中文摘要：肺炎克雷伯菌生产荚膜多糖，保护细菌细胞免受有害环境因素，如抗菌化合物和噬菌体的感染。噬菌体为了绕过这种保护屏障，编码多糖降解酶，即解聚酶，接近细菌细胞表面受体。本研究鉴定了噬菌体RAD2的特征，它能感染产生广泛、高致病性K2型荚膜多糖的肺炎克雷伯菌菌株。利用转座子定向插入测序，证明荚膜作为第一阶段受体，其产生是RAD2有效感染的绝对条件；鉴定了负责识别和降解荚膜的解聚酶，确定解聚酶在噬菌体病毒粒子尾端形成球状附属物，并以2.7埃分辨率呈现了RAD2荚膜解聚酶的低温电子显微镜结构；确定了该酶的一个假定活性位点，该活性位点由聚集的带负电荷残基组成，可促进目标多糖的水解。酶分析结合低聚糖消化产物质谱分析，进一步深入了解了酶的水解活性，当与肺炎克雷伯菌孵育时，酶会去除荚膜并使细胞对血清诱导的杀伤敏感。总的来说，这些发现扩大了对噬菌体如何靶向克雷伯菌荚膜进行感染的理解，为使用解聚酶作为抗病毒药物对抗这种医学上重要的病原体提供了一个框架。
    重点：肺炎克雷伯菌是一种医学上重要的病原体，它能产生对致病性至关重要的厚保护膜。噬菌体是细菌的天敌，许多噬菌体编码不同的“荚膜解聚酶”，专门降解宿主荚膜，这是一种可用于潜在治疗的特性。本研究已经确定了靶向临床相关K2荚膜的解聚酶的第一个结构，并确定了其假定的活性部位，为其作用机制提供了线索；还发现经重组解聚酶处理的克雷伯菌细胞被剥去荚膜，抑制了它们在血清存在下生长的能力，证明了这些强健且易于生产的酶对荚膜细菌病原体（如肺炎克雷伯菌）的抗感染潜力。
外文摘要：The production of capsular polysaccharides by Klebsiella pneumoniae protects the bacterial cell from harmful environmental factors such as antimicrobial compounds and infection by bacteriophages (phages). To bypass this protective barrier, some phages encode polysaccharide-degrading enzymes referred to as depolymerases to provide access to cell surface receptors. Here, we characterized the phage RAD2, which infects K. pneumoniae strains that produce the widespread, hypervirulence-associated K2-type capsular polysaccharide. Using transposon-directed insertion sequencing, we have shown that the production of capsule is an absolute requirement for efficient RAD2 infection by serving as a first-stage receptor. We have identified the depolymerase responsible for recognition and degradation of the capsule, determined that the depolymerase forms globular appendages on the phage virion tail tip, and present the cryoelectron microscopy structure of the RAD2 capsule depolymerase at 2.7-angstrom resolution. A putative active site for the enzyme was identified, comprising clustered negatively charged residues that could facilitate the hydrolysis of target polysaccharides. Enzymatic assays coupled with mass spectrometric analyses of digested oligosaccharide products provided further mechanistic insight into the hydrolase activity of the enzyme, which, when incubated with K. pneumoniae, removes the capsule and sensitizes the cells to serum-induced killing. Overall, these findings expand our understanding of how phages target the Klebsiella capsule for infection, providing a framework for the use of depolymerases as antivirulence agents against this medically important pathogen.
   IMPORTANCE Klebsiella pneumoniae is a medically important pathogen that produces a thick protective capsule that is essential for pathogenicity. Phages are natural predators of bacteria, and many encode diverse "capsule depolymerases" which specifically degrade the capsule of their hosts, an exploitable trait for potential therapies. We have determined the first structure of a depolymerase that targets the clinically relevant K2 capsule and have identified its putative active site, providing hints to its mechanism of action. We also show that Klebsiella cells treated with a recombinant form of the depolymerase are stripped of capsule, inhibiting their ability to grow in the presence of serum, demonstrating the anti-infective potential of these robust and readily producible enzymes against encapsulated bacterial pathogens such as K. pneumoniae.
外文关键词：Klebsiella；bacteriophages；capsular polysaccharide；cryo-EM；depolymerase
作者：Dunstan, RA；Bamert, RS；Belousoff, MJ；Short, FL；Barlow, CK；Pickard, DJ；Wilksch, JJ；Schittenhelm, RB；Strugnell, RA；Dougan, G；Lithgow, T
作者单位：Monash Univ；Wellcome Sanger Inst；Univ Cambridge；Univ Melbourne
期刊名称：MICROBIOLOGY SPECTRUM
期刊影响因子：5.465
出版年份：2021
出版刊次：1
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