RESUMEN
The Pseudomonas aeruginosa toxin ExoS, secreted by the type III secretion system (T3SS), supports intracellular persistence via its ADP-ribosyltransferase (ADPr) activity. For epithelial cells, this involves inhibiting vacuole acidification, promoting vacuolar escape, countering autophagy, and niche construction in the cytoplasm and within plasma membrane blebs. Paradoxically, ExoS and other P. aeruginosa T3SS effectors can also have antiphagocytic and cytotoxic activities. Here, we sought to reconcile these apparently contradictory activities of ExoS by studying the relationships between intracellular persistence and host epithelial cell death. Methods involved quantitative imaging and the use of antibiotics that vary in host cell membrane permeability to selectively kill intracellular and extracellular populations after invasion. Results showed that intracellular P. aeruginosa mutants lacking T3SS effector toxins could kill (permeabilize) cells when extracellular bacteria were eliminated. Surprisingly, wild-type strain PAO1 (encoding ExoS, ExoT and ExoY) caused cell death more slowly, the time extended from 5.2 to 9.5 h for corneal epithelial cells and from 10.2 to 13.0 h for HeLa cells. Use of specific mutants/complementation and controls for initial invasion showed that ExoS ADPr activity delayed cell death. Triggering T3SS expression only after bacteria invaded cells using rhamnose-induction in T3SS mutants rescued the ExoS-dependent intracellular phenotype, showing that injected effectors from extracellular bacteria were not required. The ADPr activity of ExoS was further found to support internalization by countering the antiphagocytic activity of both the ExoS and ExoT RhoGAP domains. Together, these results show two additional roles for ExoS ADPr activity in supporting the intracellular lifestyle of P. aeruginosa; suppression of host cell death to preserve a replicative niche and inhibition of T3SS effector antiphagocytic activities to allow invasion. These findings add to the growing body of evidence that ExoS-encoding (invasive) P. aeruginosa strains can be facultative intracellular pathogens, and that intracellularly secreted T3SS effectors contribute to pathogenesis.
Asunto(s)
ADP Ribosa Transferasas/metabolismo , Permeabilidad de la Membrana Celular , Exotoxinas/metabolismo , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Muerte Celular , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Proteínas Activadoras de GTPasa/metabolismo , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Mutación , Pseudomonas aeruginosa/efectos de los fármacos , Sistemas de Secreción Tipo III/metabolismo , Vacuolas/metabolismoRESUMEN
The cornea of the eye differs from other mucosal surfaces in that it lacks a viable bacterial microbiome and by its unusually high density of sensory nerve endings. Here, we explored the role of corneal nerves in preventing bacterial adhesion. Pharmacological and genetic methods were used to inhibit the function of corneal sensory nerves or their associated transient receptor potential cation channels TRPA1 and TRPV1. Impacts on bacterial adhesion, resident immune cells, and epithelial integrity were examined using fluorescent labeling and quantitative confocal imaging. TRPA1/TRPV1 double gene-knockout mice were more susceptible to adhesion of environmental bacteria and to that of deliberately-inoculated Pseudomonas aeruginosa. Supporting the involvement of TRPA1/TRPV1-expressing corneal nerves, P. aeruginosa adhesion was also promoted by treatment with bupivacaine, or ablation of TRPA1/TRPV1-expressing nerves using RTX. Moreover, TRPA1/TRPV1-dependent defense was abolished by enucleation which severs corneal nerves. High-resolution imaging showed normal corneal ultrastructure and surface-labeling by wheat-germ agglutinin for TRPA1/TRPV1 knockout murine corneas, and intact barrier function by absence of fluorescein staining. P. aeruginosa adhering to corneas after perturbation of nerve or TRPA1/TRPV1 function failed to penetrate the surface. Single gene-knockout mice showed roles for both TRPA1 and TRPV1, with TRPA1-/- more susceptible to P. aeruginosa adhesion while TRPV1-/- corneas instead accumulated environmental bacteria. Corneal CD45+/CD11c+ cell responses to P. aeruginosa challenge, previously shown to counter bacterial adhesion, also depended on TRPA1/TRPV1 and sensory nerves. Together, these results demonstrate roles for corneal nerves and TRPA1/TRPV1 in corneal resistance to bacterial adhesion in vivo and suggest that the mechanisms involve resident immune cell populations.
