Role of Pseudomonas aeruginosa ExsA in penetration through corneal epithelium in a novel in vivo model
Role of Pseudomonas aeruginosa ExsA in penetration through corneal epithelium in a novel in vivo model
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DOI：
10.1167/iovs.03-0229
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发表时间：
2003-12-01
影响因子：
4.4
通讯作者：
Fleiszig, SMJ
中科院分区：
文献类型：
--
作者：
Lee, EJ;Evans, DJ;Fleiszig, SMJ
目的。采用改良的角膜剥脱性角膜炎模型，研究铜绿假单胞菌对愈合角膜上皮的感染作用。然后使用新的模型来研究ExsA，一种铜绿假单胞菌的转录激活因子，在细菌穿透受伤和愈合的角膜上皮细胞中的作用。方法：在C57BL/6小鼠的划痕损伤角膜愈合0，6，9或12小时后接种细胞毒性(6206)或侵袭性(PAO1)铜绿假单胞菌菌株。疾病进展被监测了14天。通过荧光素染色和组织学检查，对未感染眼的愈合上皮的完整性进行了研究。在其他实验中，研究了愈合0、6或12小时后细菌exsA突变的影响。感染3小时后，用活菌计数来量化早期细菌定植水平，或在显微镜下切片以研究细菌对上皮的渗透。结果：角膜在划痕损伤后6小时而不是12小时仍对感染敏感。到6小时时，先前暴露的间质已经完全被几层上皮细胞覆盖。即使在愈合时间12小时后，荧光素染色也出人意料地出现，表明对感染的抵抗力早于上皮屏障功能的完全恢复。细菌接种后3小时，exsA突变可降低细菌定植水平和穿透上皮细胞，但仅限于愈合后6小时，且仅对细胞毒菌株(PA103)有效。侵袭株(PAO1)中exsA的突变在任何情况下都不影响3h的定植或穿透水平。结论：6h愈合性感染模型显示exsA在与角膜上皮的早期相互作用中发挥作用，这在传统的(0h)划痕模型中是检测不到的。对6小时和12小时愈合模式的比较表明，除了屏障功能之外，还有一些因素有助于抵御感染，这可以用来对角膜防御机制以及细菌绕过这些机制的方法有新的见解。
PURPOSE. The scarified cornea keratitis model was modified to study Pseudomonas aeruginosa infection of healing corneal epithelium. The new model was then used to study the role of ExsA, a transcriptional activator of P. aeruginosa, in bacterial penetration through injured and healing corneal epithelia.METHODS. Scratch-injured corneas of C57BL/6 mice were allowed to heal for 0, 6, 9, or 12 hours before inoculation with a cytotoxic (6206) or invasive (PAO1) P. aeruginosa strain. Disease progression was monitored for 14 days. The integrity of the healing epithelium was studied in uninfected eyes by fluorescein staining and by histologic examination. In other experiments, the effect of bacterial exsA mutation was studied after 0, 6, or 12 hours of healing. Three hours after infection, these eyes were used to quantify early bacterial colonization levels by viable counts, or they were sectioned to study bacterial penetration through the epithelium by microscopy.RESULTS. Corneas remained susceptible to infection 6 but not 12 hours after scratch injury. By 6 hours, the previously exposed stroma was already completely covered by several layers of epithelial cells. Fluorescein staining unexpectedly occurred even after 12 hours of healing time, showing that resistance to infection preceded full restoration of epithelial barrier function. Mutation of exsA reduced both bacterial colonization levels and penetration through the epithelium 3 hours after bacterial inoculation, but only in the 6-hour healing situation, and only for the cytotoxic strain (PA103). Mutation of exsA in the invasive strain (PAO1) had no effect on 3-hour colonization or penetration levels under any circumstances.CONCLUSIONS. The 6-hour healing infection model showed a role for ExsA in early interactions with the corneal epithelium that was not detectable with the conventional (0-hour) scratch model. Comparison of the 6- and 12-hour healing models, which showed that factors additional to barrier function contribute to defense against infection, could be used to gain new insights into corneal defense mechanisms, and the methods used by bacteria to circumvent them.