CRISPR Cas genes and Legionella pneumophila infection

CRISPR Cas基因与嗜肺军团菌感染

• 批准号: 8733513
• 负责人: NICHOLAS P CIANCIOTTO
• 金额: $ 19.31万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-12 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8733513
• 关键词: Acanthamoeba castellanii; Aerosols; Alveolar Macrophages; Amoeba genus; Animal Model; Archaea; Area; Bacteria; Bacteriophages; Biochemical; Biological Assay; Biology; Breathing; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Complement; Data; Defect; Development; Devices; Disease; Eubacterium; Fostering; Genes; Genomics; Growth; Habitats; Human; Immunity; Infection; Lead; Legionella pneumophila; Legionnaires' Disease; Link; Lung; Microbial Genetics; Mutation; Nature; Organism; Parasites; Pathogenesis; Pathogenicity; Plasmids; Play; Pneumonia; Process; Proteins; Protozoa; RNA; Research; Research Project Grants; Resistance; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleases; Risk; Role; Sequence Analysis; Staging; System; United States National Institutes of Health; Virulence; Virulent; Water; Work; design; extracellular; human BCAR1 protein; innovation; intracellular parasitism; killings; macrophage; mutant; novel; pathogen; pathogenic bacteria; programs; public health relevance; transmission process

DESCRIPTION (provided by applicant): Arguably one of the major new findings in microbial genetics in recent years is the discovery of the CRISPRCas system. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci are novel sequences found in Archaea and Bacteria that are comprised of palindromic repeats separated by unique spacer sequences. Upstream of the CRISPR array are the CRISPR-associated sequences (cas) genes. CRISPRCas loci have been primarily implicated in immunity to phage and plasmid. Overall, CRISPRCas loci have been studied in only a relatively small number of model organisms. However, genomic sequencing indicates that CRISPRCas loci are found in >400 different bacteria, including in a variety of pathogenic organisms and intracellular parasites, and the potential role of CRISPRCas in pathogenicity and intracellular infection has not been directly examined. Legionella pneumophila (Lpn) is the bacterial agent of a severe form of pneumonia called Legionnaires' disease. Humans come in contact with the bacterium by inhaling contaminated water droplets. In its aquatic habitats, Lpn is an intracellular parasite of amoebae, and infection of these protozoa is unquestionably an important step in the transmission and pathogenesis of disease. In the lung, Lpn replicates in macrophages in a process that mimics infection of amoebae. Virulent Lpn strains contain a type of CRISPRCas locus that has not been previously investigated. Given this as well as the pathogenic and intracellular nature of Lpn, we embarked on an examination of the role of CRISPRCas in Lpn biology and virulence. Data presented here show that the CRISPRCas locus of Lpn strain 130b is expressed under extracellular and intracellular growth conditions. Furthermore, loss of cas2 results in a dramatic (approx. 1000-fold) defect in Lpn infection of the amoeba Acanthamoeba castellanii. These observations lead to the unique hypothesis that the Cas protein(s) of Lpn play a heretofore unrecognized role in infection that is outside of a role in immunity to phage and plasmid. Thus, Aim 1 will utilize a full panel of CRISPRCas mutants and complemented mutants in infection assays to determine the importance of the entire CRISPRCas locus in intracellular infection. Given the now established importance of Cas2 in Lpn infection, Aim 2 will use a biochemical approach to elucidate the function of Cas2 by determining if the protein has RNase activity and if this activity is important for infection. Lastly, Aim 3 will identify the stage(s) of infection n which Cas2 plays a role; e.g., the cas2 mutant's infection defect could be indicative of diminished resistance to intracellular killing and/or impaired intracellular replication. Thus, by using Lpn a a new model organism, we will probe for novel functions of the CRISPR/Cas locus that seek to shift current research paradigms.

描述（由申请人提供）：近年来微生物遗传学的主要新发现之一是发现CRISPRCAS系统。 CRISPR（群集的定期间隔短的腔膜重复序列）是古细菌和细菌中发现的新序列，这些序列由由独特的间隔序列隔开的palindromic重复序列组成。 CRISPR阵列的上游是CRISPR相关序列（CAS）基因。 Crisprcas基因座主要与噬菌体和质粒的免疫有关。总体而言，仅在相对较少的模型生物中研究了CRISPRCAS基因座。然而，基因组测序表明在> 400种不同的细菌中发现了CRISPRCAS基因座，包括在多种致病生物和细胞内寄生虫中，并且尚未直接检查CRISPRCA在致病性和细胞内感染中的潜在作用。肺炎军团（LPN）是一种严重的肺炎形式的细菌剂，称为军团疾病。人类通过吸入污染的水滴与细菌接触。在其水生栖息地中，LPN是变形虫的细胞内寄生虫，这些原生动物的感染无疑是疾病传播和发病机理的重要一步。在肺中，LPN在模仿变形虫感染的过程中在巨噬细胞中复制。强大的LPN菌株包含一种先前未曾研究的CRISPRCA基因座。鉴于LPN的致病性和细胞内性质，我们开始研究CRISPRCA在LPN生物学和毒力中的作用。此处提供的数据表明，LPN菌株130b的CRISPRCA基因座在细胞外和细胞内生长条件下表达。此外，CAS2的损失导致变形虫Acanthamoeba Castellanii的LPN感染导致剧烈（约1000倍）。这些观察结果导致了一个独特的假设，即LPN的CAS蛋白在感染中起到了迄今未识别的作用，而在免疫中不受噬菌体和质粒的影响。因此，AIM 1将利用整个CRISPRCAS突变体和感染分析中的补体突变体，以确定整个CRISPRCAS基因座在细胞内感染中的重要性。鉴于CAS2在LPN感染中现在确定的重要性，AIM 2通过确定蛋白质是否具有RNase活性，以及​​该活性是否对感染很重要，AIM 2将使用生化方法来阐明CAS2的功能。最后，AIM 3将确定CAS2起作用的感染阶段；例如，CAS2突变体的感染缺陷可以表明对细胞内杀害和/或细胞内复制受损的抗性降低。因此，通过使用LPN A新模型生物，我们将探究试图转移当前研究范式的CRISPR/CAS基因座的新功能。