Asunto(s)
Adhesión Bacteriana , Córnea , Pseudomonas aeruginosa/metabolismo , Canal Catiónico TRPA1/metabolismo , Canales Catiónicos TRPV/metabolismo , Animales , Córnea/inervación , Córnea/metabolismo , Córnea/microbiología , Femenino , Masculino , Ratones , Ratones Noqueados , Canal Catiónico TRPA1/genética , Canales Catiónicos TRPV/genéticaRESUMEN
It is generally thought that mucosal fluids protect underlying epithelial surfaces against opportunistic infection via their antimicrobial activity. However, our published data show that human tear fluid can protect against the major opportunistic pathogen Pseudomonas aeruginosa independently of bacteriostatic activity. Here, we explored the mechanisms for tear protection, focusing on impacts of tear fluid on bacterial virulence factor expression. Results showed that tear fluid suppressed twitching motility, a type of surface-associated movement conferred by pili. Previously, we showed that twitching is critical for P. aeruginosa traversal of corneal epithelia, exit from epithelial cells after internalization, and corneal virulence. Inhibition of twitching by tear fluid was dose-dependent with dilutions to 6.25% retaining activity. Purified lactoferrin, lysozyme, and contrived tears containing these, and many other, tear components lacked the activity. Systematic protein fractionation, mass spectrometry, and immunoprecipitation identified the glycoprotein DMBT1 (Deleted in Malignant Brain Tumors 1) in tear fluid as required. DMBT1 purified from human saliva also inhibited twitching, as well as P. aeruginosa traversal of human corneal epithelial cells in vitro, and reduced disease pathology in a murine model of corneal infection. DMBT1 did not affect PilA expression, nor bacterial intracellular cyclicAMP levels, and suppressed twitching motility of P. aeruginosa chemotaxis mutants (chpB, pilK), and an adenylate cyclase mutant (cyaB). However, dot-immunoblot assays showed purified DMBT1 binding of pili extracted from PAO1 suggesting that twitching inhibition may involve a direct interaction with pili. The latter could affect extension or retraction of pili, their interactions with biotic or abiotic surfaces, or cause their aggregation. Together, the data suggest that DMBT1 inhibition of twitching motility contributes to the mechanisms by which mucosal fluids protect against P. aeruginosa infection. This study also advances our understanding of how mucosal fluids protect against infection, and suggests directions for novel biocompatible strategies to protect our surface epithelia against a major opportunistic pathogen.
Asunto(s)
Infecciones Oportunistas/microbiología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/fisiología , Pseudomonas aeruginosa/patogenicidad , Receptores de Superficie Celular/metabolismo , Antibacterianos/farmacología , Proteínas de Unión al Calcio , Células Cultivadas , Córnea/parasitología , Córnea/patología , Proteínas de Unión al ADN , Células Epiteliales/metabolismo , Células Epiteliales/patología , Epitelio Corneal/microbiología , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Queratitis/parasitología , Queratitis/patología , Receptores de Superficie Celular/genética , Proteínas Supresoras de Tumor , Virulencia , Factores de VirulenciaRESUMEN
[This corrects the article DOI: 10.1371/journal.ppat.1006392.].
RESUMEN
Research with animal models of Pseudomonas aeruginosa keratitis has shown that use of a topical corticosteroid alone against an established infection can significantly increase the number of colonizing bacteria or worsen clinical disease. Moreover, retrospective analysis has suggested that corticosteroid use in humans is associated with an increased risk of keratitis in eyes with pre-existing disease. Thus, while corticosteroids are often used to reduce ocular inflammation in the absence of infection, the risk of opportunistic infection remains a concern. However, the effect of corticosteroids on the intrinsic barrier function of uninfected corneas is unknown. Here, we tested if short-term topical corticosteroid treatment of an uninfected murine cornea would increase susceptibility to P. aeruginosa colonization or infection after epithelial injury. Topical prednisolone acetate (1%) was administered to one eye of C57BL/6 mice three times a day for 3 days; control eyes were treated with sterile PBS. Prior to inoculation with a cytotoxic P. aeruginosa corneal isolate strain 6206, corneas were subject to superficial-injury by tissue paper blotting, or scratch-injured followed by 12â¯h of healing. Previously we have shown that blotting renders mouse corneas susceptible to P. aeruginosa adhesion, but not infection, while 12â¯h healing reduces susceptibility to infection after scratching. Corneas were evaluated at 48â¯h for bacterial colonization and microbial keratitis (MK). To monitor impact on wound healing, corneal integrity was examined by fluorescein staining immediately after scarification and after 12â¯h healing. For both the tissue paper blotting and scratch-injury models, there was no significant difference in P. aeruginosa colonization at 48â¯h between corticosteroid-pretreated eyes and controls. With the blotting model, one case of MK was observed in a control (PBS-pretreated) cornea; none in corticosteroid-pretreated corneas. With the 12â¯h healing model, MK occurred in 6 of 17 corticosteroid-pretreated eyes versus 2 of 17 controls, a difference not statistically significant. Corticosteroid-pretreated eyes showed greater fluorescein staining 12â¯h after scarification injury, but this did not coincide with increased colonization or MK. Together, these data show that short-term topical corticosteroid therapy on an uninfected murine cornea does not necessarily enhance its susceptibility to P. aeruginosa colonization or infection after injury, even when it induces fluorescein staining.
