Corneal Infection: Role of Bacterial Adaptation

描述（由申请人提供）：覆盖角膜的上皮通常抵抗铜绿假单胞菌和几乎所有其他细菌的穿越。然而，软性隐形眼镜的佩戴使角膜容易受到这种病原体的感染。我们发表的数据显示，健康的大鼠角膜如果配戴被铜绿假单胞菌污染的软性隐形眼镜，就会一直死于感染，但发病时间会延迟（中位数~8天）。将晶状体从感染的大鼠眼移植到正常的大鼠眼，可将延迟时间从8天减少到2天，而在晶状体植入前浅表损伤角膜对疾病时间或严重程度没有影响。这些数据表明，大鼠发病延迟是因为细菌需要时间来适应眼表环境才能变得有毒性，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，因为铜绿假单胞菌在穿过培养的人角膜上皮之前存在显着延迟。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）效应物、蛋白酶和抗性基因的表达，然后介导角膜上皮的细菌穿越。支持这一假设的初步数据包括：

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.