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（由申请人提供）：病原菌进入组织后，被单核吞噬细胞和组成型表达孔形成蛋白Perforin-2的粒细胞摄入并杀死。我们表明，Perforin-2的遗传缺陷或siRNA敲低无法杀死细菌。通过专业吞噬细胞吞噬病原体，导致细胞内复制，致病菌也侵入上皮细胞和其他组织形成细胞并被内吞。表明角质形成细胞组成型表达 Perforin-2，并且分析的所有其他上皮细胞和组织形成细胞均通过干扰素或细菌入侵诱导表达 Perforin-2，如在吞噬细胞中一样，组织形成细胞中的 siRNA 敲低或 Perforin-2 的遗传缺陷使得这些细胞能够表达 Perforin-2。 Perforin-2 存在时可杀死病原体的细胞内复制进一步分析表明，活性氧和氮以及溶酶体水解酶增强了杀菌作用。 Perforin-2 的活性，但无法在没有 Perforin-2 的情况下清除细胞内病原体小鼠中 Perforin-2 的遗传缺陷会导致对低剂量的沙门氏菌、葡萄球菌和其他病原体的感染具有致命的易感性，而这些病原体在 Perforin-2 充足的动物中已被清除。 Perforin-2 是一种含有 MACPF 结构域的完整膜蛋白；其激活和杀伤机制非常复杂，我们表明 Perforin-2 位于分布的膜囊泡中。细菌感染导致 Perforin-2 在包围细菌的吞噬体或内体膜中快速积累，从感染细胞中重新分离细菌，电子显微镜分析显示细菌细胞壁上有 100° 宽的孔簇。致命的打击是通过穿孔素-2 的聚合和孔形成介导的，致病细菌具有逃避或破坏的机制。 Perforin-2 以便在细胞内存活在本应用中，我们将研究 Perforin-2 激活和杀伤的分子机制，这对于开发增强 Perforin-2 介导的杀伤从而克服抗生素耐药性的治疗方法是必要的。