (TTSS) mutation delayed disease progression, as illustrated by reduced severity scores and colonization levels during the first 48 h postinfection. Surprisingly, retS mutant infections then became more severe than those involving exsA mutants. By day 7, colonization levels of retS mutants even surpassed those of wild-type bacteria (more than twofold, P = 0.028). Although retS mutants caused more severe opacification of central corneas than both the wild type and the exsA mutants, neither mutant caused the peripheral ring opacity commonly associated with wild-type infection, suggesting that the TTSS was involved. Histological experiments with retS and 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 these P. aeruginosa TTSS effectors have different effects on innate immunity and that RetS influences virulence beyond its effects on the TTSS.

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Figures

FIG. 1.

Corneal infections caused by P. aeruginosa wild-type strain PA103 compared to those caused by retS (PA103_retS_::Tn_5_) and exsA (PA103_exsA_::Ω) mutants at days 1 and 2 (A) and 4 and 7 (B) postinfection with ∼106 CFU of bacteria in the 6-h healing murine model of infection. Solid arrows indicate peripheral ring infiltrates, and dashed arrows show dense central infiltrates. Within each group, the same mice were followed throughout the course of infection.

FIG. 2.

Histology (A) and pathology (B) of corneal disease at 2 days (48 h) postinoculation with P. aeruginosa strain PA103 or the retS or exsA mutant at ∼106 CFU/5 μl in the 6-h healing murine model of infection. PMN infiltration of the central cornea was observed in eyes infected with the retS and exsA mutants. However, PMN migration appeared restricted to the peripheral cornea in eyes infected with wild-type PA103 (×1,200 magnification [A]).

FIG. 3.

Corneal pathology at 2 days (48 h) postinoculation with P. aeruginosa strain PA103 or the retS, exoU, exoT, or exoU exoT mutant at ∼106 CFU/5 μl in the 6-h healing murine model of infection. Peripheral ring infiltrates (solid arrows) were observed with wild-type bacteria and the exoT mutant (still expresses ExoU). In contrast, pathology observed with the retS and exoU exoT mutants predominantly involved infiltrates in the central cornea (dashed arrows). The exoU mutant (expresses ExoT) caused pathology involving both the central and peripheral regions of the cornea.

FIG. 4.

Corneal histology at 2 days (48 h) postinoculation with P. aeruginosa strain PA103 or the exoU, exoT, or exoU exoT mutant at ∼106 CFU/5 μl in the 6-h healing murine model of infection. Extensive PMN infiltration of the central cornea was observed in eyes infected with the exoU and exoU exoT mutants. However, PMN migration was restricted to the peripheral regions of the cornea in eyes infected with wild-type PA103 and the exoT mutant (×200 magnification, left side; ×1,200 magnification, right side).

FIG. 5.

Distribution of PMNs among the peripheral, precentral, and central regions of mouse corneas at 48 h postinoculation with P. aeruginosa strain PA103 or the exoU, exoT, or exoU exoT mutant.

FIG. 6.

Thickness of peripheral and central regions of murine corneas at 48 h postinfection with P. aeruginosa strain PA103 or the exoU, exoT, or exoU exoT mutant.

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Type III secretion-dependent modulation of innate immunity as one of multiple factors regulated by Pseudomonas aeruginosa RetS - PubMed (original) (raw)