Structural biology and functional features of phage-derived depolymerase Depo32 on Klebsiella pneumoniae with K2 serotype capsular polysaccharides.
Structural biology and functional features of phage-derived depolymerase Depo32 on Klebsiella pneumoniae with K2 serotype capsular polysaccharides.
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DOI：
10.1128/spectrum.05304-22
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发表时间：
2023-09-26
影响因子：
3.7
通讯作者：
中科院分区：
文献类型：
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作者：
具有荚膜多糖的高毒力肺炎克雷伯菌（cps）引起严重的医院和社区获得性感染。噬菌体衍生解聚合酶可以降解肺炎克雷伯菌的cps以减弱细菌毒力，但其抗菌机制和临床潜力尚不清楚。在本研究中，鉴定出克雷伯菌噬菌体gh - k3衍生的解聚合酶Depo32（由基因gp32编码）能够高效地特异性降解K2血清型肺炎克雷伯菌的cps。三聚体Depo32的低温电镜结构（分辨率高达2.32 Å）揭示了两个相邻亚基的间隙中潜在的催化中心。Depo32作用下的肺炎克雷伯菌对RAW264.7细胞的吞噬更加敏感，并通过丝裂原激活的蛋白激酶信号通路激活细胞。此外，鼻内接种Depo32（单剂量200µg，每天20µg，连续3天，或与庆大霉素联合使用）可挽救感染致死剂量肺炎克雷伯菌K7的所有C57BL/6J小鼠，而不受其中和抗体的干扰。总之，这项工作详细阐述了Depo32靶向K2血清型cps降解的机制及其作为抗毒剂的潜力。目前已鉴定出20多种克雷伯菌血清型特异性解聚合酶，但大多数研究仅对解聚合酶的单剂量治疗进行评价，临床评价指标相对简单，未深入揭示解聚合酶的抗感染机制。在确定其生物学特性的基础上，利用低温电子显微镜对其结构进行了分析，并进一步确定了其潜在活性中心。此外，揭示了Depo32对巨噬细胞吞噬、信号通路激活和血清杀伤的影响，并评价了解聚合酶（单药、多药或联合庆大霉素）治疗肺炎克雷伯菌引起的急性肺炎的疗效。此外，Depo32活性位点的作用也在体外和体内研究中得到了阐明。因此，本研究通过结构生物学、细胞生物学和体内实验，论证了Depo32靶向K2血清型肺炎克雷伯菌感染的机制。
Hypervirulent Klebsiella pneumoniae with capsular polysaccharides (CPSs) causes severe nosocomial- and community-acquired infections. Phage-derived depolymerases can degrade CPSs from K. pneumoniae to attenuate bacterial virulence, but their antimicrobial mechanisms and clinical potential are not well understood. In the present study, Klebsiella phage GH-K3-derived depolymerase Depo32 (encoded by gene gp32) was identified to exhibit high efficiency in specifically degrading the CPSs of K2 serotype K. pneumoniae. The cryo-electron microscopy structure of trimeric Depo32 at a resolution up to 2.32 Å revealed potential catalytic centers in the cleft of each of the two adjacent subunits. K. pneumoniae subjected to Depo32 became more sensitive to phagocytosis by RAW264.7 cells and activated the cells by the mitogen-activated protein kinase signaling pathway. In addition, intranasal inoculation with Depo32 (a single dose of 200 µg, 20 µg daily for 3 days, or in combination with gentamicin) rescued all C57BL/6J mice infected with a lethal dose of K. pneumoniae K7 without interference from its neutralizing antibody. In summary, this work elaborates on the mechanism by which Depo32 targets the degradation of K2 serotype CPSs and its potential as an antivirulence agent. Depolymerases specific to more than 20 serotypes of Klebsiella spp. have been identified, but most studies only evaluated the single-dose treatment of depolymerases with relatively simple clinical evaluation indices and did not reveal the anti-infection mechanism of these depolymerases in depth. On the basis of determining the biological characteristics, the structure of Depo32 was analyzed by cryo-electron microscopy, and the potential active center was further identified. In addition, the effects of Depo32 on macrophage phagocytosis, signaling pathway activation, and serum killing were revealed, and the efficacy of the depolymerase (single treatment, multiple treatments, or in combination with gentamicin) against acute pneumonia caused by Klebsiella pneumoniae was evaluated. Moreover, the roles of the active sites of Depo32 were also elucidated in the in vitro and in vivo studies. Therefore, through structural biology, cell biology, and in vivo experiments, this study demonstrated the mechanism by which Depo32 targets K2 serotype K. pneumoniae infection.
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DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
4.9
作者：
Pan, Yi-Jiun;Lin, Tzu-Lung;Wang, Jin-Town
通讯作者：
Wang, Jin-Town
影响因子：
5.2
作者：
Latka, Agnieszka;Leiman, Petr G.;Briers, Yves
通讯作者：
Briers, Yves
DOI：
10.1107/s0907444909052925
发表时间：
2010-02
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Adams PD;Afonine PV;Bunkóczi G;Chen VB;Davis IW;Echols N;Headd JJ;Hung LW;Kapral GJ;Grosse-Kunstleve RW;McCoy AJ;Moriarty NW;Oeffner R;Read RJ;Richardson DC;Richardson JS;Terwilliger TC;Zwart PH
通讯作者：
Zwart PH
影响因子：
3.7
作者：
Pan YJ;Lin TL;Chen YH;Hsu CR;Hsieh PF;Wu MC;Wang JT
通讯作者：
Wang JT