Mechanisms of Sequence-Based Resistance to Viruses and Plasmids in Eubacteria

描述（由申请人提供）：近年来，已经清楚的是，许多生物体利用RNA和DNA的碱基配对潜力来实现针对病毒和其他移动的遗传元件的基于序列的抗性机制。这些机制中最著名的是RNA干扰，它使用双链RNA来触发特定基因的沉默。然而，迄今为止，这种机制仅在真核生物中有记载。最近，存在于许多真细菌和几乎所有古细菌基因组中的成簇的规则间隔短回文重复序列（CRISPR）基因座已被证明赋予针对噬菌体的基于序列的免疫力。CRISPR基因座伴随着一组cas（CRISPR相关）基因，其可能编码潜在酶机制的蛋白质组分。然而，CRISPR和cas定向干扰的生化机制尚不清楚。我们建议剖析CRISPR和cas基因功能的分子基础。基因组数据库搜索揭示了表皮葡萄球菌菌株中存在相对简单的CRISPR/cas基因座，该基因座的序列表明它不仅指定对噬菌体的抗性，而且还指定对葡萄球菌接合质粒的抗性。考虑到葡萄球菌的临床重要性和S.表皮细胞，我们将使用它作为模型系统来探索真细菌中CRISPR衍生免疫的基本方面。初步结果证实，一个S。携带CRISPR基因座的表皮葡萄球菌菌株作为质粒缀合受体是有缺陷的，而缺乏CRISPR基因座的等基因菌株则不是。这些和其他观察结果证实了CRISPR基因座在限制真细菌中水平基因转移中的作用，并为我们提供了一种简单方便的CRISPR功能测定方法。我们将使用该系统对CRISPR和cas基因功能进行遗传分析。特别是，我们将定义干扰所需的CRISPR基因座和靶质粒的序列特征，我们将测试特定cas基因在此过程中的参与。此外，我们将对先前报道的CRISPR转录本进行初步的生化分析。这些实验的结果将对CRISPR/cas功能的可行模型施加关键约束，并为深入的机制分析奠定基础。S.表皮葡萄球菌和金黄色葡萄球菌是医院感染的最常见原因，并且携带抗微生物剂抗性基因的质粒的转移有助于这些病原体的不断恶化的传播。了解CRISPR功能是开发利用这一途径阻止抗生素耐药性传播的治疗干预措施的重要一步。此外，鉴于噬菌体在致病菌进化中的重要作用，对CRISPR功能的研究将提高我们对传染病如何出现、消失和重新出现的理解。公共卫生相关性：在许多真细菌和几乎所有古细菌中，规则间隔的短回文重复序列（CRISPR）基因座赋予了对病毒和接合质粒的获得性、基于序列的抗性，但潜在的机制尚不清楚。接合性质粒上的抗生素抗性基因的转移有助于病原菌株的传播，导致对人类健康的重大威胁。这些研究将阐明CRISPR功能的机制，因此将有助于我们利用这种天然途径预防和治疗传染病。

DESCRIPTION (provided by applicant): In recent years, it has become clear that many organisms exploit the base-pairing potential of RNA and DNA to enable sequence-based resistance mechanisms against viruses and other mobile genetic elements. The best known of these mechanisms, RNA interference, uses double-stranded RNA to trigger the silencing of specific genes. However, this mechanism has thus far only been documented in eukaryotes. More recently, clustered regularly interspaced short palindromic repeat (CRISPR) loci, present in the genomes of many eubacteria and nearly all archaebacteria, have been shown to confer sequence-based immunity against bacteriophages. CRISPR loci are accompanied by a set of cas (CRISPR-associated) genes that are likely to encode protein components of the underlying enzymatic machinery. However, the biochemical mechanism of CRISPR- and cas-directed interference is unknown. We propose to dissect the molecular basis for CRISPR and cas gene function. Genome database searches have revealed the presence of a relatively simple CRISPR/cas locus in a strain of Staphylococcus epidermidis, and the sequence of the locus suggests that it specifies resistance not only to bacteriophages but also to staphylococcal conjugative plasmids. Given the clinical importance of staphylococci and the experimental tractability of S. epidermidis, we will use it as a model system to explore fundamental aspects of CRISPR-derived immunity in eubacteria. Preliminary results confirm that an S. epidermidis strain carrying the CRISPR locus is defective as a plasmid conjugation recipient, whereas an isogenic strain lacking the CRISPR locus is not. These and other observations confirm a role for CRISPR loci in restricting horizontal gene transfer in eubacteria, and provide us with a simple and convenient assay for CRISPR function. We will use this system to conduct a genetic analysis of CRISPR and cas gene function. In particular, we will define the sequence characteristics of both the CRISPR locus and the target plasmid that are needed for interference, and we will test the involvement of specific cas genes in this process. In addition, we will conduct preliminary biochemical analyses of the previously reported CRISPR transcripts. The results of these experiments will place critical constraints on viable models of CRISPR/cas function, and will set the stage for in-depth mechanistic analyses. S. epidermidis and Staphylococcus aureus are the most common causes of nosocomial infections, and the transfer of plasmids that carry antimicrobial resistance genes contributes to the ever-worsening spread of these pathogens. Understanding CRISPR function is an important step in the development of therapeutic interventions that exploit this pathway to impede the spread of antibiotic resistance. In addition, given the important role of bacteriophages in the evolution of pathogenic bacteria, the study of CRISPR function will improve our understanding of how infectious diseases emerge, disappear and re-emerge. PUBLIC HEALTH RELEVANCE: Clustered regularly interspaced short palindromic repeat (CRISPR) loci confer acquired, sequence-based resistance against viruses and conjugative plasmids in many eubacteria and nearly all archaebacteria, but the underlying mechanisms are unknown. The transfer of antibiotic resistance genes on conjugative plasmids contributes to the spread of pathogenic bacterial strains, leading to significant threats to human health. The proposed studies will clarify the mechanisms of CRISPR function, and will therefore contribute to our ability to exploit this natural pathway to prevent and treat infectious disease.