Using CRISPR immunity to prevent the spread of virulence traits among pathogens

利用 CRISPR 免疫来防止病原体毒力特征的传播

基本信息

批准号：
8356936
负责人：
Luciano A Marraffini
金额：
$ 250.52万
依托单位：
ROCKEFELLER UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-30 至 2017-06-30
项目状态：
已结题

项目摘要

DESCRIPTION (Provided by the applicant) Abstract: In modern times there has been a relentless battle against bacterial pathogens that constantly evolve to evade our efforts to eradicate them. Pathogens not only have become more virulent but also have acquired resistance to virtually all known antibiotics. The rise of antimicrobial-resistant strains in the hospital setting has become a major health care crisis. To limit the spread of such antibiotic-resistant pathogens, a greater understanding of their means of emergence and survival is required. The major route for the acquisition of virulence factors and antimicrobial resistance is the exchange of genetic material between related or unrelated bacterial species, known as horizontal gene transfer (HGT). The study of HGT and its barriers will help us understand and limit the emergence of new pathogenic strains. Recently, clustered regularly interspaced short palindromic repeat (CRISPR) loci have been revealed as a programmable barrier to HGT. CRISPR loci, and their associated cas (CRISPR associated) genes, encode a sequence-specific defense mechanism against bacteriophages and plasmids, the main vehicles of HGT, known as CRISPR interference. CRISPR/Cas systems are extremely diverse. There are 45 different cas gene families associated with CRISPR loci that can be classified into ten different CRISPR/Cas subtypes. The biological significance of this complexity remains unknown as only a few CRISPR/Cas subtypes have been characterized at the molecular level. In addition, it is not known whether CRISPR interference can prevent HGT during bacterial infection. To address both issues, we plan to study CRISPR interference in the human pathogen Streptococcus pneumoniae. The pneumococcus is famous for its ability to engage in gene transfer, both in vitro and during infection. In addition, the accumulation of almost a century of pneumococcal research has led to the development of simple techniques for the genetic manipulation of this organism. Important for this proposal, S. pneumoniae lacks CRISPR loci, therefore we plan to ectopically express the diverse CRISPR/Cas subtypes in this bacterium and compare them in their ability to prevent different routes of HGT. This will allow us to identify the CRISPR/Cas subtypes that are most adequate to prevent HGT and test them in vivo, in a mouse model for the transfer of virulence or antibiotic resistance genes during pneumococcal infection. This study will substantially advance our understanding of the molecular mechanisms underlying CRISPR interference, of the impact of these loci on the evolution of bacterial pathogens, and of the potential of this pathway as a tool to limit the rais of more virulent strains. Public Health Relevance: Clustered, regularly interspaced, short, palindromic repeat (CRISPR) loci specify an adaptive, heritable, RNAdirected interference pathway that confers immunity against viruses and conjugative plasmids in many bacteria. However, the mechanism of CRISPR interference is poorly understood. The transfer of virulence and antibiotic resistance genes is mediated by viruses and conjugative plasmids and contributes to the raise of increasingly virulent bacterial pathogens, leading to significant threats to human health. The proposed studies will clarify the molecular basis for CRISPR function and will therefore contribute to our ability to exploit this natural pathway to prevent or treat infectious disease.

描述（由申请人提供）摘要：在现代，人们与细菌病原体进行了无情的斗争，这些细菌病原体不断进化，以逃避我们根除它们的努力。病原体不仅变得更加致命，而且对几乎所有已知的抗生素都产生了耐药性。医院环境中抗菌药物耐药菌株的增加已成为重大的医疗保健危机。为了限制此类抗生素抗性病原体的传播，需要更好地了解它们的出现和生存方式。获得毒力因子和抗菌素耐药性的主要途径是相关或不相关细菌物种之间遗传物质的交换，称为水平基因转移（HGT）。 HGT及其障碍的研究将有助于我们了解和限制新致病菌株的出现。最近，聚集的规则间隔短回文重复 (CRISPR) 位点已被揭示为 HGT 的可编程障碍。 CRISPR 位点及其相关 cas（CRISPR 相关）基因编码针对噬菌体和质粒（HGT 的主要载体）的序列特异性防御机制，称为 CRISPR 干扰。 CRISPR/Cas 系统极其多样化。有 45 个不同的 cas 基因家族与 CRISPR 位点相关，可分为 10 种不同的 CRISPR/Cas 亚型。这种复杂性的生物学意义仍然未知，因为只有少数 CRISPR/Cas 亚型在分子水平上得到了表征。此外，尚不清楚CRISPR干扰是否可以阻止细菌感染期间的HGT。为了解决这两个问题，我们计划研究 CRISPR 对人类病原体肺炎链球菌的干扰。肺炎球菌以其在体外和感染过程中进行基因转移的能力而闻名。此外，近一个世纪的肺炎球菌研究积累导致了对该生物体基因操作的简单技术的发展。对于这一提议来说重要的是，肺炎链球菌缺乏 CRISPR 位点，因此我们计划在该细菌中异位表达不同的 CRISPR/Cas 亚型，并比较它们预防不同途径 HGT 的能力。这将使我们能够识别最适合预防 HGT 的 CRISPR/Cas 亚型，并在小鼠模型中进行体内测试，以在肺炎球菌感染期间转移毒力或抗生素抗性基因。这项研究将极大地增进我们对 CRISPR 干扰的分子机制、这些位点对细菌病原体进化的影响以及该途径作为限制更具毒性菌株的工具的潜力的理解。公共卫生相关性：成簇、规则间隔、短、回文重复 (CRISPR) 位点指定了一种适应性、可遗传、RNA 定向的干扰途径，可赋予许多细菌针对病毒和接合质粒的免疫力。然而，人们对 CRISPR 干扰的机制知之甚少。毒力和抗生素抗性基因的转移由病毒和接合质粒介导，导致毒力日益增强的细菌病原体的产生，对人类健康造成重大威胁。拟议的研究将阐明 CRISPR 功能的分子基础，从而有助于我们利用这种自然途径来预防或治疗传染病的能力。