Staphylococcus aureus exploitation of autophagy promotes latent infection

金黄色葡萄球菌利用自噬促进潜伏感染

基本信息

批准号：
8511238
负责人：
Alice S Prince
金额：
$ 22.88万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-03-01 至 2015-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8511238
关键词：
Address Antibiotics Atopic Dermatitis Automobile Driving Autophagocytosis Autophagosome Bioinformatics Biopsy Cells Characteristics Chronic Clinical Data Development Diabetic wound Drug Combinations Drug Targeting Electron Transport Endosomes Fostering Frequencies Generations Genes Genetic Techniques Genotype Goals Hemolysin Human Immune Immune response In Vitro Individual Infection Infectious Skin Diseases Laboratories Mediating Modeling Molecular Mutation Organism Osteomyelitis Oxidative Stress Oxygen Pathway interactions Phagocytes Pharmaceutical Preparations Phase Polyamines Predisposition Production Reagent Recurrence Resolution Role SCID-hu Mice Signal Pathway Signal Transduction Site Skin Skin Tissue Skin graft Small Interfering RNA Soft Tissue Infections Spermidine Staphylococcus aureus System Testing Therapeutic Therapeutic Agents Time United States Virulence Vitamin K 2 Vitamin K 3 auxotrophy extracellular genetic manipulation genome sequencing in vivo inhibitor/antagonist keratinocyte killings latent infection methicillin resistant Staphylococcus aureus mutant pathogen pressure prevent public health relevance response sensor skin xenograft uptake

项目摘要

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.

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。尽管多种抗生素在体外对耐甲氧西林金黄色葡萄球菌 (MRSA) 菌株具有活性，但 SA 感染通常是持续存在的，并在长时间潜伏后重新激活。由于针对生物体的治疗策略未能成功根除这些感染，因此该项目的目标是针对宿主——识别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了这样一个受保护的生态位，用于选择SA突变体，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。在该项目的R21阶段，我们将全面表征与SA持久性相关的细胞内区室，并确定SA激活诱导自噬体形成的信号通路。由于自噬通常起到根除细胞内病原体的作用，因此我们预测特定的 SA 突变体会被选择来响应这种环境，从而避免溶酶体融合和根除，并能够耐受该区室内的氧化应激。我们将使用生物信息学方法，将选择用于适应自噬体的实验室菌株的全基因组测序数据与来自慢性感染的临床分离株的基因型进行比较。 R21 组件的目标是确定特定的 SA 突变体被主动选择以响应自噬体，并在角质形成细胞内建立潜伏感染的病灶。在R33组件中，我们将开发人类皮肤感染的模型；首先使用人类原代角质形成细胞的器官型培养物，确定药理学和遗传技术可削弱角质形成细胞自噬的诱导，从而预防慢性 SA 感染的发生。使用靶向 AMPK-TORC1 通路的药物或抑制特定自噬基因的 siRNA，我们期望阻断自噬并阻止选择可以在此环境中生存的 SA 突变体。然后，带有人类皮肤异种移植物的 SCID:hu 小鼠将被用作在存在完整免疫反应的情况下人类皮肤感染的原理证明。体外鉴定出的最有效的试剂将在人类皮肤移植物上进行测试，以确定它们阻止 SA 适应人类角质形成细胞的能力。