Genetic and Proteomic Approaches to Reveal Bacterial Vulnerabilities to Phage Predation

项目总结 我们正在进入一个抗生素后的世界，在这个世界里，了解抗菌策略的机制并不是 足以确保有效的治疗。我们还必须考虑抵抗机制并加以解决。 在设计过程中。我们将使用遗传学和蛋白质组学来发现细菌是如何对抗噬菌体的 (噬菌体)裂解。推动这一目标的是对抗噬菌体抗性机制的愿望，从而使细菌 更容易被噬菌体捕食。我们首先使用最先进的遗传方法来解决这个问题 询问必需基因在限制噬菌体复制和细菌裂解中的作用。使用CRISPR 转录干扰(CRISPRi)，我们将在人类病原体假单胞菌中进行首次研究 确定必要基因的部分敲除是否能对噬菌体复制产生积极影响。 我们假设，抑制某些必需基因不仅会限制细菌的适应性，而且还会 可能提高任何噬菌体-宿主配对的成功，无论先验状态是否为 噬菌体抵抗力或敏感性。换句话说，即使已经在给定宿主中复制的噬菌体也可以做得更好。 我们还将利用CRISPRi筛选的简便性来识别限制噬菌体的非必需基因 以依赖菌株的方式复制，更类似于规范的高变量免疫系统(例如CRISPR)。 为了进一步理解噬菌体抗性的物理基础，我们将创建一张物理地图 利用全细胞分离蛋白质组学研究噬菌体-宿主蛋白-蛋白质相互作用。这一点尤其关键，因为 许多噬菌体蛋白的功能未知，与我们鉴定必需蛋白的目标一致 与噬菌体因子相互作用的复合体。我们将验证从基因和基因识别的因素的重要性 在单基因敲除过程中用噬菌体复制试验进行蛋白质组学分析以评估 观察到的现象。最后，我们怀疑噬菌体的“辅助基因”(即不严格意义上的高变位基因 对于所有宿主中的噬菌体复制是必不可少的)代表宿主的抑制物和调节剂的宝库 过程，这可能是有用的遗传饲料，以加强未来的噬菌体疗法。辅助基因有 生物信息鉴定和宿主结合伙伴将通过传统的亲和纯化进行鉴定- 质谱仪，并用噬菌体复制试验验证。我们的研究策略结合了 三名研究人员的互补专业知识：约瑟夫·邦迪-德诺米，噬菌体生物学和细菌专家 以及丹妮尔·斯万尼，她是一位生物分析质谱学专家，她利用自己的技能 定义许多人类病原体的宿主-病原体蛋白质-蛋白质网络；以及微生物学家杰森·彼得斯 具有细菌遗传学和致病机理方面的专业知识。

PROJECT SUMMARY We are entering a post antibiotic world where understanding the mechanism of an antibacterial strategy is not sufficient to ensure an effective therapy. We must also consider the mechanisms of resistance and address them during the design process. We will use genetics and proteomics to discover how bacteria combat bacteriophage (phage) lysis. Driving this goal is the desire to combat phage resistance mechanisms so as to make bacteria more susceptible to phage predation. We first tackle the problem by employing state of the art genetic methods to interrogate the role of essential genes in limiting phage replication and bacterial lysis. Using CRISPR transcriptional interference (CRISPRi), we will conduct the first studies in the human pathogen Pseudomonas aeruginosa to determine whether partial knockdown of essential genes can positively impact phage replication. We hypothesize that inhibition of certain essential genes will not only limit bacterial fitness, but also has the potential to enhance the success of any phage-host pairing, regardless of whether the a priori state is one of phage resistance or sensitivity. Put another way, even phages that already replicate in a given host can do better. We will additionally harness the ease of CRISPRi screening to identify non-essential genes that limit phage replication in strain-dependent manners, more akin to canonical hypervariable immune systems (e.g. CRISPR). To further our understanding of the physical underpinnings of phage resistance, we will create a physical map of phage-host protein-protein interactions using whole cell fractionation proteomics. This is particularly critical as many phage proteins are of unknown function and is in line with our goal of identifying essential protein complexes interacting with phage factors. We will validate the importance of factors identified from genetic and proteomic assays with phage replication assays during single gene knockdown to assess generalities of phenomena observed. Lastly, we suspect that phage “accessory genes” (i.e. hypervariable loci not strictly essential for phage replication in all hosts) represent a treasure trove of inhibitors and modulators of host processes, which could be useful genetic fodder for enhancing future phage therapeutics. Accessory genes have been bioinformatically identified and host binding partners will be identified with conventional affinity purification- mass spectrometry, and validated with phage replication assays. Our research strategy combines the complementary expertise of three investigators: Joseph Bondy-Denomy, an expert in phage biology and bacterial immune systems; and Danielle Swaney, an expert in bioanalytical mass spectrometry who has used her skills to define the host-pathogen protein-protein network for many human pathogens; and Jason Peters, a microbiologist with expertise in bacterial genetics and pathogenesis.