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，每年的费用约为$ 25B。尽管付出了巨大的努力，理解 人类慢性伤口涉及的机制取得了有限的成功，主要是因为 我们无法在人类慢性伤口进行实验，并且因为当前的动物模型不足。我们已经发展了 一种新型的小鼠模型，用于密切模仿人类的糖尿病慢性伤口。高水平的氧化应激 （OS）对于慢性伤口发育很重要。人类慢性伤口具有高水平的OS。使用糖尿病小鼠， 我们可以通过治疗后立即产生高水平的OS来产生慢性伤口100％的时间 具有针对两种抗氧化酶的抑制剂。伤口在治疗后20天内完全慢性 保持慢性直至鼠标死亡，有时> 100天。鼠标模型中的伤口具有相同的所有 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，角质形成细胞迁移受损（可能抑制重新上皮化），慢性 炎症，缺乏适当的血管生成和基质沉积。同样重要的是，小鼠的慢性伤口 模型通过消除非生物膜形成的细菌从皮肤微生物组上存在的细菌发展出生物膜 有利于生物膜形成物种。这些生物膜形成的细菌也存在于人的皮肤上，并出现在 人类糖尿病慢性伤口。所有这些特征表明PI的鼠标模型模仿了关键方面 人类慢性伤口。我们假设高OS水平会影响伤口的微环境，从而导致 作战OS的基因的表达，与粘附蛋白有关和法定人数传感分子的表达和表达 铜绿假单胞菌有利于生物膜开发的毒物。为了检验这一假设，我们将：目标＃1：隔离纯 我们慢性伤口小鼠模型中的生物膜培养物培养并对其基因组进行测序。我们已经孤立了 PA从这样的伤口中。 AIM＃2：使用RNASEQ，在感染的无菌伤口中进行实验 在存在或不存在高OS和A.的情况下，仅P.a确定已知是否涉及的P.a基因 对高水平的OS的反应，对表面的粘附，体外的群体传感分子的产生和毒力因子的响应 在PA从皮肤微生物组中的非生物膜形成的PA过渡期间，在CW床的体内也表达 在CW中形成生物膜。 B.确定PA在伤口床与非生物表面中表达的新基因， 如果时间允许或将来的资金确定它们在PA过渡到生物膜中是否重要 成型。我们的建议具有重要意义和创新性，因为使用了我们的新型DB/DB - / - 慢性伤口模型，我们 将确定在慢性伤口的高OS环境中，P.A如何形成生物膜。我们还将确定 P.A分子在伤口床上通过这种细菌有助于生物膜发育。最重要的是，我们的工作 会影响医疗保健，因为它可能会识别生物膜开发至关重要的生物标志物 通过糖尿病伤口的P.A。这种生物标志物在人类中验证时具有客观指导治疗的潜力 清创术后，以防止生物膜返回。目前，伤口床评估是主观的。