RESUMEN
Pseudomonas aeruginosa is invasive or cytotoxic to host cells, depending on the type III secretion system (T3SS) effectors encoded. While the T3SS is known to be involved in disease in vivo, how it participates remains to be clarified. Here, mouse models of superficial epithelial injury (tissue paper blotting with EGTA treatment) and immunocompromise (MyD88 deficiency) were used to study the contribution of the T3SS transcriptional activator ExsA to epithelial traversal. Corneas of excised eyeballs were inoculated with green fluorescent protein (GFP)-expressing PAO1 or isogenic exsA mutants for 6 h ex vivo before bacterial traversal and epithelial thickness were quantified by using imaging. In the blotting-EGTA model, exsA mutants were defective in capacity for traversal. Accordingly, an â¼16-fold variability in exsA expression among PAO1 isolates from three sources correlated with epithelial loss. In contrast, MyD88-/- epithelia remained susceptible to P. aeruginosa traversal despite exsA mutation. Epithelial lysates from MyD88-/- mice had reduced antimicrobial activity compared to those from wild-type mice with and without prior antigen challenge, particularly 30- to 100-kDa fractions, for which mass spectrometry revealed multiple differences, including (i) lower baseline levels of histones, tubulin, and lumican and (ii) reduced glutathione S-transferase, annexin, and dermatopontin, after antigen challenge. Thus, the importance of ExsA in epithelial traversal by invasive P. aeruginosa depends on the compromise enabling susceptibility, suggesting that strategies for preventing infection will need to extend beyond targeting the T3SS. The data also highlight the importance of mimicking conditions allowing susceptibility in animal models and the need to monitor variability among bacterial isolates from different sources, even for the same strain.
Asunto(s)
Proteínas Bacterianas/genética , Sistemas de Secreción Bacterianos/fisiología , Lesiones de la Cornea/microbiología , Epitelio Corneal/microbiología , Interacciones Huésped-Patógeno/inmunología , Factor 88 de Diferenciación Mieloide/genética , Pseudomonas aeruginosa/patogenicidad , Transactivadores/genética , Animales , Anexinas/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Epitelio Corneal/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Infecciones Bacterianas del Ojo/microbiología , Glutatión Transferasa/metabolismo , Proteínas Fluorescentes Verdes , Histonas/metabolismo , Sulfato de Queratano/metabolismo , Lumican , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infecciones por Pseudomonas/microbiología , Proteínas Recombinantes de Fusión/genética , Tubulina (Proteína)/metabolismoRESUMEN
PURPOSE: Pseudomonas aeruginosa keratitis is a sight-threatening complication of contact lens wear, yet mechanisms by which lenses predispose to infection remain unclear. Here, we tested the hypothesis that tear fluid at the posterior contact lens surface can lose antimicrobial activity over time during lens wear. METHODS: Daily disposable lenses were worn for 1, 2, 4, 6, or 8 hours immediately after removal from their packaging or after presoaking in sterile saline for 2 days to remove packaging solution. Unworn lenses were also tested, some coated in tears "aged" in vitro for 1 or 8 hours. Lenses were placed anterior surface down into tryptic soy agar cradles containing gentamicin (100 µg/mL) to kill bacteria already on the lens and posterior surfaces inoculated with gentamicin-resistant P. aeruginosa for 3 hours. Surviving bacteria were enumerated by viable counts of lens homogenates. RESULTS: Posterior surfaces of lenses worn by patients for 8 hours supported more P. aeruginosa growth than lenses worn for only 1 hour, if lenses were presoaked before wear (â¼ 2.4-fold, p = 0.01). This increase was offset if lenses were not presoaked to remove packaging solution (p = 0.04 at 2 and 4 hours). Irrespective of presoaking, lenses worn for 8 hours showed more growth on their posterior surface than unworn lenses coated with tear fluid that was aged for 8 hours in vitro (â¼ 8.6-fold, presoaked, p = 0.003; â¼ 5.4-fold from packaging solution, p = 0.004). Indeed, in vitro incubation did not impact tear antimicrobial activity. CONCLUSIONS: This study shows that postlens tear fluid can lose antimicrobial activity over time during contact lens wear, supporting the idea that efficient tear exchange under a lens is critical for homeostasis. Additional studies are needed to determine applicability to other lens types, wearing modalities, and relevance to contact lens-related infections.