Staphylococcus aureus exploitation of autophagy promotes latent infection

描述（由申请人提供）：金黄色葡萄球菌（SA）是皮肤和软组织感染的最常见原因。尽管多种抗生素对耐甲氧西林金黄色葡萄球菌（MRSA）具有体外活性，但SA感染通常持续存在，并在长时间潜伏期后重新激活。由于针对生物体的治疗策略不能成功根除这些感染，因此本项目的目标是针对宿主-识别角化细胞的成分，这些成分被利用并作为持续SA感染的病灶。我们假设角化细胞的自噬体为SA突变体的选择提供了这样一个受保护的生态位，这些突变体已经适应了这些细胞内的氧化应激条件，并耐受局部浓度的有毒多胺。在本项目的R21阶段，我们将充分表征与SA持久性相关的细胞内腔室，并确定SA激活的诱导自噬体形成的信号通路。由于自噬通常具有根除细胞内病原体的功能，我们预测特异性SA突变体是为了应对这种环境而选择的，这种环境可以避免溶酶体融合和根除，并且可以耐受细胞内的氧化应激。我们将使用生物信息学方法，比较实验室菌株适应自噬体的全基因组测序数据与慢性感染临床分离株的基因型。R21成分的目的是确定特异性SA突变体在响应自噬体时被主动选择，并在角化细胞内建立潜伏感染病灶。在R33部分，我们将开发一个人体皮肤感染模型；首先使用人原代角质形成细胞的器官型培养，以确定药理学和遗传技术削弱角质形成细胞自噬的诱导将阻止慢性SA感染的建立。使用靶向AMPK-TORC1通路或抑制特异性自噬基因的siRNA的药物，我们期望阻断自噬并阻止SA突变体在这种环境下存活的选择。移植了人类皮肤的SCID:hu小鼠将在存在完整免疫反应的情况下作为人类皮肤感染的原理证明。在体外鉴定出的最有效的试剂将在人皮肤移植物上进行测试，以确定其阻止SA对人角质形成细胞适应的能力。

DESCRIPTION (provided by applicant): Staphylococcus aureus (SA) is the most common cause of skin and soft tissue infection. Despite multiple classes of antibiotics with in vitro actiity against even the methicillin resistant S. aureus (MRSA) strains, SA infections are often persistent and reactivate after long periods of latency. As therapeutic strategies targeting the organism are not successful in eradicating these infections, the goal of this project is to target the host - to identify components of the keratinocyte that are exploited and serve as a nidus for persistent SA infection. We postulate that the autophagosome of keratinocytes represents such a protected niche for the selection of SA mutants that have adapted to the conditions of oxidative stress within these cells and tolerate local concentrations of toxic polyamines. In the R21 phase of this project, we will fully characterize the intracellular compartment associated with SA persistence and identify the signaling pathways activated by SA to induce autophagosome formation. As autophagy normally functions to eradicate intracellular pathogens, we predict that specific SA mutants are selected in response to this milieu that avoid lysosomal fusion and eradication and can tolerate the oxidative stress within this compartment. We will use a bioinformatics approach, comparing whole genome sequencing data from laboratory strains selected for adaptation to the autophagosome with the genotypes of clinical isolates from chronic infections. The goals of the R21 component are to establish that specific SA mutants are actively selected in response to the autophagosome and establish a nidus of latent infection within keratinocytes. In the R33 component, we will develop a model of human skin infection; first using organotypic cultures of human primary keratinocytes, to establish that pharmacologic and genetic techniques to impair the induction of autophagy in keratinocytes will prevent the establishment of chronic SA infection. Using drugs that target the AMPK-TORC1 pathway or siRNA inhibiting specific autophagy genes, we expect to block autophagy and prevent the selection of SA mutants that can survive in this setting. A SCID:hu mouse with human skin xenografts will then be used as proof of principle in the setting of human skin infection in the presence of an intact immune response. The most effective reagents identified in vitro will be tested on human skin grafts for their ability to prevent the adaptation of SA to the human keratinocyte.