Staphylococcus aureus exploitation of autophagy promotes latent infection

描述(申请人提供)：金黄色葡萄球菌(SA)是最常见的皮肤和软组织感染的原因。尽管多种抗生素对耐甲氧西林金黄色葡萄球菌(MRSA)菌株具有体外活性，但金黄色葡萄球菌感染通常是持久的，并在长时间潜伏期后重新激活。由于针对机体的治疗策略在根除这些感染方面并不成功，本项目的目标是针对宿主-识别被利用的角质形成细胞成分，并作为持续SA感染的核心。我们推测，角质形成细胞的自噬小体代表了这样一个受保护的利基环境，用于选择已适应这些细胞内氧化应激条件并耐受局部浓度有毒多胺的SA突变体。在这个项目的R21阶段，我们将充分描述与SA持久性相关的细胞内间隔，并确定SA激活的诱导自噬小体形成的信号通路。由于自噬通常起到消除细胞内病原体的作用，我们预测会选择特定的SA突变体来响应这种环境，避免溶酶体的融合和根除，并能够耐受这个隔室内的氧化应激。我们将使用生物信息学方法，将实验室菌株的全基因组测序数据与慢性感染的临床分离株进行比较，这些菌株被选为适应自噬小体。R21组分的目标是确定特定的SA突变体被主动选择以响应自噬小体，并在角质形成细胞内建立潜伏感染的病灶。在R33组件中，我们将建立一个人类皮肤感染的模型；首先使用人原代角质形成细胞的器官型培养，以建立药理学和遗传学技术来削弱诱导角质形成细胞的自噬，以防止建立慢性SA感染。使用针对AMPK-TORC1途径的药物或抑制特定自噬基因的siRNA，我们希望阻止自噬，并阻止选择能够在这种情况下存活的SA突变体。然后，一只移植了人皮肤的SCID：HU小鼠将被用作在存在完整免疫反应的情况下设定人类皮肤感染的原则证据。在体外确定的最有效的试剂将在人类皮肤移植物上测试它们阻止SA适应人类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.