Asunto(s)
Lesiones de la Cornea/microbiología , Infecciones Bacterianas del Ojo/microbiología , Glucocorticoides/uso terapéutico , Prednisolona/análogos & derivados , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/aislamiento & purificación , Administración Oftálmica , Animales , Córnea/efectos de los fármacos , Lesiones de la Cornea/diagnóstico , Úlcera de la Córnea/diagnóstico , Úlcera de la Córnea/microbiología , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Epitelio Corneal/lesiones , Infecciones Bacterianas del Ojo/diagnóstico , Femenino , Fluoresceína/metabolismo , Colorantes Fluorescentes/metabolismo , Ratones , Ratones Endogámicos C57BL , Prednisolona/uso terapéutico , Premedicación , Infecciones por Pseudomonas/diagnóstico , Estudios Retrospectivos , Cicatrización de HeridasRESUMEN
Cell surface glycosylation is thought to be involved in barrier function against microbes at mucosal surfaces. Previously we showed that the epithelium of healthy mouse corneas becomes vulnerable to Pseudomonas aeruginosa adhesion if it lacks the innate defense protein MyD88 (myeloid differentiation primary response gene 88), or after superficial injury by blotting with tissue paper. Here we explored their effect on corneal surface glycosylation using a metabolic label, tetra-acetylated N-azidoacetylgalactosamine (Ac4GalNAz). Ac4GalNAz treatment labeled the surface of healthy mouse corneas, leaving most cells viable, and bacteria preferentially associated with GalNAz-labeled regions. Surprisingly, corneas from MyD88-/- mice displayed similar GalNAz labeling to wild-type corneas, but labeling was reduced and patchy on IL-1 receptor (IL-1R)-knockout mouse corneas (P < 0.05, ANOVA). Tissue paper blotting removed GalNAz-labeled surface cells, causing DAPI labeling (permeabilization) of underlying cells. MS of material collected on the tissue paper blots revealed 67 GalNAz-labeled proteins, including intracellular proteins. These data show that the normal distribution of surface glycosylation requires IL-1R, but not MyD88, and is not sufficient to prevent bacterial binding. They also suggest increased P. aeruginosa adhesion to MyD88-/- and blotted corneas is not due to reduction in total surface glycosylation, and for tissue paper blotting is likely due to cell permeabilization.-Jolly, A. L., Agarwal, P., Metruccio, M. M. E., Spiciarich, D. R., Evans, D. J., Bertozzi, C. R., Fleiszig, S. M. J. Corneal surface glycosylation is modulated by IL-1R and Pseudomonas aeruginosa challenge but is insufficient for inhibiting bacterial binding.
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Córnea/microbiología , Córnea/fisiología , Factor 88 de Diferenciación Mieloide/metabolismo , Receptores de Interleucina-1/metabolismo , Animales , Adhesión Bacteriana , Femenino , Adhesivo de Tejido de Fibrina , Regulación de la Expresión Génica/fisiología , Glicoproteínas , Glicosilación , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Pseudomonas aeruginosa , Receptores de Interleucina-1/genéticaRESUMEN
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.
