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

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 ϳ10 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.Recently, we reported that retS, a gene encoding a novel Pseudomonas aeruginosa hybrid sensor-response regulator protein, is involved in corneal virulence in vivo during the first 24 h of the disease (42). In vitro analysis revealed that mutation of retS in strain PA103 caused loss of type III secretion and a reduction in multiple epithelium-associated virulence mechanisms, including; twitching motility, epithelial association, invasion, and intracellular survival (42). Two other recent publications by others show that RetS (regulator of exopolysaccharide and type III secretion), also designated RtsM (regulator of type III secretion), participates in virulence during acute pneumonia (16 to 18 h postinoculation) (20,27). These studies also showed loss of the ExsA-regulated type III secretion system (TTSS) in retS and rtsM mutants in vitro. This could be restored by overexpression of ExsA (27). One of these studies also showed that RetS positively and negatively affects the expression of many genes in addition to regulation of type III secretion (20). F...

Mutation of the phospholipase catalytic domain of the Pseudomonas aeruginosa cytotoxin ExoU abolishes colonization promoting activity and reduces corneal disease severity

“…A peripheral ring infiltrate, which we have previously reported to require ExoU, was observed only in those eyes infected with bacteria expressing intact exoU (Zolfaghar et al, 2006).…”

“…We have previously shown that ExoU is involved in the ability of P. aeruginosa to colonize the cornea in vivo and to cause pathology (Lee et al, 2003) which involves inhibition of phagocyte infiltration of the central cornea (Zolfaghar et al, 2006). This followed our earlier work showing that ExoU was responsible for the acute cytotoxicity of cytotoxic strains of P. aeruginosa towards mammalian cells in vitro (Finck-Barbancon et al, 1997;Fleiszig et al, 1997).…”

“…We previously described the inflammatory infiltrate induced by PA103ΔUT, wild-type PA103 and mutants in known TTSS effectors a murine model of infectious keratitis (Zolfaghar et al, 2006). In that study, ExoU was responsible for repression of phagocyte infiltration of infected corneas at 48 h in vivo, correlating with promotion of bacterial colonization, and with disease severity scores and appearance.…”

“…Overall disease pathology in the murine scarification model of P. aeruginosa keratitis at 24 h and 48 h after inoculation with 5 μl of cell culture medium containing ~1 × 10 6 cfu strain PA103ΔexoUexoT::Tc (PA103ΔUT) complemented with pUCP18, pUCPexoU, or the phospholipase mutant pUCPexoUD344A. Note the ring infiltrate of the eyes infected with bacteria expressing exoU (Zolfaghar et al, 2006). *24 h images were equally adjusted for brightness and contrast using Adobe Photoshop to match backgrounds of 48 h data.…”

“…Cytotoxic isolates can also kill epithelial cells on the surface of intact corneas ex vivo (Fleiszig et al, 1998). Using a murine scarification model of keratitis, we previously demonstrated that ExoU also makes a significant contribution to ocular colonization and corneal disease, and that it represses phagocyte infiltration of the central region of infected corneas in vivo (Lee et al, 2003;Zolfaghar et al, 2006). Others have shown that complementation of exoU into an invasive strain of P. aeruginosa enhances its virulence in a murine model of acute pneumonia (Allewelt et al, 2000).…”