Characterisation of Bacteriophage-Encoded Depolymerases Selective for Key Klebsiella pneumoniae Capsular Exopolysaccharides.
Characterisation of Bacteriophage-Encoded Depolymerases Selective for Key Klebsiella pneumoniae Capsular Exopolysaccharides.
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DOI：
10.3389/fcimb.2021.686090
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发表时间：
2021
影响因子：
5.7
通讯作者：
Taylor PW
中科院分区：
文献类型：
--
作者：
Blundell-Hunter G;Enright MC;Negus D;Dorman MJ;Beecham GE;Pickard DJ;Wintachai P;Voravuthikunchai SP;Thomson NR;Taylor PW
荚膜多糖使临床上重要的肺炎克雷伯氏菌克隆在易感宿主中引起严重的全身感染。噬菌体编码的胶囊解聚酶有可能提供一种替代治疗模式的患者时，多药耐药性已经侵蚀了传统的抗生素化疗的疗效。调查164 K。来自泰国重症监护患者的肺炎链球菌显示了大量低丰度的不同K型，但有四种（K2，K51，K1，K10）的频率至少为5%。为了确定解聚酶的能力，以降解胶囊与这些常见的K-型，62裂解噬菌体从泰国医院的污水中分离，使用K1，K2和K51分离株作为主机;噬菌体噬菌斑，无一例外，显示光晕指示胶囊降解酶的存在。噬菌体基因组的大小范围为41-348 kb，具有50 - 535个预测编码序列（CDS）。使用定制的噬菌体蛋白质数据库，我们成功地将注释应用于这些CDS的30 - 70%（平均值= 58%）。最大的基因组，即所谓的巨型噬菌体，携带多个tRNA以及CRISPR重复序列和间隔序列。发现其中一个较小的噬菌体基因组含有推定的Cas 1 E型基因，表明这些专性裂解噬菌体中宿主DNA获得的历史。全基因组测序（WGS）表明，一些噬菌体展示了一个扩展的主机范围，由于存在多个解聚酶基因，在总共42个候选解聚酶基因被确定为多达8个在一个单一的基因组。根据宿主范围、噬菌体形态和WGS，选择了七种不同的病毒体进行进一步研究。表达K1、K2和K51解聚酶的候选基因，并纯化为his 6标记的可溶性蛋白，并证明了对K. pneumoniae荚膜多糖的凝胶电泳和Anton-Paar滚球粘度法测定。解聚酶以K-型特异性方式从K中完全除去了囊。肺炎细胞。我们的结论是，广泛的宿主范围的噬菌体携带多种酶，每一种酶都有能力降解一种单一的K型，这些酶作为治疗剂的任何未来用途将需要酶的鸡尾酒效用对一系列的K。肺炎感染。
Capsular polysaccharides enable clinically important clones of Klebsiella pneumoniae to cause severe systemic infections in susceptible hosts. Phage-encoded capsule depolymerases have the potential to provide an alternative treatment paradigm in patients when multiple drug resistance has eroded the efficacy of conventional antibiotic chemotherapy. An investigation of 164 K. pneumoniae from intensive care patients in Thailand revealed a large number of distinct K types in low abundance but four (K2, K51, K1, K10) with a frequency of at least 5%. To identify depolymerases with the capacity to degrade capsules associated with these common K-types, 62 lytic phage were isolated from Thai hospital sewage water using K1, K2 and K51 isolates as hosts; phage plaques, without exception, displayed halos indicative of the presence of capsule-degrading enzymes. Phage genomes ranged in size from 41–348 kb with between 50 and 535 predicted coding sequences (CDSs). Using a custom phage protein database we were successful in applying annotation to 30 - 70% (mean = 58%) of these CDSs. The largest genomes, of so-called jumbo phage, carried multiple tRNAs as well as CRISPR repeat and spacer sequences. One of the smaller phage genomes was found to contain a putative Cas type 1E gene, indicating a history of host DNA acquisition in these obligate lytic phage. Whole-genome sequencing (WGS) indicated that some phage displayed an extended host range due to the presence of multiple depolymerase genes; in total, 42 candidate depolymerase genes were identified with up to eight in a single genome. Seven distinct virions were selected for further investigation on the basis of host range, phage morphology and WGS. Candidate genes for K1, K2 and K51 depolymerases were expressed and purified as his6-tagged soluble protein and enzymatic activity demonstrated against K. pneumoniae capsular polysaccharides by gel electrophoresis and Anton-Paar rolling ball viscometry. Depolymerases completely removed the capsule in K-type-specific fashion from K. pneumoniae cells. We conclude that broad-host range phage carry multiple enzymes, each with the capacity to degrade a single K-type, and any future use of these enzymes as therapeutic agents will require enzyme cocktails for utility against a range of K. pneumoniae infections.
影响因子：
3.6
作者：
Leggate DR;Bryant JM;Redpath MB;Head D;Taylor PW;Luzio JP
通讯作者：
Luzio JP