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（重复的规律间隔的短回文重复序列）基因座是在细菌和细菌中发现的新序列，其由通过独特的间隔序列分开的回文重复序列组成。CRISPR阵列的上游是CRISPR相关序列（cas）基因。CRISPRCas基因座主要涉及对噬菌体和质粒的免疫。总体而言，CRISPRCas基因座仅在相对少量的模式生物中进行了研究。然而，基因组测序表明，CRISPRCas基因座存在于>400种不同的细菌中，包括多种病原生物和细胞内寄生虫，并且CRISPRCas在致病性和细胞内感染中的潜在作用尚未直接研究。嗜肺军团菌（Lpn）是一种称为军团菌病的严重肺炎的细菌病原体。人类通过吸入受污染的水滴与细菌接触。在其水生栖息地，Lpn是阿米巴原虫的细胞内寄生虫，这些原生动物的感染无疑是疾病传播和发病的重要步骤。在肺中，Lpn在巨噬细胞中复制，其过程类似于阿米巴感染。毒力Lpn菌株含有一种先前未研究过的CRISPRCas基因座。鉴于这一点以及Lpn的致病性和细胞内性质，我们开始研究CRISPRCas在Lpn生物学和毒力中的作用。本文提供的数据显示，Lpn菌株130 b的CRISPRCas基因座在细胞外和细胞内生长条件下表达。此外，cas 2的丢失导致了戏剧性的（约。1000倍）的Lpn感染的卡氏阿米巴原虫的缺陷。这些观察结果导致了一个独特的假设，即Lpn的Cas蛋白在感染中起着迄今为止未被认识到的作用，其在对噬菌体和质粒的免疫中的作用之外。因此，Aim 1将在感染测定中利用一组完整的CRISPRCas突变体和互补突变体，以确定整个CRISPRCas基因座在细胞内感染中的重要性。鉴于目前已确定Cas 2在Lpn感染中的重要性，Aim 2将使用生物化学方法通过确定蛋白质是否具有RNA酶活性以及该活性是否对感染重要来阐明Cas 2的功能。最后，目标3将确定Cas 2发挥作用的感染阶段;例如，Cas 2突变体的感染缺陷可以指示对细胞内杀伤的抗性降低和/或细胞内复制受损。因此，通过使用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，以寻求改变当前的研究范式。

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.