Staphylococcus aureus exploitation of autophagy promotes latent infection

描述（由申请方提供）：金黄色葡萄球菌（SA）是皮肤和软组织感染的最常见原因。尽管有多种抗生素对耐甲氧西林的沙门氏菌具有体外活性。金黄色葡萄球菌（MRSA）菌株，SA感染往往是持久性的，并在长时间的潜伏期后重新激活。由于针对生物体的治疗策略在根除这些感染方面并不成功，因此该项目的目标是针对宿主-鉴定被利用并作为持续性SA感染病灶的角质形成细胞的成分。我们假设，角质形成细胞的自噬体代表这样一个受保护的小生境选择SA突变体，已适应这些细胞内的氧化应激条件，并容忍当地浓度的有毒多胺。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区室，并确定SA激活的诱导自噬体形成的信号通路。由于自噬通常用于根除细胞内病原体，因此我们预测选择特定的SA突变体来响应这种环境，避免溶酶体融合和根除，并且可以耐受该隔室内的氧化应激。我们将使用生物信息学方法，比较选择适应自噬体的实验室菌株的全基因组测序数据与慢性感染临床分离株的基因型。R21组分的目标是确定响应于自噬体主动选择特定SA突变体，并在角质形成细胞内建立潜伏感染的病灶。在R33组分中，我们将开发人皮肤感染的模型;首先使用人原代角质形成细胞的器官型培养物，以确定削弱角质形成细胞中自噬诱导的药理学和遗传技术将防止慢性SA感染的建立。使用靶向AMPK-TORC 1通路的药物或抑制特定自噬基因的siRNA，我们希望阻断自噬并阻止选择可以在这种环境中存活的SA突变体。然后，在存在完整免疫应答的情况下，将具有人皮肤异种移植物的SCID：hu小鼠用作人皮肤感染背景下的原理证明。将在人皮肤移植物上测试在体外鉴定的最有效的试剂，以测试其防止SA适应人角质形成细胞的能力。

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.