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Adhesión Bacteriana/fisiología , Lentes de Contacto Hidrofílicos/microbiología , Pseudomonas aeruginosa/fisiología , Lágrimas/fisiología , Adulto , Antibacterianos/farmacología , Recuento de Colonia Microbiana , Equipos Desechables , Farmacorresistencia Bacteriana/fisiología , Femenino , Gentamicinas/farmacología , Humanos , Masculino , Pseudomonas aeruginosa/aislamiento & purificación , Factores de Tiempo , Adulto JovenRESUMEN
BACKGROUND: To determine the relationship between type three secretion genotype and fluoroquinolone resistance for P. aeruginosa strains isolated from microbial keratitis during the Steroids for Corneal Ulcers Trial (SCUT) and for two laboratory strains, PA103 and PAO1. METHODS: Confirmed P. aeruginosa isolates from the SCUT were divided into exoU(+) or exoU(-). The exoU(+) strains contained the gene encoding ExoU, a powerful phospholipase toxin delivered into host cells by the type three secretion system. Isolates were then assessed for susceptibility to fluoroquinolone, cephalosporin, and aminoglycoside antibiotics using disk diffusion assays. Etest was used to determine the MIC of moxifloxacin and other fluoroquinolones. Laboratory isolates in which the exoU gene was added or deleted were also tested. RESULTS: A significantly higher proportion of exoU(+) strains were resistant to ciprofloxacin (p = 0.001), gatifloxacin (p = 0.003), and ofloxacin (p = 0.002) compared to exoU(-) isolates. There was no significant difference between exoU(+) or exoU(-) negative isolates with respect to susceptibility to other antibiotics except gentamicin. Infections involving resistant exoU(+) strains trended towards worse clinical outcome. Deletion or acquisition of exoU in laboratory isolates did not affect fluoroquinolone susceptibility. CONCLUSIONS: Fluoroquinolone susceptibility of P. aeruginosa isolated from the SCUT is consistent with previous studies showing elevated resistance involving exoU encoding (cytotoxic) strains, and suggest worse clinical outcome from infections involving resistant isolates. Determination of exoU expression in clinical isolates of P. aeruginosa may be helpful in directing clinical management of patients with microbial keratitis.
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Córnea/microbiología , Úlcera de la Córnea/microbiología , Farmacorresistencia Bacteriana , Infecciones Bacterianas del Ojo/microbiología , Fluoroquinolonas/uso terapéutico , Glucocorticoides/uso terapéutico , Pseudomonas aeruginosa/patogenicidad , Anciano , Córnea/patología , Úlcera de la Córnea/tratamiento farmacológico , Úlcera de la Córnea/patología , ADN Bacteriano/genética , Infecciones Bacterianas del Ojo/tratamiento farmacológico , Infecciones Bacterianas del Ojo/patología , Femenino , Genotipo , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Pronóstico , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/aislamiento & purificación , Agudeza VisualRESUMEN
: Clinician-scientists bridge the gap between basic research and patient care. At the 2012 Annual Meeting, a symposium highlighting the application of cutting-edge optometric research within the anterior segment was held to present and discuss some of the recent basic scientific advances that will both shape and guide the development of future clinical care practices. This article summarizes this work, bringing together four experts, all clinician-scientists in the field of cornea and ocular surface. Collectively, this work provides new insights to clinicians and researchers alike, as well as brings forth a greater appreciation of the impact of ongoing optometric bench research in advancing clinical care.
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Investigación Biomédica/organización & administración , Enfermedades de la Conjuntiva/terapia , Enfermedades de la Córnea/terapia , Síndromes de Ojo Seco/terapia , Infecciones del Ojo/terapia , Enfermedades de los Párpados/terapia , Optometría/organización & administración , Investigación Biomédica/tendencias , Humanos , Optometría/tendenciasRESUMEN
Purpose: Contact lens wear induces corneal parainflammation involving increased immune cell numbers after 24 hours' (CD11c+, Lyz2+, γδ-T cells) and six days' (Ly6G+ cells) wear. We investigated the time course of onset and resolution of these responses. Methods: LysMcre or C57BL/6J mice were fitted with a contact lens (four to 48 hours). Contralateral eyes did not wear lenses. After lens removal, Lyz2+, MHC-II+ or Ly6G+ cells were examined by quantitative imaging. RT-qPCR determined cytokine gene expression. Results: Lens wear for 24 hours increased corneal Lyz2+ cells versus contralateral eyes approximately two-fold. Corneas remained free of visible pathology. The Lyz2+ response was not observed after four or 12 hours' wear, nor after 12 hours' wear plus 12 hours' no wear. Lens removal after 24 hours' wear further increased Lyz2+ cells (â¼48% after one day), which persisted for four days, returning to baseline by seven days. Lyz2+ cells in contralateral eyes remained at baseline. MHC-II+ cells showed a similar response but without increasing after lens removal. Lens wear for 48 hours showed reduced Lyz2+ cells versus 24 hours' wear with one day discontinuation, correlating with reduced IL-1ß and IL-18 gene expression. Lens wear for 24 hours did not induce Ly6G+ responses six days after removal. Conclusions: Lens-induced corneal parainflammation involving Lyz2+ cells requires 24 hours' wear but persists after lens discontinuation, requiring seven days for reversal. Lens wear for 48 hours may suppress initial Lyz2+ cell and cytokine responses. The significance of parainflammation during and after lens wear remains to be determined.
