A phage protein-derived antipathogenic peptide that targets type IV pilus assembly

Phage-inspired antibacterial discovery is a new approach that recruits phages in search for antibacterials with new molecular targets, in that phages are the biological entities well adapted to hijack host bacterial physiology in favor of their own thrive. We previously observed that phage-mediated twitching motility inhibition was effective to control the acute infections caused by Pseudomonas aeruginosa and that the motility inhibition was attributed to the delocalization of PilB, the type IV pilus (TFP) assembly ATPase by binding of the 136-amino acid (aa) phage protein, Tip. Here, we created a series of truncated and point-mutant Tip proteins to identify the critical residues in the Tip bioactivity: N-terminal 80-aa residues were dispensable for the Tip activity; we identified that Asp82, Leu84, and Arg85 are crucial in the Tip function. Furthermore, a synthetic 15-aa peptide (P1) that corresponds to Leu73 to Ala87 is shown to suffice for PilB delocalization, twitching inhibition, and virulence attenuation upon exogenous administration. The transgenic flies expressing the 15-aa peptide were resistant to P. aeruginosa infections as well. Taken together, this proof-of-concept study reveals a new antipathogenic peptide hit targeting bacterial motility and provides an insight into antibacterial discovery targeting TFP assembly.

以噬菌体（phage）为灵感的抗菌药物发现是一种新兴策略，该策略通过招募噬菌体来搜寻具备全新分子靶点的抗菌物质——其核心逻辑在于噬菌体是一类高度适配宿主细菌生理系统、可劫持宿主生理机能以实现自身增殖的生物实体。我们此前的研究发现，噬菌体介导的菌毛运动抑制可有效控制铜绿假单胞菌（Pseudomonas aeruginosa）引发的急性感染，而该运动抑制效应源于由136个氨基酸（amino acid, aa）组成的噬菌体蛋白Tip结合IV型菌毛（type IV pilus, TFP）组装ATP酶PilB后，使其发生异位分布。本研究中，我们构建了一系列截短型与点突变型Tip蛋白，以鉴定Tip生物活性所需的关键残基：实验结果表明，Tip蛋白的N端80个氨基酸残基对其活性并非必需；同时我们确认，Asp82、Leu84与Arg85这三个位点是Tip发挥功能的关键残基。进一步研究发现，一段对应Leu73至Ala87区域的合成15肽（P1）即可满足PilB异位分布、菌毛运动抑制以及外源给药后实现毒力衰减的全部效应。表达该15肽的转基因果蝇同样对铜绿假单胞菌感染具备抗性。综上，本概念验证研究揭示了一类靶向细菌运动系统的新型抗病原肽候选物，并为靶向IV型菌毛组装的抗菌药物发现提供了全新研究思路。