The Evolutionary Dynamics of Plasmid Uptake in Pathogenic Bacteria

描述(申请人提供)：尽管经过数十年的研究和数十种抗生素，但由于抗生素耐药性的快速演变，细菌病原体再次成为西方国家的主要死亡原因之一。获得质粒是抗生素耐药性在细菌中传播的主要原因。然而，在我们对如何有力地阻止质粒获取的理解上存在着根本性的差距，特别是在抗生素提供的选择性压力下。这项提议的长期目标是开发信息，允许设计新的疗法和方法来限制质粒摄取，大大延长最后一线抗生素药物的寿命。重要的是，屏蔽的自然蓝图 在已测序的细菌基因组中，大约50%的基因组中似乎存在质粒获取。CRISPR被称为簇状规则间隔短回文重复序列(CRISPR)，是一种基因电路，它切割和破坏以前遇到的特定质粒和病毒序列，从而抑制病毒和质粒在CRISPR+细胞中建立其遗传物质。CRISPR可能是以降低其细菌宿主的基因组可塑性为代价的，这些宿主不能再使用获得质粒来快速进化。因此，这项提案的具体目标是确定允许细菌维持CRISPR的机制和选择压力，尽管它的成本很高。这一目标意义重大，因为CRISPR有能力阻止抗生素耐药性传播的主要途径，即获得质粒。我们假设CRISPR保存在细菌群体中是因为它提供了持续的抗病毒免疫。为了验证这一假设，提出了一项跨学科的计算和实验研究。首先，将设计一个新的随机模型，在不同的病毒压力和定期抗生素治疗下竞争CRISPR+和CRISPR-细菌。该模型将使我们能够量化潜在的参数机制，在这些参数机制中，抗病毒免疫在很长一段时间内维持CRISPR+细胞，尽管无法获得质粒的成本。此外，该模型将确定是否存在最佳治疗方案，在这些方案中，减少抗生素剂量可以通过保留CRISPR+共生储存库来改善患者的预后。CRISPR提供对抗病毒的健康优势和对抗抗生素的健康劣势的基本假设随后将在肠球菌中进行实验测试，肠球菌是耐药医院感染的主要原因。通过实验检测CRISPR在肠球菌基因组中的抗质粒和抗病毒效果，明确检测CRISPR的抗病毒免疫选择能否抑制致命性多重耐药病原体的出现。

DESCRIPTION (provided by applicant): Despite decades of research and dozens of antibiotics, bacterial pathogens are again among the leading causes of death in the West due to the rapid evolution of antibiotic resistance. Plasmid acquisition is the leading cause of the spread of antibiotic resistance in bacteria. Yet, there is a fundamental gap in our understanding of how to robustly block plasmid acquisition, especially with the selective pressure that antibiotics provide. The long-term goal of this proposal is to develop the information that will allow new therapies and approaches to be designed that limit plasmid uptake, substantially extending the lifetime of last-line antibiotic drugs. Importantly, a natural blueprint for blocking plasmid acquisition appears to exist in approximately 50% of sequenced bacterial genomes. Known as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), CRISPR is a genetic circuit that cleaves and destroys specific previously encountered plasmid and viral sequences, thus inhibiting viruses and plasmids from establishing their genetic material in CRISPR+ cells. CRISPR comes at a likely cost of reduced genomic plasticity for its bacterial hosts, who can no longer use plasmid acquisition to rapidly evolve. The specific goal of this proposal is thus to define the mechanisms and selective pressures allowing bacteria to maintain CRISPR despite its costs. This goal is significant because of CRISPR's ability to block the prime route of antibiotic resistance spread, plasmid acquisition. We hypothesize that CRISPR is preserved in bacterial populations because it provides sustained antiviral immunity. To test this hypothesis, an interdisciplinary computational and experimental study is proposed. First, a novel stochastic model will be designed to compete CRISPR+ and CRISPR- bacteria under varying viral pressure and periodic antibiotic treatment. The model will enable us to quantify potential parameter regimes in which antiviral immunity maintains CRISPR+ cells for extended periods of time despite the cost of being unable to acquire plasmids. Further, the model will pinpoint whether optimal treatment regimes exist in which reducing antibiotic dosages can improve patient outcomes by preserving CRISPR+ commensal reservoirs. The basic assumptions that CRISPR provides a fitness advantage against viruses and a fitness disadvantage against antibiotics will then be tested experimentally in Enterococcus, a leading cause of antibiotic resistant hospital infections. By experimentally assaying the anti-plasmid and anti-viral efficienc of CRISPR in an Enterococcus genome, I will definitively test whether selection for CRISPR's antiviral immunity can inhibit the emergence of deadly multi-drug resistant pathogens.