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Lentes de Contacto , Cristalino , Ratones , Animales , Ratones Endogámicos C57BL , Lentes de Contacto/efectos adversos , Córnea , Citocinas/genéticaRESUMEN
Microbial keratitis is a sight-threatening complication associated with contact lenses. The introduction of silicone hydrogel lens materials with increased oxygen transmission to the ocular surface has not significantly altered the incidence of microbial keratitis. These data suggest that alternate, or additional, predisposing factors involving lens wear must be addressed to reduce or eliminate these infections. The contact lens can provide a surface for microbial growth in situ and can also influence ocular surface homeostasis through effects on the tear fluid and corneal epithelium. Thus, it is intuitive that future contact lens materials could make a significant contribution to preventing microbial keratitis. Design of the "right" material to prevent microbial keratitis requires understanding the effects of current materials on bacterial virulence in the cornea and on ocular surface innate defenses. Current knowledge in each of these areas will be presented with a discussion of future directions needed to understand the influence of lens material on the pathogenesis of microbial keratitis.
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Infecciones Bacterianas/etiología , Lentes de Contacto Hidrofílicos/efectos adversos , Queratitis/microbiología , Lentes de Contacto Hidrofílicos/microbiología , Córnea/metabolismo , Humanos , Hidrogeles , Oxígeno/metabolismo , Factores de Riesgo , Siliconas , Lágrimas/metabolismoRESUMEN
Purpose: Contact lens wear can induce corneal parainflammation involving CD11c+ cell responses (24 hours), γδ T cell responses (24 hours and 6 days), and IL-17-dependent Ly6G+ cell responses (6 days). Topical antibiotics blocked these CD11c+ responses. Because corneal CD11c+ responses to bacteria require transient receptor potential (TRP) ion-channels (TRPA1/TRPV1), we determined if these channels mediate lens-induced corneal parainflammation. Methods: Wild-type mice were fitted with contact lenses for 24 hours or 6 days and compared to lens wearing TRPA1 (-/-) or TRPV1 (-/-) mice or resiniferatoxin (RTX)-treated mice. Contralateral eyes were not fitted with lenses. Corneas were examined for major histocompatibility complex (MHC) class II+, CD45+, γδ T, or TNF-α+ cell responses (24 hours) or Ly6G+ responses (6 days) by quantitative imaging. The quantitative PCR (qPCR) determined cytokine gene expression. Results: Lens-induced increases in MHC class II+ cells after 24 hours were abrogated in TRPV1 (-/-) but not TRPA1 (-/-) mice. Increases in CD45+ cells were unaffected. Increases in γδ T cells after 24 hours of wear were abrogated in TRPA1 (-/-) and TRPV1 (-/-) mice, as were 6 day Ly6G+ cell responses. Contralateral corneas of TRPA1 (-/-) and TRPV1 (-/-) mice showed reduced MHC class II+ and γδ T cells at 24 hours. RTX inhibited lens-induced parainflammatory phenotypes (24 hours and 6 days), blocked lens-induced TNF-α and IL-18 gene expression, TNF-α+ cell infiltration (24 hours), and reduced baseline MHC class II+ cells. Conclusions: TRPA1 and TRPV1 mediate contact lens-induced corneal parainflammation after 24 hours and 6 days of wear and can modulate baseline levels of resident corneal immune cells.
