Genome-wide phage susceptibility analysis in Acinetobacter baumannii reveals capsule modulation strategies that determine phage infectivity.
Genome-wide phage susceptibility analysis in Acinetobacter baumannii reveals capsule modulation strategies that determine phage infectivity.
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DOI：
10.1371/journal.ppat.1010928
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发表时间：
2023-06
期刊：
影响因子：
6.7
通讯作者：
中科院分区：
文献类型：
--
作者：
噬菌体作为耐药病原体鲍氏不动杆菌院内感染的一种有效治疗方法，已重新受到关注。我们对A.鲍曼不动杆菌对噬菌体的防御仍然有限，尽管这一信息可能导致改进的抗微生物疗法。为了解决这个问题，我们确定了噬菌体易感性的全基因组决定因素。鲍曼不动杆菌，使用Tn-seq.这些研究集中在裂解性噬菌体Loki上，其通过未知机制靶向不动杆菌。我们确定了41个候选位点，增加对洛基的易感性时，中断，和10个，降低易感性。结合自发电阻图，我们的结果支持Loki使用K3胶囊作为必需受体的模型，并且胶囊调制提供了A。鲍曼不动杆菌与控制对噬菌体的脆弱性的策略。这种控制的一个关键中心是由全局调节因子BfmRS对荚膜合成和噬菌体毒力的转录调节。超激活BfmRS的突变同时增加了包膜水平、Loki吸附、Loki复制和宿主杀伤，而BfmRS失活突变具有相反的效果，减少了包膜并阻断了Loki感染。我们确定了新的BfmRS激活突变，包括敲除T2 RNA酶蛋白和二硫键形成酶DsbA，使细菌对噬菌体挑战超敏。我们进一步发现，已知改变荚膜结构和细菌毒力的糖基转移酶的突变也可以引起完全的噬菌体抗性。最后，包括脂寡糖和Lon蛋白酶在内的其他因子独立于胶囊调节而起作用以干扰Loki感染。这项工作表明，胶囊的调节和结构调节，已知改变A。鲍曼不动杆菌毒力也是噬菌体易感性的主要决定因素。鲍曼不动杆菌的抗生素耐药性感染是重症监护病房的一个主要问题，并且在COVID-19大流行期间频率增加。这些感染的毒力取决于细菌周围的多糖荚膜。噬菌体，或杀死细菌的病毒，代表了一种有希望的替代疗法，以对付高度耐药的A。鲍曼不动杆菌感染;鲍曼不动杆菌特异性噬菌体通常靶向荚膜。在这里，我们使用高通量遗传学来分析A。鲍曼不动杆菌防御噬菌体并鉴定增强其杀伤活性的方法。我们发现，以导致胶囊产量增加的方式对细菌施加压力也会导致对噬菌体的超敏感性。相反，关闭应激反应或突变胶囊结构会导致完全的噬菌体抗性。改变另一种表面结构、脂寡糖或细胞内蛋白酶也会增强噬菌体的攻击。已知调节荚膜多糖的量或组成会影响A.鲍曼不动杆菌。因此，这项工作揭示了噬菌体压力和A.鲍曼不动杆菌，并确定了控制机制，可用于改善未来的噬菌体为基础的抗微生物疗法。
Phage have gained renewed interest as an adjunctive treatment for life-threatening infections with the resistant nosocomial pathogen Acinetobacter baumannii. Our understanding of how A. baumannii defends against phage remains limited, although this information could lead to improved antimicrobial therapies. To address this problem, we identified genome-wide determinants of phage susceptibility in A. baumannii using Tn-seq. These studies focused on the lytic phage Loki, which targets Acinetobacter by unknown mechanisms. We identified 41 candidate loci that increase susceptibility to Loki when disrupted, and 10 that decrease susceptibility. Combined with spontaneous resistance mapping, our results support the model that Loki uses the K3 capsule as an essential receptor, and that capsule modulation provides A. baumannii with strategies to control vulnerability to phage. A key center of this control is transcriptional regulation of capsule synthesis and phage virulence by the global regulator BfmRS. Mutations hyperactivating BfmRS simultaneously increase capsule levels, Loki adsorption, Loki replication, and host killing, while BfmRS-inactivating mutations have the opposite effect, reducing capsule and blocking Loki infection. We identified novel BfmRS-activating mutations, including knockouts of a T2 RNase protein and the disulfide formation enzyme DsbA, that hypersensitize bacteria to phage challenge. We further found that mutation of a glycosyltransferase known to alter capsule structure and bacterial virulence can also cause complete phage resistance. Finally, additional factors including lipooligosaccharide and Lon protease act independently of capsule modulation to interfere with Loki infection. This work demonstrates that regulatory and structural modulation of capsule, known to alter A. baumannii virulence, is also a major determinant of susceptibility to phage. Antibiotic-resistant infections with Acinetobacter baumannii are a major problem in critical care units and have increased in frequency during the COVID-19 pandemic. The virulence of these infections depends on a polysaccharide capsule surrounding the bacterium. Phage, or viruses that kill bacteria, represent a promising alternative therapy against highly antibiotic-resistant A. baumannii infections, and A. baumannii-specific phage often target the capsule. Here, we use high-throughput genetics to analyze how A. baumannii defends against phage and identify ways to potentiate their killing activity. We found that stressing the bacteria in ways that cause augmented production of capsule also causes hyper-susceptibility to phage. By contrast, turning off the stress response, or mutating the capsule structure, causes complete phage resistance. Altering another surface structure, lipooligosaccharide, or an intracellular protease also enhances phage attack. Modulating the amounts or makeup of capsular polysaccharide is known to influence virulence in A. baumannii. This work thus uncovers a connection between phage pressure and the evolution of virulence in A. baumannii, and it identifies control mechanisms that may be leveraged for improving future phage-based antimicrobial therapies.
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