Role of<i>Pseudomonas aeruginosa</i>ExsA in Penetration through Corneal Epithelium in a Novel In Vivo Model

Lee, Ellen J; Evans, David J.; Fleiszig, Suzanne M. J.

doi:10.1167/iovs.03-0229

Cited by 54 publications

(57 citation statements)

References 43 publications

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“…The 6-h healing murine model of corneal infection was used since we had previously used it to demonstrate the role of ExsA in P. aeruginosa corneal penetration in vivo and the contribution of RetS to early (Ͻ24 h) corneal virulence (29,42). Following anesthesia, three full-thickness epithelial abrasions were produced on the left corneas of 6-week old female C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) with a sterile 26-guage needle.…”

Section: Methodsmentioning

confidence: 99%

“…Type III secretion contributes to the pathogenesis of septic shock and the virulence of P. aeruginosa in the cornea (28,29), burn wounds (24,25), and the airways (1,2,11,26,30,32,33,36,39). Four known effector proteins are delivered into mammalian cells by this TTSS: ExoS, ExoT, ExoU, and ExoY.…”

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confidence: 99%

“…We hypothesized that loss of the TTSS would account for the reduced virulence of P. aeruginosa retS mutants. To test this hypothesis, we compared retS mutant infections to disease caused by wild-type P. aeruginosa strain PA103 and that caused by an isogenic exsA (transcriptional activator of the TTSS), exoU, exoT, or exoU exoT mutant by using the 6-h healing model of corneal infection (29). Infected eyes were examined for disease severity and bacterial colonization and by histological methods during the first 7 days postinfection.…”

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confidence: 99%

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

et al. 2006

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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...

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Section: Methodsmentioning

confidence: 99%

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

et al. 2006

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“…Recently, Sun et al showed that the exposure of C 57 BL/6 mouse corneal epithelium to S. aureus was found to induce neutrophil recruitment to the corneal stroma and increased corneal thickness and haze in a TLR2 dependent manner [15]. Since interactions between bacteria and epithelium are one of the early events in the establishment of the infection [16], it is important to identify the bacterial virulence factors and host cell components that contribute to infection and inflammation.…”

Section: Introductionmentioning

confidence: 99%

Staphylococcus aureus lipoproteins trigger human corneal epithelial innate response through toll-like receptor-2

Kumar

Gui

et al. 2008

Microbial Pathogenesis

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Bacterial lipoproteins (LP) are a family of cell wall components found in a wide variety of bacteria. In this study, we characterized the response of HUCL, a telomerase-immortalized human corneal epithelial cell (HCEC) line, to LP isolated from Staphylococcus (S) aureus. S. aureus LP (saLP) prepared by Triton X-114 extraction stimulated the activation of NF-kB, JNK, and P38 signaling pathways in HUCL cells. The extracts failed to stimulate NF-kB activation in HUCL cells after lipoprotein lipase treatment and in cell lines expressing TLR4 or TLR9, but not TLR2, indicating lipoprotein nature of the extracts. saLP induced the up-regulation of a variety of inflammatory cytokines and chemokines (IL-6, IL-8, ICAM-1), antimicrobial molecules (hBD-2, LL-37, and iNOS), and homeostasis genes (Mn-SOD) at both the mRNA level and protein level. Similar inflammatory response to saLP was also observed in primarily cultured HCECs using the production of IL-6 as readout. Moreover, TLR2 neutralizing antibody blocked the saLP-induced secretion of IL-6, IL-8 and hBD2 in HUCL cells. Our findings suggest that saLP activates TLR2 and triggers innate immune response in the cornea to S. aureus infection via production of proinflammatory cytokines and defense molecules. r

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“…For example, in the cornea, a multilayered epithelium protects the underlying stroma. Indeed, P. aeruginosa corneal infection does not occur in the absence of fullthickness epithelial injury or contact lens wear (32,43,54). For this reason, much of what we understand about host-microbe interactions in vivo has been derived from experimental models that deliberately bypass the epithelial barrier (9,18).…”

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Role of Defensins in Corneal Epithelial Barrier Function against Pseudomonas aeruginosa Traversal

et al. 2011

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Studies have shown that epithelium-expressed antimicrobial peptides (AMPs), e.g., ␤-defensins, play a role in clearing bacteria from mouse corneas already infected with Pseudomonas aeruginosa. Less is known about the role of AMPs in allowing the cornea to resist infection when healthy. We previously reported that contact lens exposure, a major cause of P. aeruginosa keratitis, can inhibit the upregulation of human ␤-defensin 2 (hBD-2) by corneal epithelial cells in response to P. aeruginosa antigens in vitro. Here, we studied the role of AMPs in maintaining the corneal epithelial barrier to P. aeruginosa penetration using both in vitro (human) and in vivo (mouse) experiments. Results showed that preexposing human corneal epithelial multilayers to bacterial antigens in a culture supernatant (known to upregulate AMP expression) reduced epithelial susceptibility to P. aeruginosa traversal up to 6-fold (P < 0.001). Accordingly, small interfering RNA (siRNA) knockdown of any one of four AMPs expressed by human epithelia promoted P. aeruginosa traversal by more than 3-fold (P < 0.001). The combination knockdown of AMPs further enhanced susceptibility to bacterial traversal by ϳ8-fold (P < 0.001). In vivo experiments showed that the loss of murine ␤-defensin 3 (mBD-3), a murine ortholog of hBD-2, enhanced corneal susceptibility to P. aeruginosa. The uninjured ocular surface of mBD-3 ؊/؊ mice showed a reduced capacity to clear P. aeruginosa, and their corneal epithelia were more susceptible to bacterial colonization, even when inoculated ex vivo to exclude tear fluid effects. Together, these in vitro and in vivo data show functional roles for AMPs in normal corneal epithelial cell barrier function against P. aeruginosa.

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Role ofPseudomonas aeruginosaExsA in Penetration through Corneal Epithelium in a Novel In Vivo Model

Cited by 54 publications

References 43 publications

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

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

Staphylococcus aureus lipoproteins trigger human corneal epithelial innate response through toll-like receptor-2

Role of Defensins in Corneal Epithelial Barrier Function against Pseudomonas aeruginosa Traversal

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