Laboratory of Transcription, Center for DNA Fingerprinting and Diagnostics, Uppal, Hyderabad, India; Graduate Studies, Manipal Institute of Higher Education, Manipal, Karnataka, India.
Laboratory of Transcription, Center for DNA Fingerprinting and Diagnostics, Uppal, Hyderabad, India.
J Biol Chem. 2021 Jan-Jun;296:100653. doi: 10.1016/j.jbc.2021.100653. Epub 2021 May 15.
The transcription terminator Rho regulates many physiological processes in bacteria, such as antibiotic sensitivity, DNA repair, RNA remodeling, and so forth, and hence, is a potential antimicrobial target, which is unexplored. The bacteriophage P4 capsid protein, Psu, moonlights as a natural Rho antagonist. Here, we report the design of novel peptides based on the C-terminal region of Psu using phenotypic screening methods. The resultant 38-mer peptides, in addition to containing mutagenized Psu sequences, also contained plasmid sequences, fused to their C termini. Expression of these peptides inhibited the growth of Escherichia coli and specifically inhibited Rho-dependent termination in vivo. Peptides 16 and 33 exhibited the best Rho-inhibitory properties in vivo. Direct high-affinity binding of these two peptides to Rho also inhibited the latter's RNA-dependent ATPase and transcription termination functions in vitro. These two peptides remained functional even if eight to ten amino acids were deleted from their C termini. In silico modeling and genetic and biochemical evidence revealed that these two peptides bind to the primary RNA-binding site of the Rho hexamer near its subunit interfaces. In addition, the gene expression profiles of these peptides and Psu overlapped significantly. These peptides also inhibited the growth of Mycobacteria and inhibited the activities of Rho proteins from Mycobacterium tuberculosis, Xanthomonas, Vibrio cholerae, and Salmonella enterica. Our results showed that these novel anti-Rho peptides mimic the Rho-inhibition function of the ∼42-kDa dimeric bacteriophage P4 capsid protein, Psu. We conclude that these peptides and their C-terminal deletion derivatives could provide a basis on which to design novel antimicrobial peptides.
转录终止因子 Rho 调控细菌的许多生理过程，如抗生素敏感性、DNA 修复、RNA 重塑等，因此是一个潜在的抗菌靶点，尚未被探索。噬菌体 P4 衣壳蛋白 Psu 兼职作为天然 Rho 拮抗剂。在这里，我们使用表型筛选方法基于 Psu 的 C 端区域设计了新型肽。所得的 38 肽除了包含突变的 Psu 序列外，还在其 C 末端融合了质粒序列。这些肽的表达抑制了大肠杆菌的生长，并特异性地抑制了体内 Rho 依赖性终止。肽 16 和 33 在体内表现出最好的 Rho 抑制特性。这两种肽直接与 Rho 高亲和力结合，也抑制了后者在体外的 RNA 依赖性 ATP 酶和转录终止功能。即使从 C 端删除 8 到 10 个氨基酸，这两种肽仍然保持功能。计算机建模和遗传及生化证据表明，这两种肽结合在 Rho 六聚体的主要 RNA 结合位点附近，靠近其亚基界面。此外，这些肽和 Psu 的基因表达谱有很大的重叠。这些肽还抑制分枝杆菌的生长，并抑制结核分枝杆菌、黄单胞菌、霍乱弧菌和肠炎沙门氏菌的 Rho 蛋白的活性。我们的结果表明，这些新型抗 Rho 肽模拟了约 42 kDa 二聚噬菌体 P4 衣壳蛋白 Psu 的 Rho 抑制功能。我们得出结论，这些肽及其 C 端缺失衍生物可以为设计新型抗菌肽提供基础。
