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

描述（由申请人提供）：近年来，很明显，许多生物利用RNA和DNA的碱基配对潜力来实现针对病毒和其他可移动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，这种机制迄今为止只在真核生物中有记载。最近，在许多真细菌和几乎所有的古细菌的基因组中存在的聚集规律间隔的短回文重复（CRISPR）位点已被证明具有基于序列的噬菌体免疫。CRISPR基因座伴随着一组cas （CRISPR相关）基因，这些基因可能编码潜在酶机制的蛋白质成分。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。基因组数据库检索显示，在一株表皮葡萄球菌中存在一个相对简单的CRISPR/cas位点，该位点的序列表明，它不仅对噬菌体有抗性，而且对葡萄球菌结合质粒也有抗性。鉴于葡萄球菌的临床重要性和表皮葡萄球菌的实验易感性，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，携带CRISPR基因座的表皮葡萄球菌作为质粒结合受体存在缺陷，而缺乏CRISPR基因座的等基因菌株则没有缺陷。这些和其他观察结果证实了CRISPR基因座在真细菌中限制基因水平转移的作用，并为我们提供了一种简单方便的检测CRISPR功能的方法。我们将利用该系统进行CRISPR和cas基因功能的遗传分析。特别是，我们将定义干扰所需的CRISPR位点和靶质粒的序列特征，我们将测试特定cas基因在这一过程中的参与。此外，我们将对之前报道的CRISPR转录本进行初步的生化分析。这些实验的结果将对CRISPR/cas功能的可行模型提出关键的限制，并将为深入的机制分析奠定基础。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，携带抗微生物药物耐药性基因的质粒的转移导致这些病原体的传播日益恶化。了解CRISPR的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的古细菌中，聚集规律间隔的短回文重复序列（CRISPR）位点赋予获得性、基于序列的对病毒和共轭质粒的抗性，但其潜在机制尚不清楚。抗生素耐药基因在结合质粒上的转移有助于致病菌株的传播，对人类健康造成重大威胁。拟议的研究将阐明CRISPR功能的机制，因此将有助于我们利用这种自然途径来预防和治疗传染病的能力。

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.