Liu Yannan, Mi Zhiqiang, Mi Liyuan, Huang Yong, Li Puyuan, Liu Huiying, Yuan Xin, Niu Wenkai, Jiang Ning, Bai Changqing, Gao Zhancheng
Department of Respiratory and Critical Care Medicine, Peking University People's Hospital, Beijing, China.
Department of Respiratory and Critical Care Medicine, 307th Hospital of PLA, Beijing, China.
PeerJ. 2019 Jan 14;7:e6173. doi: 10.7717/peerj.6173. eCollection 2019.
The emergence of multidrug- or extensively drug-resistant has made it difficult to treat and control infections caused by this bacterium. It is urgently necessary to search for alternatives to conventional antibiotics for control of severe infections. In recent years, bacteriophages and their derivatives, such as depolymerases, showed great potential as antibacterial or antivirulence agents against bacterial infections. Nonetheless, unlike broad-spectrum bactericidal antibiotics, phage-encoded depolymerase targets only a limited number of bacterial strains. Therefore, identification of novel depolymerases and evaluation of their ability to control infections is important.
A bacteriophage was isolated from hospital sewage using an extensively drug-resistant strain as the host bacterium, and the phage's plaque morphology and genomic composition were studied. A polysaccharide depolymerase (Dpo48) was expressed and identified, and the effects of pH and temperature on its activity were determined. Besides, a serum killing assay was conducted, and amino acid sequences homologous to those of putative polysaccharide depolymerases were compared.
Phage IME200 yielded clear plaques surrounded by enlarged halos, with polysaccharide depolymerase activity against the host bacterium. A tail fiber protein with a Pectate_lyase_3 domain was identified as Dpo48 and characterized . Dpo48 was found to degrade the capsule polysaccharide of the bacterial surface, as revealed by Alcian blue staining. Dpo48 manifested stable activity over a broad range of pH (5.0-9.0) and temperatures (20-70 °C). Results from serum killing assays indicated that 50% serum was sufficient to cause a five log reduction of overnight enzyme-treated bacteria, with serum complement playing an important role in these killing assays. Moreover, Dpo48 had a spectrum of activity exactly the same as its parental phage IME200, which was active against 10 out of 41 strains. Amino acid sequence alignment showed that the putative tail fiber proteins had a relatively short, highly conserved domain in their N-terminal sequences, but their amino acid sequences containing pectate lyase domains, found in the C-terminal regions, were highly diverse.
Phage-encoded capsule depolymerases may become promising antivirulence agents for preventing and controlling infections.
多重耐药或广泛耐药菌的出现使得治疗和控制由该细菌引起的感染变得困难。迫切需要寻找传统抗生素的替代品来控制严重感染。近年来，噬菌体及其衍生物，如解聚酶，作为抗细菌感染的抗菌或抗毒力剂显示出巨大潜力。然而，与广谱杀菌抗生素不同，噬菌体编码的解聚酶仅靶向有限数量的细菌菌株。因此，鉴定新型解聚酶并评估其控制感染的能力很重要。
以广泛耐药菌株作为宿主菌，从医院污水中分离出一株噬菌体，并对其噬菌斑形态和基因组组成进行研究。表达并鉴定了一种多糖解聚酶（Dpo48），并测定了pH和温度对其活性的影响。此外，进行了血清杀菌试验，并比较了与假定多糖解聚酶氨基酸序列同源的序列。
噬菌体IME200产生了边缘有扩大晕圈的清晰噬菌斑，对宿主菌具有多糖解聚酶活性。一种具有果胶酸裂解酶_3结构域的尾丝蛋白被鉴定为Dpo48并进行了表征。阿尔辛蓝染色显示，Dpo48可降解细菌表面的荚膜多糖。Dpo48在较宽的pH范围（5.0 - 9.0）和温度范围（20 - 70°C）内表现出稳定的活性。血清杀菌试验结果表明，50%的血清足以使经酶处理过夜的细菌数量减少5个对数级，血清补体在这些杀菌试验中起重要作用。此外，Dpo48的活性谱与其亲本噬菌体IME200完全相同，对41株菌株中的10株有活性。氨基酸序列比对显示，假定的尾丝蛋白在其N端序列中有一个相对较短、高度保守的结构域，但在C端区域发现的含有果胶酸裂解酶结构域的氨基酸序列高度多样。
噬菌体编码的荚膜解聚酶可能成为预防和控制感染的有前景的抗毒力剂。
