Exploiting Pf phage superinfection to lower Pseudomonas aeruginosa virulence via evolutionary tradeoffs

项目总结 许多临床相关的病原体在它们的染色体中整合了噬菌体基因组。 (前驱体)，这会对细菌的表型和适应性有很大影响。因为噬菌体的适合性是平局 为了适应细菌宿主，噬菌体可以通过减轻它们对宿主的负担而变得更适合宿主。但是，当 细菌被多个噬菌体感染(即重叠感染)，对宿主资源的竞争可能选择噬菌体 它们与其他噬菌体相比更具竞争力，即使它们的宿主负担增加了。这样做的目的是 建议理解并学会利用细胞内噬菌体之间的进化权衡 在双重感染期间的竞争力和细菌适应性。了解这种权衡将使我们有更好的洞察力 研究前驱体如何影响其细菌宿主的表型和适合性，并可能为一部小说铺平道路 噬菌体疗法中的一种方法，它利用这种权衡来使细菌感染更容易治疗。 我们建议使用铜绿假单胞菌(PA)及其原噬菌体PF噬菌体作为更好的模型系统 了解噬菌体竞争性和细菌适合度之间的进化权衡。超过一半的 PA携带着PF预言体。此外，慢性感染PA的囊性纤维化(CF)患者更多 与急性感染的患者相比，PA患者更有可能具有PF前驱体。这提示Pf噬菌体是一个完整的 部分PA的进化史和发病机制。本项目的第一部分旨在了解帕斯是否 毒力因子的产生与PA染色体中携带的Pf前体数有关。 从之前资助的一项研究中，我们收集了33名携带以下病毒的CF患者的&gt；100pa临床分离株 零个、一个或两个PF先知。使用这个收集和实验室的PA菌株，我们将测试Pf之间的相关性 拷贝数和毒力因子的产生，如绿青素和吡咯烷酮。在第二个目标中，我们将 用原噬菌体抑制基因突变的Pf噬菌体超级感染PaLab株PA14。这个Pf噬菌体 突变体在宿主细胞内以高种群快速复制，创造了有利于 缺乏衣壳基因的缺陷干扰(DI)噬菌体的选择。这些DI噬菌体是利用 全长噬菌体，用于公共资源，如衣壳，以自私地繁殖。PF衣壳蛋白与临床相关 铜绿假单胞菌的表型，如生物被膜的稳定性和对抗生素的耐受性。因此，衣壳基因的丢失不仅是 PF噬菌体变得比其他PF噬菌体更具竞争力，但也会影响宿主的适应性。我们计划 在生物被膜和抗生素筛选下进化感染PA14的自私PF噬菌体，以检测衣壳是否 随着时间的推移，基因会丢失，破坏生物膜的稳定性和抗生素的耐受性。 该项目的完成将提供关于前驱体对其细菌的影响的有价值的信息 寄主的表型和适合度。利用噬菌体竞争力和宿主之间的进化权衡 健身可能会为噬菌体疗法中的一种新方法铺平道路，类似于基因驱动，它利用 这样的权衡使细菌感染更容易治疗。

PROJECT SUMMARY Many clinically relevant pathogens have bacteriophage (phage) genomes integrated in their chromosome (prophages), which can have large effects on the bacteria’s phenotype and fitness. Since phage fitness is tied to bacterial host fitness, phages can become fitter by decreasing their burden on their host. However, when bacteria are infected by multiple phages (i.e., superinfection), competition for host resources may select phages that are more competitive against other phages, even at the increased burden to their host. The objective of this proposal is to understand and learn to exploit the evolutionary tradeoff between phage intracellular competitiveness and bacterial fitness during superinfection. Understanding this tradeoff will give better insight into how prophages influence their bacterial host’s phenotype and fitness, and potentially pave way for a novel approach in phage therapy that utilizes such tradeoffs to make bacterial infections easier to treat. We propose to use Pseudomonas aeruginosa (Pa) and its prophage, Pf phage, as a model system for better understanding the evolutionary tradeoff between phage competitiveness and bacterial fitness. More than half of Pa carry Pf prophages. Furthermore, cystic fibrosis (CF) patients who are chronically infected with Pa were more likely to have Pa that have Pf prophages than acutely infected patients. This hints that Pf phages are an integral part of Pa evolutionary history and pathogenesis. The first part of this project aims to understand whether Pa virulence factor production is correlated with the number of Pf prophages that Pa carries in its chromosome. From a previously funded study, we have a collection of >100 Pa clinical isolates from 33 CF patients that carry zero, one, or two Pf prophages. Using this collection and lab Pa strains, we will test for correlations between Pf copy number and the production of virulence factors like pyocyanin and pyoverdine. In the second aim, we will superinfect Pa lab strain PA14 with Pf phage that has a mutation in the prophage repressor gene. This Pf phage mutant replicates quickly and at high populations within the host cell, creating an environment that favors selection for defective interfering (DI) phages that lack capsid genes. These DI phages are cheaters that exploit full-length phages for public resources, like capsid, to selfishly propagate. Pf capsid is tied to clinically relevant phenotypes of Pa, such as biofilm robustness and antibiotic tolerance. Thus, the loss of capsid genes is not only a way Pf phages become more competitive against other Pf phages but can affect host fitness as well. We plan to evolve PA14 infected with this selfish Pf phage under biofilm and antibiotic selection to test whether capsid genes become lost over time, undermining biofilm stability and antibiotic tolerance. Completion of this project will provide valuable information on the influence prophages have on their bacterial host’s phenotype and fitness. Exploiting the evolutionary tradeoff between phage competitiveness and host fitness may potentially pave way for a novel approach in phage therapy, analogous to a gene drive, that exploits such tradeoffs to make bacterial infections easier to treat.