Zhang Zheng, Tian Changyu, Zhao Jiangtao, Chen Xiao, Wei Xiao, Li Huan, Lin Weishi, Feng Ruo, Jiang Aimin, Yang Wenhui, Yuan Jing, Zhao Xiangna
College of Food Science, South China Agricultural University, Guangzhou, China.
Institute of Disease Control and Prevention, China PLA, Beijing, China.
Front Microbiol. 2018 Mar 15;9:450. doi: 10.3389/fmicb.2018.00450. eCollection 2018.
phage phiAxp-3, an N4-like bacteriophage, specifically recognize lipopolysaccharide (LPS) as its receptor. PhiAxp-3 tail sheath protein (TSP, ORF69) shares 54% amino acid sequence identity with the TSP of phage N4 (gp65); the latter functions as a receptor binding protein and interacts with the outer membrane receptor NfrA of its host bacterium. Thus, we hypothesized that ORF69 is the receptor-binding protein of phiAxp-3. In the present study, a series of ORF69 truncation variants was constructed to identify the part(s) of this protein essential for binding to LPS. Phage adsorption and enzyme-linked immunosorbent assay showed that amino acids 795-1195 of the TSP, i.e., ORF69(795-1195), are sufficient and essential for receptor and binding. The optimum temperature and pH for the functions of ORF69 and ORF69(795-1195) are 4/25°C and 7, respectively. cytotoxicity assays showed that ORF69 and ORF69(795-1195) were respectively toxic and non-toxic to a human immortalized normal hepatocyte cell line (LO2; doses: 0.375-12 μg). The potential of this non-toxic truncated version of phiASP-3 TSP for clinical applications is discussed.
噬菌体phiAxp-3是一种N4样噬菌体，它特异性识别脂多糖（LPS）作为其受体。PhiAxp-3尾鞘蛋白（TSP，ORF69）与噬菌体N4的TSP（gp65）具有54%的氨基酸序列同一性；后者作为受体结合蛋白，与其宿主细菌的外膜受体NfrA相互作用。因此，我们推测ORF69是phiAxp-3的受体结合蛋白。在本研究中，构建了一系列ORF69截短变体，以确定该蛋白中与LPS结合必不可少的部分。噬菌体吸附和酶联免疫吸附测定表明，TSP的氨基酸795-1195，即ORF69(795-1195)，对于受体结合是足够且必不可少的。ORF69和ORF69(795-1195)功能的最适温度和pH分别为4/25°C和7。细胞毒性试验表明，ORF69和ORF69(795-1195)对人永生化正常肝细胞系（LO2；剂量：0.375-12μg）分别具有毒性和无毒性。讨论了phiASP-3 TSP这种无毒截短形式在临床应用中的潜力。
