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（由适用提供）：覆盖角膜的上皮通常会通过铜绿假单胞菌和几乎所有其他细菌来抵抗遍历遍历。然而，软接触透镜佩戴了角膜与这种病原体的视力威胁感染。我们发表的数据表明，尽管健康的大鼠角膜始终屈服于铜绿假单胞菌污染的软接触镜，但疾病发作的延迟（中位数〜8天）。将镜头从感染到Na的眼睛转移到大鼠眼睛，将延迟从〜8减少到〜2天，而在晶状体结合之前，超级会损伤角膜，对疾病时机或其严重程度没有影响。这些数据表明，大鼠的疾病发作延迟发生是因为细菌需要时间来适应眼部表面环境才能变得有毒，这与长时间佩戴隐形眼镜的人的感染风险增加一致。人（体外）数据还支持细菌适应的作用，因为铜绿假单胞菌遍历培养的人角膜上皮存在很大的延迟。假设是，暴露于泪液或角膜上皮细胞中的宿主抗菌剂会触发铜绿假单胞菌3型分泌系统（T3SS）的表达（T3SS）的作用，蛋白酶和耐药参加，然后介导角膜上皮细菌的细菌遍历遍历。支持此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，还可以使细菌适应性促进体内促进上皮遍历。 2）穿越人角膜上皮的多层后 细胞，铜绿假单胞菌显示出遍及细胞的增强（〜100-1000倍）的能力，与在细胞内部生存的能力增加并损害了跨多个耐药性（TER）。 3）上皮遍历改变铜绿假单胞菌基因表达。 4）受影响基因的子集调节遍历遍历，使用细菌突变体显示。 5）这些基因中的至少一个是细菌T3SS的新型调节剂。 6）T3SS培养体的体外（人细胞）和体内（小鼠）。 7）对铜绿假单胞菌T3SS的表达进行了更新，以暴露于人泪液或人角膜上皮细胞裂解物。 8）与T3SS无关的因素也可以给定较长的暴露，或者如果托管先天防御受到损害（例如MyD88淘汰赛），则可以中值遍历。 AIM 1将确定宿主暴露期间中位遍历的细菌适应。我们将使用RNA-Seq研究细菌转录组如何随着眼表面因子的暴露而变化，而TN-Seq缩小了这些变化中的哪种变化会调节上皮遍历。 AIM 2将检查使用细菌突变体在体外遍历分析和小鼠角膜中参与的机制。 AIM 3将使用QRT-PCR来监测暴露于内源性宿主抗菌剂的影响，从而探索可实现关键细菌适应的宿主触发器。由于上皮遍历是感染发病机理的早期一步，研究细菌适应和宿主触发因素，使细菌能够做到这一点可能导致新的策略来防止，而不是简单地治疗感染。