Oxidative Stress-induced mechanisms of biofilms development in chronic wounds colonized with Pseudomonas aeruginosa

铜绿假单胞菌定植的慢性伤口中氧化应激诱导的生物膜发育机制

• 批准号: 10320028
• 负责人: MANUELA M. MARTINS-GREEN
• 金额: $ 21.98万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-17 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10320028
• 关键词: Adhesions; Affect; Aftercare; Animal Model; Bacteria; Bacterial Adhesins; Beds; Biological Markers; Cell Adhesion Molecules; Cell Death; Characteristics; Chronic; Complex; DNA Damage; Debridement; Deposition; Development; Diabetic mouse; Environment; Epithelial; Excision; Funding; Future; Gene Expression; Genes; Genome; Healthcare; Human; Human Characteristics; Impairment; In Vitro; Inflammation; Injury; Lead; Lipids; Microbial Biofilms; Modeling; Molecular; Mus; Mutation; Obese Mice; Oncogene Deregulation; Oxidative Stress; Persons; Process; Production; Proteins; Pseudomonas aeruginosa; Regulation; Skin; Sterility; Surface; Testing; Time; Virulence Factors; Virulent; Work; Wound Infection; Wound models; angiogenesis; antioxidant enzyme; base; biological adaptation to stress; catalase; chronic wound; combat; cost; diabetic; diabetic ulcer; experimental study; healing; in vivo; inhibitor; innovation; keratinocyte; leptin receptor; microbiome; migration; mouse model; novel; prevent; quorum sensing; response; skin microbiome; success; transcriptome sequencing; wound; wound bed; wound biofilm; wound treatment

Chronic wounds impact ~6.5M people and cost ~$25B/year in the US alone. Despite significant effort, understanding the mechanisms involved in development of chronic wounds in humans has met with limited success, primarily because we cannot experiment in human chronic wounds and because current animal models are inadequate. We have developed a novel mouse model for diabetic chronic wounds that closely mimics those of humans. High levels of oxidative stress (OS) are important for chronic wound development. Human chronic wounds have high levels of OS. Using diabetic mice, we can generate chronic wounds 100% of the time by creating high levels of OS immediately after wounding by treating with inhibitors specific to two antioxidant enzymes. The wounds become fully chronic within 20 days after treatment and remain chronic until the mouse dies, sometimes >100 days. The wounds in the mouse model feature all of the same problems observed in human chronic diabetic wounds: high levels of OS lead to DNA damage, gene deregulation, protein and lipid damage, cell death, impaired keratinocyte migration (potentially inhibiting re-epithelialization), chronic inflammation, lack of proper angiogenesis and matrix deposition. Equally important, the chronic wounds in the mouse model develop a biofilm from the bacteria present on the skin microbiome by elimination of non-biofilm-forming bacteria in favor of the biofilm-forming species. These biofilm-forming bacteria are also present on human skin and appear in human diabetic chronic wounds. All of these characteristics indicate that the PI's mouse model mimics key aspects of human chronic wounds. We hypothesize that high OS levels affect the microenvironment of the wound resulting in expression of genes that combat OS, that are involved in adhesin and expression of quorum sensing molecules and virulent factors that favor biofilm development by P. aeruginosa. To test this hypothesis, we will: Aim#1: Isolate a pure culture of PA from the biofilms in our chronic wound mouse model and sequence its genome. We already isolated PA from one such wound. Aim#2: Using RNAseq, perform experiments in sterile wounds infected with the isolated P.A alone in the presence or absence of high OS and: A. Determine whether P.A genes known to be involved in response to high levels of OS, adhesion to surfaces, production of quorum sensing molecules and virulence factors in vitro are also expressed in in vivo in the CW bed during the transition of PA from non-biofilm-forming in the skin microbiome to biofilm-forming in the CW. B. Identify new genes that are expressed by PA in the wound bed versus abiotic surfaces, and if time permits or with future funding determine whether they may be important in the transition of PA to biofilm forming. Our proposal is significant and innovative because with the use of our novel db/db-/- chronic wound model, we will determine how P.A becomes biofilm-forming in the high OS environment of a chronic wound. We will also identify P.A molecules that contribute to biofilm development by this bacterium in the wound bed. Most importantly, our work will impact health care because it will potentially identify biomarkers that are critical for initiation of biofilm development by P.A in diabetic wounds. Such biomarkers have the potential, when verified in humans, to objectively guide treatment after debridement to prevent return of biofilm. Currently, wound bed assessment is subjective.

慢性伤口影响约650万人，仅在美国每年花费约250亿美元。尽管付出了巨大的努力， 涉及人类慢性伤口发展的机制取得的成功有限，主要是因为 我们不能在人类慢性伤口中进行实验，因为目前的动物模型是不够的。我们已经开发 一种新型的糖尿病慢性伤口小鼠模型，非常接近人类的伤口。高水平的氧化应激 (OS)对慢性伤口的发展很重要人类慢性伤口具有高水平的OS。使用糖尿病小鼠， 我们可以通过在创伤后立即产生高水平的OS， 用两种抗氧化酶的特异性抑制剂。伤口在治疗后20天内完全变成慢性， 保持慢性直到小鼠死亡，有时>100天。小鼠模型中的伤口都具有相同的特征 在人类慢性糖尿病伤口中观察到的问题：高水平的OS导致DNA损伤，基因失调，蛋白质 和脂质损伤、细胞死亡、受损的角质形成细胞迁移（潜在地抑制上皮再生）、慢性 炎症、缺乏适当的血管生成和基质沉积。同样重要的是，老鼠的慢性伤口 模型通过消除非生物膜形成细菌，从皮肤微生物组上存在的细菌中形成生物膜 有利于形成生物膜的物种。这些生物膜形成细菌也存在于人类皮肤上， 人类糖尿病慢性伤口。所有这些特征表明，PI的小鼠模型模拟了 人类慢性创伤我们假设高OS水平影响伤口的微环境， 与OS对抗的基因的表达，这些基因参与粘附素和群体感应分子的表达， 有利于铜绿假单胞菌生物膜形成的毒力因子。为了验证这个假设，我们将：目标#1：分离一个纯的 在我们的慢性创伤小鼠模型中从生物膜中培养PA并对其基因组测序。我们已经隔离了 一个这样的伤口。目标#2：使用RNAseq，在感染了分离的 在存在或不存在高OS的情况下单独使用P. A，以及：A.确定是否已知参与P.A基因 对高水平OS的反应，对表面的粘附，群体感应分子和体外毒力因子的产生 在PA从皮肤微生物组中的非生物膜形成转变期间，也在CW床中体内表达 生物膜的形成。B。鉴定PA在伤口床与非生物表面中表达的新基因， 如果时间允许或有未来的资金，确定它们是否在PA向生物膜的过渡中重要 成形我们的建议是重要的和创新的，因为使用我们的新的db/db-/-慢性伤口模型，我们 将决定P. A如何在慢性伤口的高OS环境中形成生物膜。我们还将确定 P.A分子，有助于伤口床中细菌的生物膜发展。最重要的是，我们的工作 将影响医疗保健，因为它可能会识别生物标志物，这些生物标志物对生物膜的形成至关重要 在糖尿病伤口中的应用这些生物标志物在人类中得到验证后，有可能客观地指导治疗 以防止生物膜的返回。目前，伤口床评估是主观的。