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Lentes de Contacto , Factor de Necrosis Tumoral alfa , Animales , Ratones , Córnea/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Canales Iónicos , Canal Catiónico TRPA1/genética , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Pathogenesis of Pseudomonas aeruginosa infections can include bacterial survival inside epithelial cells. Previously, we showed this involves multiple roles played by the type three-secretion system (T3SS), and specifically the effector ExoS. This includes ExoS-dependent inhibition of a lytic host cell response that subsequently enables intracellular replication. Here, we studied the underlying cell death response to intracellular P. aeruginosa, comparing wild-type to T3SS mutants varying in capacity to induce cell death and that localize to different intracellular compartments. Results showed that corneal epithelial cell death induced by intracellular P. aeruginosa lacking the T3SS, which remains in vacuoles, correlated with activation of NF-κB as measured by p65 relocalization and TNFα transcription and secretion. Deletion of caspase-4 through CRISPR-Cas9 mutagenesis delayed cell death caused by these intracellular T3SS mutants. Caspase-4 deletion also countered more rapid cell death caused by T3SS effector-null mutants still expressing the TSSS apparatus that traffic to the host cell cytoplasm, and in doing so rescued intracellular replication normally dependent on ExoS. While HeLa cells lacked a lytic death response to T3SS mutants, it was found to be enabled by interferon gamma treatment. Together, these results show that epithelial cells can activate the noncanonical inflammasome pathway to limit proliferation of intracellular P. aeruginosa, not fully dependent on bacterially-driven vacuole escape. Since ExoS inhibits the lytic response, the data implicate targeting of caspase-4, an intracellular pattern recognition receptor, as another contributor to the role of ExoS in the intracellular lifestyle of P. aeruginosa.
RESUMEN
Pathogenesis of Pseudomonas aeruginosa infections can include bacterial survival inside epithelial cells. Previously, we showed that this involves multiple roles played by the type three secretion system (T3SS), and specifically the effector ExoS. This includes ExoS-dependent inhibition of a lytic host cell response that subsequently enables intracellular replication. Here, we studied the underlying cell death response to intracellular P. aeruginosa, comparing wild-type to T3SS mutants varying in capacity to induce cell death and that localize to different intracellular compartments. Results showed that corneal epithelial cell death induced by intracellular P. aeruginosa lacking the T3SS, which remains in vacuoles, correlated with the activation of nuclear factor-κB as measured by p65 relocalization and tumor necrosis factor alpha transcription and secretion. Deletion of caspase-4 through CRISPR-Cas9 mutagenesis delayed cell death caused by these intracellular T3SS mutants. Caspase-4 deletion also countered more rapid cell death caused by T3SS effector-null mutants still expressing the T3SS apparatus that traffic to the host cell cytoplasm, and in doing so rescued intracellular replication normally dependent on ExoS. While HeLa cells lacked a lytic death response to T3SS mutants, it was found to be enabled by interferon gamma treatment. Together, these results show that epithelial cells can activate the noncanonical inflammasome pathway to limit proliferation of intracellular P. aeruginosa, not fully dependent on bacterially driven vacuole escape. Since ExoS inhibits the lytic response, the data implicate targeting of caspase-4, an intracellular pattern recognition receptor, as another contributor to the role of ExoS in the intracellular lifestyle of P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa can exhibit an intracellular lifestyle within epithelial cells in vivo and in vitro. The type three secretion system (T3SS) effector ExoS contributes via multiple mechanisms, including extending the life of invaded host cells. Here, we aimed to understand the underlying cell death inhibited by ExoS when P. aeruginosa is intracellular. Results showed that intracellular P. aeruginosa lacking T3SS effectors could elicit rapid cell lysis via the noncanonical inflammasome pathway. Caspase-4 contributed to cell lysis even when the intracellular bacteria lacked the entire T33S and were consequently unable to escape vacuoles, representing a naturally occurring subpopulation during wild-type infection. Together, the data show the caspase-4 inflammasome as an epithelial cell defense against intracellular P. aeruginosa, and implicate its targeting as another mechanism by which ExoS preserves the host cell replicative niche.
