Skin microbial-based mechanisms of accelerated wound healing

项目摘要 对于不愈合的伤口，需要改进的治疗方法，因为它们呈现出主要的 由于治疗费用以及发病率和死亡率的增加，对卫生保健系统构成了挑战。 皮肤微生物组存在于所有皮肤伤口的界面处，但其作为一种新型治疗方法的潜力 目标仍未锁定因此，我们的长期目标是了解宿主-微生物之间的相互作用， 伤口愈合，所以我们可以利用这些机制来确定伤口愈合治疗。启动该 调查，我们的实验室以前进行了宏基因组分析的伤口样本，从非感染 糖尿病足溃疡（DFU），并出土了一个令人惊讶的普遍伤口居民粪产碱菌。非常 在创伤的背景下，对该物种知之甚少，因此我们试图研究A.粪 对伤口愈合的影响我们观察到令人惊讶的发现，用A.粪便加快了 在体内的伤口愈合和激活角质形成细胞的促上皮化表型。因此，中心目标 本研究的目的是确定A.粪便介导加速伤口愈合。亲 炎性细胞因子信号传导是促进上皮再形成所必需的，特别是IL-6， 显示在愈合过程中诱导角质形成细胞的活化。因此，我测试了A。粪便诱导的细胞因子 在角质形成细胞中的应答，并发现用A.粪条件培养基。 细菌可以调节宿主反应的主要机制是通过产生分泌的 分子。我发现A.粪便，而不是细菌细胞表面分子， 角质形成细胞迁移和IL-6产生。总之，这些发现导致我的假设，A。粪 产生分泌的分子，通过增强角质形成细胞IL-6信号传导来改善上皮再形成。要求1 将决定A的宿主机制。通过结合以下措施，粪便诱导加速愈合 伤口愈合测定和转录谱分析。目的二是确定A. 产生促愈合分泌分子所必需的粪便。我将用比较基因组学的方法 鉴定与促伤口愈合表型分离的基因组基因座。为了实现这一目标，我将利用 收藏44 A粪肠球菌临床DFU分离株，并进行基因组多重比对。我会把这个 使用伤口愈合测定的表型筛选的遗传筛选，以确定哪个基因组位点 与促愈合表型分离。 结合这些实验目标，我还将参加一个严格的培训计划， 在我的博士导师的指导下，我在宾夕法尼亚大学攻读博士学位。本培训计划 我将通过以下目标促进我作为未来医生科学家的发展：加强 独立作为一个调查员，发展计算和湿实验室技术，扩大知识， 微生物组和伤口愈合领域，改善科学交流，并从事指导。

PROJECT SUMMARY Improved therapeutic approaches are needed for non-healing wounds, as they present a major challenge to the healthcare system by increasing treatment costs as well as rates of morbidity and mortality. The skin microbiome exists at the interface of all cutaneous wounds, but its potential as a novel therapeutic target remains untapped. Therefore, our long-term goal is to understand host-microbial interactions during wound healing, so we may leverage these mechanisms to identify wound healing treatments. To initiate this investigation, our lab previously performed a metagenomic analysis on wound samples from non-infected diabetic foot ulcers (DFUs) and unearthed a surprisingly prevalent wound inhabitant Alcaligenes faecalis. Very little is known about this species in the context of wounds, so we sought to investigate the effect of A. faecalis on wound healing. We observed the surprising finding that treating wounds with A. faecalis accelerates the rate of wound healing in vivo and activates a pro-epithelialization phenotype in keratinocytes. Thus, the central goal of this study is to identify the mechanism by which A. faecalis mediates accelerated wound healing. Pro- inflammatory cytokine signaling is necessary to promote re-epithelialization, and IL-6 in particular has been shown to induce activation of keratinocytes during healing. Therefore, I tested if A. faecalis induced a cytokine response in keratinocytes and found robust IL-6 production after treatment with A. faecalis conditioned media. A primary mechanism by which bacteria can modulate host responses is through production of secreted molecules. I found that sterile supernatant of A. faecalis, rather than bacterial-cell surface molecules, promotes keratinocyte migration and IL-6 production. Together, these findings lead to my hypothesis that A. faecalis produces secreted molecules that improve re-epithelialization by enhancing keratinocyte IL-6 signaling. Aim 1 will determine the host mechanisms of A. faecalis-induced accelerated healing through a combination of wound healing assays and transcriptional profiling. Aim 2 will identify the microbial genetic determinants of A. faecalis necessary to produce the pro-healing secreted molecule. I will use comparative genomics approach to identify genomic loci that segregate with a pro-wound healing phenotype. To complete this aim, I will leverage our collection of 44 A. faecalis clinical DFU isolates and perform a genomic multiple alignment. I will pair this genetic screen with a phenotypic screen using wound healing assays to determine which genomic locus segregates with the pro-healing phenotype. In conjunction with these experimental aims, I will also engage in a rigorous training plan at the University of Pennsylvania under the guidance of the MD/PhD Program and my PhD advisor. This training plan will foster my development as a future physician scientist through the following goals: strengthen independence as an investigator, develop computational and wet lab techniques, expand knowledge in the microbiome and wound healing fields, improve scientific communication, and engage in mentorship.