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

描述(由申请人提供) 摘要：在现代，与细菌病原体进行了一场无情的斗争，这些病原体不断进化，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抗药性。抗菌素耐药菌株在医院环境中的上升已成为一大医疗危机。为了限制这种抗药性病原体的传播，需要更多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无关细菌物种之间的遗传物质交换，称为水平基因转移(HGT)。对HGT及其屏障的研究将有助于我们了解和限制新的致病株的出现。最近，聚集的规则间隔短回文重复(CRISPR)基因座被发现是HGT的可编程障碍。CRISPR基因座及其相关的CAS(CRISPR相关)基因编码一种针对噬菌体和质粒的序列特异性防御机制，这是HGT的主要载体，称为CRISPR干扰。CRISPR/CAS系统极为多样化。与CRISPR基因座相关的Cas基因家族有45个，可分为10个不同的CRISPR/Cas亚型。这种复杂性的生物学意义尚不清楚，因为只有少数CRISPR/CA亚型在分子水平上得到了表征。此外，目前尚不清楚CRISPR干扰能否在细菌感染过程中预防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期间进行基因转移的能力而闻名。此外，近一个世纪以来肺炎球菌研究的积累导致了对这种有机体进行基因操作的简单技术的发展。重要的是，肺炎链球菌缺乏CRISPR基因座，因此我们计划在这种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足以预防HGT的CRISPR/CAS亚型，并在体内测试它们，在肺炎球菌感染期间转移毒力或抗生素耐药基因的小鼠模型中。这项研究将极大地促进我们对CRISPR干扰的分子机制、这些基因座对细菌病原体进化的影响以及该途径作为限制更强毒力菌株RAI的潜力的理解。 公共卫生相关性：簇状、规则间隔、短回文重复(CRISPR)基因座指定了一种适应性、可遗传的、RNA定向的干扰途径，使许多细菌对病毒和接合质粒具有免疫力。然而，CRISPR干扰的机制尚不清楚。毒力和抗生素耐药基因的转移是由病毒和接合质粒介导的，并导致越来越毒力的细菌病原体的增加，导致对人类健康的严重威胁。拟议的研究将阐明CRISPR功能的分子基础，因此将有助于我们利用这一自然途径来预防或治疗传染病。

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.