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Inflamasomas , Pseudomonas aeruginosa , Humanos , Células HeLa , Pseudomonas aeruginosa/fisiología , Inflamasomas/metabolismo , Células Epiteliales/microbiología , Vacuolas/microbiologíaRESUMEN
PURPOSE: Previously, using a murine model, we reported that contact lens (CL) wear induced corneal parainflammation involving CD11c+ cells after 24 h and Ly6G+ cells (neutrophils) after 5-6 days. Here, we investigated the role of IL-17 and γδ T cells in the CL-induced neutrophil response. METHODS: CL-wearing C57BL/6 wild-type (WT) mice were compared to lens-wearing IL-17A/F single or double gene knock-out mice, or mice treated with UC7-13D5 monoclonal antibody to functionally deplete γδ T cells. Contralateral eyes served as no lens wear controls. Corneal Ly6G+ and γδ T cell responses were quantified as was expression of genes encoding pro-inflammatory cytokines IL-17A/F, IL-ß, IL-18 and expression of IL-17A/F protein. RESULTS: After 6 days lens wear, WT corneas showed Ly6G+ cell infiltration while remaining free of visible pathology. In contrast, lens-wearing corneas of IL-17AF (-/-), IL-17A (-/-) mice and γδ T cell-depleted mice showed little or no Ly6G+ cell infiltration. No Ly6G+ cell infiltration was detected in contralateral eye controls. Lens-wearing WT corneas also showed a significant increase in γδ T cells after 24 h that was maintained after 6 days of wear, and significantly increased cytokine gene expression after 6 days versus contralateral controls: IL-18 & IL-17A (â¼3.9 fold) and IL-23 (â¼6.5-fold). Increased IL-17A protein (â¼4-fold) was detected after 6 days lens wear. γδ T cell-depletion abrogated these lens-induced changes in cytokine gene and protein expression. CONCLUSION: Together, these data show that IL-17A and γδ T cells are required for Ly6G+ cell (neutrophil) infiltration of the cornea during contact lens-induced parainflammation.
Asunto(s)
Lentes de Contacto , Interleucina-17 , Ratones , Animales , Interleucina-17/genética , Interleucina-17/metabolismo , Interleucina-18/metabolismo , Ratones Endogámicos C57BL , Linfocitos T/metabolismo , Córnea/metabolismo , Citocinas/metabolismo , Ratones NoqueadosRESUMEN
Previously we reported contact lens-induced CD11c+ cell responses in healthy mouse corneas, a phenomenon that also occurs in humans. To test involvement of ocular-associated bacteria, the impact of topical antibiotics on corneal CD11c+ cell populations during 24 h of lens wear was examined. Corneas were treated with gentamicin and ofloxacin (0.3%) or gentamicin alone, some also treated prior to lens wear (24 h). Contralateral PBS-treated eyes served as controls. CD11c-YFP (Yellow Fluorescent Protein) mice allowed CD11c+ cell visualization. Viable bacteria, on the ocular surface or contact lens, were labeled using FISH (16S rRNA-targeted probe) or click-chemistry (alkDala). Antibiotic treatment reduced baseline CD11c+ cell numbers without lens wear and suppressed CD11c+ cell responses to lens wear if corneas were both pretreated and treated during wear. Few bacteria colonized corneas or lenses under any circumstances. Conjunctival commensals were significantly reduced by antibiotics with or without lens wear, but minimally impacted by lens wear alone. Deliberate inoculation with conjunctival commensals triggered CD11c+ cell responses irrespective of antibiotic pretreatment. These results suggest that while lens wear does not necessarily increase quantifiable numbers of conjunctival commensals, those neutralized by antibiotics play a role in lens-associated CD11c+ cell responses and maintaining baseline CD11c+ cell populations.
Asunto(s)
Antibacterianos , Lentes de Contacto , Animales , Antibacterianos/metabolismo , Antibacterianos/farmacología , Bacterias , Antígeno CD11c/metabolismo , Recuento de Células , Córnea/metabolismo , Gentamicinas/metabolismo , Gentamicinas/farmacología , Ratones , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismoRESUMEN
Within epithelial cells, Pseudomonas aeruginosa depends on its type III secretion system (T3SS) to escape vacuoles and replicate rapidly in the cytosol. Previously, it was assumed that intracellular subpopulations remaining T3SS-negative (and therefore in vacuoles) were destined for degradation in lysosomes, supported by data showing vacuole acidification. Here, we report in both corneal and bronchial human epithelial cells that vacuole-associated bacteria can persist, sometimes in the same cells as cytosolic bacteria. Using a combination of phase-contrast, confocal, and correlative light-electron microscopy (CLEM), we also found they can demonstrate biofilm-associated markers: cdrA and cyclic-di-GMP (c-di-GMP). Vacuolar-associated bacteria, but not their cytosolic counterparts, tolerated the cell-permeable antibiotic ofloxacin. Surprisingly, use of mutants showed that both persistence in vacuoles and ofloxacin tolerance were independent of the biofilm-associated protein CdrA or exopolysaccharides (Psl, Pel, alginate). A T3SS mutant (ΔexsA) unable to escape vacuoles phenocopied vacuole-associated subpopulations in