Corneal Infection: Role of Bacterial Adaptation

角膜感染：细菌适应的作用

• 批准号: 8774092
• 负责人: Suzanne MJ FLEISZIG
• 金额: $ 39.14万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8774092
• 关键词: Adenylate Cyclase; Bacteria; Bacterial Adhesion; Bacterial Typing; Bioinformatics; Biological Assay; Cells; Contact Lenses; Cornea; Corneal Diseases; DNA Sequence; Data; Disease; Environment; Epithelial; Epithelial Cells; Epithelium; Exposure to; Extended-Wear Contact Lenses; Eye; Gene Expression; Gene Expression Profile; Genes; Growth; Human; Hydrophilic Contact Lenses; In Situ; In Vitro; Infection; Infection prevention; Integration Host Factors; Knock-out; Lead; Liquid substance; Mediating; Microbial Biofilms; Monitor; Morphology; Mus; Nature; Onset of illness; Pathogenesis; Pathway interactions; Peptide Hydrolases; Pseudomonas aeruginosa; Publishing; Rattus; Resistance; Risk; Risk Factors; Role; Severities; System; Testing; Time; Virulent; Vision; antimicrobial; cell motility; corneal epithelium; experience; extracellular; in vivo; injured; lens; mutant; novel; novel strategies; ocular surface; pathogen; prevent; public health relevance; resistance factors; transcriptome sequencing

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.

描述（由申请人提供）：覆盖角膜的上皮通常会抵抗铜绿假单胞菌和几乎所有其他细菌的穿过。然而，软性接触透镜的佩戴使角膜易于受到这种病原体的感染。我们发表的数据显示，虽然健康的大鼠角膜在配戴被铜绿假单胞菌污染的软性角膜接触镜时始终会受到感染，但疾病发作会延迟（中位数约为8天）。将镜片从感染的大鼠眼睛转移到未感染的大鼠眼睛，将延迟从约8天减少到约2天，而在透镜验配之前表面损伤角膜对疾病时间或其严重程度没有影响。这些数据表明，大鼠中的疾病发作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一致。人体（体外）数据也支持细菌适应的作用，因为铜绿假单胞菌穿过培养的人角膜上皮细胞之前有一个显着的延迟。假设是暴露于泪液或角膜上皮细胞中的宿主抗菌剂触发铜绿假单胞菌3型分泌系统（T3 SS）效应物、蛋白酶和抗性基因的表达，然后介导细菌穿过角膜上皮。支持这一假设的初步数据包括： 1)暴露于体内因素，而不仅仅是透镜上的生物膜生长，能够使细菌适应促进体内上皮穿越。2)在穿过多层人类角膜上皮后 在细胞中，铜绿假单胞菌表现出增强的（~100-1000倍）穿过细胞的能力，这与在细胞内存活和损害跨上皮电阻（TER）的能力增加相关。3)上皮穿越改变铜绿假单胞菌基因表达。4)一个子集的影响基因调节遍历，显示使用细菌突变体。5)这些基因中至少有一个是细菌T3 SS的新型调节因子。6)T3 SS介导体外（人细胞）和体内（小鼠）的穿越。7)铜绿假单胞菌T3 SS的表达在暴露于人泪液或人角膜上皮细胞裂解物时上调。8)T3 SS-独立的因素也可以介导穿越给予较长的暴露，或者如果宿主先天防御受损（例如MyD 88敲除）。目的1将确定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表因素而变化，并使用Tn-seq来缩小这些变化中哪些调节上皮穿越。目的2将研究其参与人类体外穿越试验和小鼠角膜原位使用细菌突变体的机制。目的3将探索宿主触发因素，使关键的细菌适应，使用qRT-PCR监测暴露于内源性宿主抗菌剂的影响。由于上皮穿越是感染发病机制的早期步骤，因此研究细菌适应性和使细菌能够做到这一点的宿主触发因素可能会导致预防而不仅仅是治疗感染的新策略。