Metruccio Matteo M E, Evans David J, Gabriel Manal M, Kadurugamuwa Jagath L, Fleiszig Suzanne M J
School of Optometry, University of California Berkeley, CA, USA.
School of Optometry, University of CaliforniaBerkeley, CA, USA; College of Pharmacy, Touro University CaliforniaVallejo, CA, USA.
Front Microbiol. 2016 Jun 3;7:871. doi: 10.3389/fmicb.2016.00871. eCollection 2016.
Pseudomonas aeruginosa is a leading cause of human morbidity and mortality that often targets epithelial surfaces. Host immunocompromise, or the presence of indwelling medical devices, including contact lenses, can predispose to infection. While medical devices are known to accumulate bacterial biofilms, it is not well understood why resistant epithelial surfaces become susceptible to P. aeruginosa. Many bacteria, including P. aeruginosa, release outer membrane vesicles (OMVs) in response to stress that can fuse with host cells to alter their function. Here, we tested the hypothesis that mucosal fluid can trigger OMV release to compromise an epithelial barrier. This was tested using tear fluid and corneal epithelial cells in vitro and in vivo. After 1 h both human tear fluid, and the tear component lysozyme, greatly enhanced OMV release from P. aeruginosa strain PAO1 compared to phosphate buffered saline (PBS) controls (∼100-fold). Transmission electron microscopy (TEM) and SDS-PAGE showed tear fluid and lysozyme-induced OMVs were similar in size and protein composition, but differed from biofilm-harvested OMVs, the latter smaller with fewer proteins. Lysozyme-induced OMVs were cytotoxic to human corneal epithelial cells in vitro and murine corneal epithelium in vivo. OMV exposure in vivo enhanced Ly6G/C expression at the corneal surface, suggesting myeloid cell recruitment, and primed the cornea for bacterial adhesion (∼4-fold, P < 0.01). Sonication disrupted OMVs retained cytotoxic activity, but did not promote adhesion, suggesting the latter required OMV-mediated events beyond cell killing. These data suggest that mucosal fluid induced P. aeruginosa OMVs could contribute to loss of epithelial barrier function during medical device-related infections.
铜绿假单胞菌是导致人类发病和死亡的主要原因，常侵袭上皮表面。宿主免疫功能低下或存在包括隐形眼镜在内的留置医疗器械会增加感染风险。虽然已知医疗器械会积累细菌生物膜，但对于抗性上皮表面为何会变得易受铜绿假单胞菌感染，人们还了解甚少。许多细菌，包括铜绿假单胞菌，在应激时会释放外膜囊泡（OMV），这些囊泡可与宿主细胞融合以改变其功能。在此，我们检验了这样一个假设：黏膜液体会触发OMV释放，从而破坏上皮屏障。我们在体外和体内分别使用泪液和角膜上皮细胞对此进行了测试。1小时后，与磷酸盐缓冲盐水（PBS）对照相比，人泪液以及泪液成分溶菌酶均大大增强了铜绿假单胞菌PAO1菌株的OMV释放（约100倍）。透射电子显微镜（TEM）和SDS - PAGE显示，泪液和溶菌酶诱导产生的OMV在大小和蛋白质组成上相似，但与从生物膜收获的OMV不同，后者较小且蛋白质较少。溶菌酶诱导产生的OMV在体外对人角膜上皮细胞以及在体内对小鼠角膜上皮具有细胞毒性。体内OMV暴露会增强角膜表面Ly6G/C的表达，提示髓样细胞募集，并使角膜对细菌黏附产生预激发作用（约4倍，P < 0.01）。超声处理破坏的OMV仍保留细胞毒性活性，但不促进黏附，这表明后者需要OMV介导的除细胞杀伤之外的事件。这些数据表明，黏膜液体诱导产生的铜绿假单胞菌OMV可能在与医疗器械相关的感染过程中导致上皮屏障功能丧失。
