Corneal Infection: Role of Bacterial Adaptation

描述(申请人提供)：覆盖角膜的上皮通常能抵抗铜绿假单胞菌和几乎所有其他细菌的侵袭。然而，配戴软性隐形眼镜使角膜容易受到这种病原体的威胁-感染。我们公布的数据显示，尽管健康的大鼠角膜如果戴上受铜绿假单胞菌污染的软性隐形眼镜，总是会死于感染，但疾病发作的时间会延迟(中位数~8天)。将晶状体从感染的大鼠眼移植到NA�ve大鼠眼内，将延迟从~8天减少到~2天，而在配戴晶状体之前对角膜进行表面损伤对疾病的时间和严重程度没有影响。这些数据表明，大鼠发病延迟是因为细菌需要时间适应眼表环境才能变得有毒，这与长时间佩戴隐形眼镜的人感染风险增加是一致的。人体(体外)数据也支持细菌适应的作用，因为在铜绿假单胞菌穿过培养的人角膜上皮之前有显著的延迟。假设暴露于泪液或角膜上皮细胞中的宿主抗菌剂会触发铜绿假单胞菌3型分泌系统(T3SS)效应器、蛋白酶和耐药基因的表达，然后介导细菌穿越角膜上皮。支持这一假设的初步数据包括： 1)暴露于体内因素，而不仅仅是晶状体上的生物膜生长，能够使细菌适应促进体内上皮细胞的穿越。2)穿过多层人角膜上皮细胞后 在细胞中，铜绿假单胞菌表现出增强的(~100-1000倍)穿越细胞的能力，这与在细胞内生存的能力增加和降低跨上皮阻力(TER)有关。3)上皮穿越改变了铜绿假单胞菌的基因表达。4)利用细菌突变体显示，受影响的基因的子集调节遍历。5)这些基因中至少有一个是细菌T3SS的新调控基因。6)T3SS在体外(人细胞)和体内(小鼠)介导穿透。7)人泪液或人角膜上皮细胞裂解物可上调铜绿假单胞菌T3SS的表达。8)T3SS非依赖因子也可以在较长时间暴露或宿主固有防御受损(例如MyD88基因敲除)的情况下介导穿越。目标1将确定在宿主暴露期间介导穿越的细菌适应。我们将使用RNA-SEQ来研究细菌转录组如何随着暴露于眼睛表面因素而变化，并使用TN-SEQ来缩小这些变化中哪些影响上皮细胞穿越的范围。目的2将利用细菌突变体研究它们参与人类体外遍历实验和小鼠角膜原位实验的机制。目标3将探索使关键细菌适应的宿主触发因素，使用qRT-PCR来监测接触内源性宿主抗菌剂的影响。由于上皮遍历是感染发病机制的早期步骤，研究使细菌能够这样做的细菌适应和宿主触发因素可能会导致预防感染的新策略，而不仅仅是治疗感染。

DESCRIPTION (provided by applicant): The epithelium covering the cornea usually resists traversal by Pseudomonas aeruginosa and almost all other bacteria. Yet, soft contact lens wear predisposes the cornea to sight threating-infections with this pathogen. Our published data show that while healthy rat corneas consistently succumb to infection if fitted with P. aeruginosa-contaminated soft contact lenses, there is a delay in disease onset (median ~8 days). Transferring lenses from infected to na�ve rat eyes, reduces the delay from ~8 to ~2 days, while superficially injuring the cornea before lens fitting has no impact on disease timing or its severity. These data suggest that the disease onset delay in rats occurs because bacteria need time to adapt to the ocular surface environment to become virulent, consistent with the increased risk of infection in people who wear contact lenses for extended time periods. Human (in vitro) data also support a role for bacterial adaptation, as there is a significant delay befor P. aeruginosa traverses cultured human corneal epithelium. The hypothesis is that exposure to host antimicrobials in tear fluid or corneal epithelial cells triggers expression of P. aeruginosa Type 3 Secretion System (T3SS) effectors, proteases and resistance genes that then mediate bacterial traversal of the corneal epithelium. Preliminary data supporting this hypothesis include: 1) Exposure to in vivo factors, not simply biofilm growth on a lens, enables bacterial adaptations promoting epithelial traversal in vivo. 2) After traversing multilayers of human corneal epithelial cells, P. aeruginosa demonstrates an enhanced (~100-1000-fold) capacity to traverse cells, correlating with an increased capacity to survive inside cells and compromise transepithelial resistance (TER). 3) Epithelial traversal alters P. aeruginosa gene expression. 4) A subset of the impacted genes modulate traversal, shown using bacterial mutants. 5) At least one of these genes is a novel regulator of the bacterial T3SS. 6) The T3SS mediates traversal in vitro (human cells) and in vivo (mouse). 7) Expression of the P. aeruginosa T3SS is upregulated on exposure to human tear fluid or human corneal epithelial cell lysates. 8) T3SS-independent factors can also mediate traversal given longer exposure, or if host innate defenses are compromised (e.g. MyD88 knockout). Aim 1 will determine bacterial adaptations during host exposure that mediate traversal. We will use RNA-seq to study how the bacterial transcriptome changes with exposure to ocular surface factors, and Tn-seq to narrow down which of these changes modulate epithelial traversal. Aim 2 will examine the mechanisms for their involvement in a human in vitro traversal assay and in the mouse cornea in situ using bacterial mutants. Aim 3 will explore host triggers enabling the key bacterial adaptations, using qRT-PCR to monitor the impact of exposure to endogenous host antimicrobials. Since epithelial traversal is an early step in the pathogenesis of infection, studying bacterial adaptations and host triggers that enable bacteria to do it could lead to novel strategies for preventing, not simply treating, infection.