Killing of intracellular bacteria by Perforin-2

Perforin-2 杀死细胞内细菌

基本信息

批准号：
9193612
负责人：
GEORGE Patrick MUNSON
金额：
$ 38.38万
依托单位：
UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-12-01 至 2019-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9193612
关键词：
Animals Antibiotic Resistance Bacteria Bacterial Antibiotic Resistance Bacterial Infections Biochemical Cell Cycle Cell Wall Cell membrane Cells Complex Cullin Proteins Cytoplasmic Tail Cytoprotection Cytosol Data Dissection Dose Electrons Embryo Endosomes Enzymes Epithelial Epithelial Cells Event Fibroblasts Functional disorder Genes Genetic Genus Mycobacterium Genus staphylococcus Goals Health Homeostasis Hydrolase Immune In Vitro Infection Ingestion Integral Membrane Protein Interferons Invaded Knock-out Lymphocyte Measures Mediating Membrane Microscopic Molecular Mononuclear Muramidase Mus Mutagenesis Mycobacterium tuberculosis Nitrogen Oxygen Partner in relationship Pasteurella pseudotuberculosis Pathogenicity Phagocytes Phagocytosis Phagosomes Pharmaceutical Preparations Phosphorylation Predisposition Proteasome Inhibitor Proteins Risk Role Salmonella Site-Directed Mutagenesis Small Interfering RNA Testing Tissues Tuberculosis VDAC1 gene Vesicle Wild Type Mouse antimicrobial bactericide cell killing germ free condition granulocyte in vivo inhibitor/antagonist keratinocyte killings knock-down macrophage multicatalytic endopeptidase complex pathogen pathogenic bacteria perforin 2 polymerization prevent public health relevance therapy development ubiquitin-protein ligase

项目摘要

DESCRIPTION (provided by applicant): After entering tissues, pathogenic bacteria are ingested and killed by mononuclear phagocytes and by granulocytes that constitutively express the pore-forming protein Perforin-2. We show that genetic deficiency or siRNA knock down of Perforin-2 disables killing of the pathogen by professional phagocytes resulting in intracellular replication. Pathogenic bacteria also invade and are endocytosed by epithelial cells and other tissue forming cells. We show that keratinocytes express Perforin-2 constitutively and that all other epithelial and tissue forming cells analyzed are induced to express Perforin-2 by interferons or bacterial invasion. As in phagocytes, siRNA knock down or genetic deficiency of Perforin-2 in tissue forming cells enables intracellular replication of the pathogen which is kille in the presence of Perforin-2. Further analysis reveals that reactive oxygen and nitrogen species and lysosomal hydrolases enhance the bactericidal activity of Perforin-2 but are unable to clear intracellular pathogens without Perforin-2. Genetic deficiency of Perforin-2 in mice causes lethal susceptibility to infection with low doses of Salmonella, Staphylococcus and other pathogens that are cleared in Perforin-2 sufficient animals. Perforin-2 is a MACPF domain containing, integral membrane protein; its activation and killing mechanisms is highly complex. We show that Perforin-2 is localized in membrane vesicles that are distributed throughout the cytosol in uninfected cells. Bacterial infection causes rapid accumulation of Perforin-2 in the phagosomal or endosomal membrane enclosing the bacterium. Re-isolation of bacteria from infected cells and electron microscopic analysis reveals clusters of 100 � wide pores on bacterial cell walls suggesting that the lethal hit is mediated by polymerization and pore-formation by Perforin-2. Pathogenic bacteria have mechanisms to evade or subvert Perforin-2 in order to survive inside cells. In this application we will study the molecular mechanisms of Perforin-2 activation and killing, which is requisite for development of treatments to enhance Perforin-2 mediated killing that may overcome antibiotic resistance.

描述（由应用提供）：进入组织后，致病细菌被单核吞噬细胞摄入和杀死，并被组成型表达孔形成蛋白质蛋白质2的粒细胞摄入和杀死。我们表明，遗传缺陷或siRNA击倒Perforin-2通过专业吞噬细胞杀死病原体，导致细胞内复制。致病细菌也侵入并被上皮细胞和其他组织形成细胞内吞。我们表明角质形成细胞组成性地表达perforin-2，并且所有其他分析的上皮和组织形成细胞都被诱导通过干扰素或细菌浸润表达perforin-2。与吞噬细胞一样，siRNA在组织形成细胞的组织中敲低或遗传缺乏症，可以在perforin-2存在下杀死病原体的细胞内复制。进一步的分析表明，活性氧和氮种和溶酶体水解酶增强了穿孔蛋白-2的细菌化活性，但没有perforin-2，无法清除细胞内病原体。小鼠穿孔蛋白-2的遗传缺乏会导致致命的敏感性，以低剂量的沙门氏菌，葡萄球菌和其他在Puretin-2足够动物中清除的病原体感染。 Perforin-2是一个含有整体膜蛋白的MACPF结构域。它的激活和杀戮机制非常复杂。我们表明，穿孔蛋白2位于未感染细胞中整个细胞质中分布的膜蔬菜中。细菌感染会导致源自细菌的吞噬或内体膜中穿孔蛋白-2的快速加速。从感染细胞和电子显微镜分析中对细菌的重新分离表明细菌细胞壁上有100张孔的簇表明致命的击中是通过perforin-2的聚合和孔形成介导的。致病细菌具有逃避或颠覆穿孔2的机制，以便在细胞内生存。在此应用中，我们将研究穿孔-2激活和杀伤的分子机制，这是开发治疗所必需的，以增强可能克服抗生素耐药性的穿孔蛋白-2介导的杀戮。