Chemical Probes Unravel an Antimicrobial Defense Response Triggered by Binding of the Human Opioid Dynorphin to a Bacterial Sensor Kinase

Host–microbe communication via small molecule signals is important for both symbiotic and pathogenic relationships, but is often poorly understood at the molecular level. Under conditions of host stress, levels of the human opioid peptide dynorphin are elevated, triggering virulence in the opportunistic pathogenic bacterium Pseudomonas aeruginosa via an unknown pathway. Here we apply a multilayered chemical biology strategy to unravel the mode of action of this putative interkingdom signal. We designed and applied dynorphin-inspired photoaffinity probes to reveal the protein targets of the peptide in live bacteria via chemical proteomics. ParS, a largely uncharacterized membrane sensor of a two-component system, was identified as the most promising hit. Subsequent full proteome studies revealed that dynorphin(1–13) induces an antimicrobial peptide-like response in Pseudomonas, with specific upregulation of membrane defense mechanisms. No such response was observed in a parS mutant, which was more susceptible to dynorphin-induced toxicity. Thus, P. aeruginosa exploits the ParS sensing machinery to defend itself against the host in response to dynorphin as a signal. This study highlights interkingdom communication as a potential essential strategy not only for induction of P. aeruginosa virulence but also for maintaining viability in the hostile environment of the host.

通过小分子信号介导的宿主-微生物（host–microbe）交流，在共生与致病双方的相互作用中均发挥关键作用，但其分子层面的调控机制迄今仍鲜有清晰阐释。当宿主处于应激状态时，人体内的阿片肽类物质强啡肽（dynorphin）水平会升高，并通过一条尚未阐明的信号通路，触发机会致病菌铜绿假单胞菌（Pseudomonas aeruginosa）的毒力表达。本研究采用多层化化学生物学策略，解析这一推定的跨界信号的作用机制。我们设计并合成了基于强啡肽的光亲和探针（photoaffinity probes），借助化学蛋白质组学（chemical proteomics）技术，在活细菌中鉴定该肽段的蛋白靶点。双组分系统（two-component system）中一个此前几乎未被表征的膜传感器ParS，被鉴定为最具潜力的候选靶点。后续的全蛋白质组学研究显示，强啡肽(1–13)可在铜绿假单胞菌中诱导类抗菌肽（antimicrobial peptide）应答，并特异性上调膜防御相关机制的表达。而parS突变体中未出现此类应答，且其对强啡肽诱导的毒性更为敏感。综上，铜绿假单胞菌借助ParS感知系统，以强啡肽为信号，抵御宿主环境的压力。本研究表明，跨界信号交流不仅是铜绿假单胞菌诱导毒力的关键策略，也是其在宿主内的恶劣环境中维持生存的必要手段。