wild-type PAO1-infected cells, with results revealing that epithelial cell death depended upon bacterial viability. Intravital confocal imaging of infected mouse corneas confirmed that P. aeruginosa formed similar intracellular subpopulations within epithelial cells in vivo. Together, these results show that P. aeruginosa differs from other pathogens by diversifying intracellularly into vacuolar and cytosolic subpopulations that both contribute to pathogenesis. Their different gene expression and behavior (e.g., rapid replication versus slow replication/persistence) suggest cooperation favoring both short- and long-term interests and another potential pathway to treatment failure. How this intracellular diversification relates to previously described "acute versus chronic" virulence gene-expression phenotypes of P. aeruginosa remains to be determined. IMPORTANCE Pseudomonas aeruginosa can cause sight- and life-threatening opportunistic infections, and its evolving antibiotic resistance is a growing concern. Most P. aeruginosa strains can invade host cells, presenting a challenge to therapies that do not penetrate host cell membranes. Previously, we showed that the P. aeruginosa type III secretion system (T3SS) plays a pivotal role in survival within epithelial cells, allowing escape from vacuoles, rapid replication in the cytoplasm, and suppression of host cell death. Here, we report the discovery of a novel T3SS-negative subpopulation of intracellular P. aeruginosa within epithelial cells that persist in vacuoles rather than the cytoplasm and that tolerate a cell-permeable antibiotic (ofloxacin) that is able to kill cytosolic bacteria. Classical biofilm-associated markers, although demonstrated by this subpopulation, are not required for vacuolar persistence or antibiotic tolerance. These findings advance our understanding of how P. aeruginosa hijacks host cells, showing that it diversifies into multiple populations with T3SS-negative members enabling persistence while rapid replication is accomplished by more vulnerable T3SS-positive siblings. Intracellular P. aeruginosa persisting and tolerating antibiotics independently of the T3SS or biofilm-associated factors could present additional challenges to development of more effective therapeutics.
Asunto(s)
Proteínas Bacterianas , Pseudomonas aeruginosa , Animales , Ratones , Humanos , Proteínas Bacterianas/metabolismo , Pseudomonas aeruginosa/genética , Sistemas de Secreción Tipo III/metabolismo , Bacterias/metabolismo , Ofloxacino/metabolismo , Antibacterianos/metabolismo , Regulación Bacteriana de la Expresión GénicaRESUMEN
Studies have shown that epithelium-expressed antimicrobial peptides (AMPs), e.g., ß-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 ß-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 â¼8-fold (P < 0.001). In vivo experiments showed that the loss of murine ß-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.
Asunto(s)
Defensinas/metabolismo , Epitelio Corneal/metabolismo , Epitelio Corneal/microbiología , Pseudomonas aeruginosa/fisiología , Animales , Línea Celular , Regulación de la Expresión Génica/fisiología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , beta-Defensinas/genética , beta-Defensinas/metabolismoRESUMEN
Flagella and pili are appendages that modulate attachment of Pseudomonas aeruginosa to solid surfaces. However, previous studies have mostly reported absolute attachment. Neither the dynamic roles of these appendages in surface association nor those of attachment phenotypes have been quantified. We used video microscopy to address this issue. Unworn, sterile, soft contact lenses were placed in a laminar-flow optical chamber. Initial lens association kinetics for P. aeruginosa strain PAK were assessed in addition to lens-surface association phenotypes. Comparisons were made to strains with mutations in flagellin (fliC) or pilin (pilA) or those in flagellum (motAB) or pilus (pilU) function. PAK and its mutants associated with the contact lens surface at a constant rate according to first-order kinetics. Nonswimming mutants associated â¼30 to 40 times slower than the wild type. PAK and its pilA mutant associated at similar rates, but each â¼4 times faster than the pilU mutant. Lens attachment by wild-type PAK induced multiple phenotypes (static, lateral, and rotational surface movement), each showing only minor detachment. Flagellin (fliC) and flagellar-motility (motAB) mutants did not exhibit surface rotation. Conversely, strains with mutations in pilin (pilA) and pilus retraction (pilU) lacked lateral-surface movement but displayed enhanced surface rotation. Slower surface association of swimming-incapable P. aeruginosa mutants was ascribed to lower convective-diffusion-arrival rates, not to an inability to adhere. Flagellum function (swimming) enhanced lens association, attachment, and rotation; hyperpiliation hindered lens association. P. aeruginosa bound through three different adhesion sites: flagellum, pili, and body. Reduction of bacterial attachment to contact lenses thus requires blockage of multiple adhesion phenotypes.