CRISPR Cas genes and Legionella pneumophila infection

描述(由申请人提供)：可以说，近年来微生物遗传学的主要新发现之一是CRISPRCas系统的发现。CRISPR(簇状规则间隔短回文重复序列)基因座是在古生菌和细菌中发现的新序列，由独特的间隔区序列分隔的回文重复组成。CRISPR阵列的上游是CRISPR相关序列(CA)基因。CRISPRCas基因座主要与对噬菌体和质粒的免疫有关。总体而言，CRISPRCas基因座只在数量相对较少的模式生物中进行了研究。然而，基因组测序表明，CRISPRCas基因座在不同的细菌中被发现，包括在各种病原体和细胞内寄生虫中，并且CRISPRCas基因在致病性和细胞内感染中的潜在作用尚未被直接研究。嗜肺军团菌(LPN)是一种被称为军团病的严重肺炎的细菌剂。人类通过吸入受污染的水滴接触到这种细菌。在其水生生境中，LPN是一种阿米巴的胞内寄生虫，这些原虫的感染无疑是疾病传播和发病过程中的重要一步。在肺部，LPN在巨噬细胞中复制，复制过程类似于阿米巴感染。强毒LPN株含有一种以前未被研究过的CRISPRCas基因座。鉴于这一点以及LPN的致病和细胞内性质，我们开始研究CRISPRCas在LPN生物学和毒力中的作用。本文提供的数据表明，LPN菌株130b的CRISPRCas基因在胞外和胞内生长条件下都有表达。此外，失去Cas2会导致戏剧性的(约卡氏棘阿米巴LPN感染的1000倍缺陷。这些观察结果导致了一种独特的假设，即LPN的CaS蛋白(S)在感染过程中发挥了迄今未被认识的作用，而不是在对噬菌体和质粒的免疫中发挥作用。因此，AIM 1将在感染检测中利用完整的CRISPRCas突变体和互补突变体来确定整个CRISPRCas基因座在细胞内感染中的重要性。鉴于Cas2在LPN感染中的重要性，Aim 2将使用生化方法通过确定该蛋白是否具有核糖核酸酶活性以及该活性是否对感染重要来阐明Cas2的功能。最后，目标3将确定Cas2起作用的感染阶段(S)；例如，Cas2突变体的感染缺陷可能表明对细胞内杀伤的抵抗力减弱和/或细胞内复制受损。因此，通过利用LPN作为一种新的模式生物，我们将探索CRISPR/CAS基因座的新功能，试图改变目前的研究范式。

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.