Table_2_Characterisation of Bacteriophage-Encoded Depolymerases Selective for Key Klebsiella pneumoniae Capsular Exopolysaccharides.docx

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.

荚膜多糖（capsular polysaccharides）可使肺炎克雷伯菌（Klebsiella pneumoniae）的临床重要克隆株在易感宿主中引发严重全身性感染。当多重耐药（multiple drug resistance）削弱了常规抗生素化疗的疗效时，噬菌体编码的荚膜解聚酶（phage-encoded capsule depolymerases）有望为患者提供一种全新的治疗范式。对泰国重症监护病房患者分离的164株肺炎克雷伯菌开展的调查显示，存在大量丰度较低的独特K型，但其中4种K型（K2、K51、K1、K10）的检出频率至少达5%。为筛选可降解上述常见K型相关荚膜的解聚酶，研究人员以K1、K2及K51菌株为宿主，从泰国医院污水中分离得到62株裂解性噬菌体（lytic phage）；所有噬菌斑（phage plaques）均无一例外地呈现出表明存在荚膜降解酶的晕圈（halos）。噬菌体基因组大小介于41~348 kb之间，预测编码序列（predicted coding sequences, CDSs）数量为50~535个。借助定制噬菌体蛋白质数据库，我们成功为其中30%~70%（平均58%）的CDSs完成了功能注释。所谓巨型噬菌体（jumbo phage）的大型基因组携带有多份转运RNA（transfer RNAs, tRNAs）以及CRISPR重复序列与间隔序列（CRISPR repeat and spacer sequences）。研究还发现，一株小型噬菌体基因组中含有推定的Cas1E型基因（Cas type 1E gene），表明这类专性裂解性噬菌体曾发生过宿主DNA获取事件。全基因组测序（whole-genome sequencing, WGS）结果显示，部分噬菌体因携带多套解聚酶基因而拥有更广的宿主范围；研究共鉴定出42个候选解聚酶基因，单株噬菌体基因组中最多可携带8个此类基因。基于宿主范围、噬菌体形态及全基因组测序结果，研究人员选取了7种不同的噬菌体粒子（virions）开展后续研究。针对K1、K2及K51型解聚酶的候选基因被表达并纯化为带有his6标签的可溶性蛋白，通过凝胶电泳（gel electrophoresis）与安东帕滚球粘度法（Anton-Paar rolling ball viscometry）证实了其针对肺炎克雷伯菌荚膜多糖的酶活性。解聚酶可按K型特异性方式完全去除肺炎克雷伯菌细胞表面的荚膜。本研究得出结论：宽宿主范围噬菌体携带有多种酶，每种酶仅能降解一种K型荚膜；若未来将这类酶用作治疗制剂，则需组合使用多种酶以覆盖多株肺炎克雷伯菌感染。