The Evolutionary Dynamics of Plasmid Uptake in Pathogenic Bacteria

描述（由申请人提供）：尽管数十年的研究和数十种抗生素，由于抗生素耐药性的快速演变，细菌病原体再次成为西方死亡的主要原因之一。质粒获得是细菌抗生素耐药性传播的主要原因。然而，在我们对如何强有力地阻止质粒获得的理解方面存在根本性的差距，特别是在抗生素提供的选择压力下。该提案的长期目标是开发信息，允许设计限制质粒摄取的新疗法和方法，大大延长最后一线抗生素药物的寿命。重要的是， 质粒获得似乎存在于约50%的测序细菌基因组中。CRISPR被称为规则间隔短回文重复序列（CRISPR），是一种基因回路，可以切割和破坏先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的代价可能是其细菌宿主的基因组可塑性降低，这些宿主无法再利用质粒获得来快速进化。因此，该提案的具体目标是定义允许细菌维持CRISPR的机制和选择压力，尽管其成本很高。这一目标意义重大，因为CRISPR能够阻断抗生素耐药性传播的主要途径，即质粒获得。我们假设CRISPR在细菌群体中得以保留，因为它提供了持续的抗病毒免疫。为了验证这一假设，提出了一个跨学科的计算和实验研究。首先，将设计一种新的随机模型来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，在这些机制中，抗病毒免疫可以长时间维持CRISPR+细胞，尽管无法获得质粒的成本很高。此外，该模型将确定是否存在最佳治疗方案，其中减少抗生素剂量可以通过保留CRISPR+骨髓库来改善患者的预后。CRISPR提供了针对病毒的适应性优势和针对抗生素的适应性劣势的基本假设将在肠球菌中进行实验测试，肠球菌是抗生素耐药性医院感染的主要原因。通过实验分析CRISPR在肠球菌基因组中的抗质粒和抗病毒效率，我将明确测试CRISPR抗病毒免疫的选择是否可以抑制致命的多药耐药病原体的出现。

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.