中文摘要：细胞内病原体对全球公共卫生构成严重威胁。源于噬菌体的肽聚糖水解酶，即细胞溶解酶,杀菌能力强,风险低，有望替代传统抗生素。然而，大多数蛋白溶酶因为大小排斥，不能穿透哺乳动物细胞膜。此前，我们报道了一种广谱嵌合细胞溶解酶ClyR，它含有来自PlyC溶酶半胱氨酸、组氨酸依赖的酰胺水解酶/肽酶催化结构域和来自PlySs2溶酶的SH-3b细胞壁结合结构域。本文进一步报道在ClyR的两个组成结构域连接区域预测到一种新的细胞内穿透肽（CPP），通过该肽介导，ClyR可以经由小窝蛋白依赖的内吞作用被上皮细胞内化，从而靶向细胞内病原体。研究发现CPP的K153、P154、R169和R188残基对ClyR介导的内化和细胞内杀伤至关重要。RNA序列分析进一步显示，上皮细胞暴露于100 mu g/ml ClyR 24 h后，其转录和代谢谱存在微小差异。综上所述，本研究证明了ClyR的一种新的内化机制，为合理设计下一代细胞溶解酶靶向细胞外和细胞内病原体提供了新的理念。
外文摘要：Intracellular pathogens pose serious challenges to the public health worldwide. Lysin, peptidoglycan hydrolase from phage, is promising alternative to conventional antibiotics because of its high bactericidal activity and low risk of resistance. However, most proteinaceous lysins cannot penetrate the mammalian cell membrane because of size exclusion. Previously, we reported a broad-spectrum chimeric lysin, ClyR, with a cysteine, histidine-dependent amidohydrolase/peptidase catalytic domain from PlyC lysin and an SH-3b cell-wall binding domain from PlySs2 lysin. Herein, we further report that a novel internal cell-penetrating peptide (CPP) is predicted in the junction region of the two constitutive domains of ClyR, mediated by which ClyR can be internalized by epithelial cells through caveolin-dependent endocytosis to target intracellular pathogens. Residues K153, P154, R169, and R188 of the internal CPP were found to be essential for ClyR-mediated internalization and intracellular killing. RNA-seq analysis further showed that there are minor differences in transcript and metabolic profiles from epithelial cells exposed to 100 mu g/ml ClyR for 24 h. Taken together, our findings demonstrate a novel mechanism of internalization by ClyR, providing new insights into the rational designing of the next-generation lysins to target both extracellular and intracellular pathogens.
外文关键词：Bacteriophage lysin；Chimeric lysin；Lysin therapy；Intracellular pathogen； Cell-penetrating peptide
作者：Yang, H；Xu, JJ；Gong, YJ；Tang, Y；Li, WY；Zheng, ZH；Li, YH；He, J；Wei, HP
作者单位：Chinese Acad Sci； Univ Chinese Acad Sci；Wuhan Univ；Huazhong Agr Univ
期刊名称：INTERNATIONAL JOURNAL OF PHARMACEUTICS
期刊影响因子：4.845
出版年份：2021
出版刊次：599
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