Liu Yang Sylvia, Zhang Chengqian, Khoo Bee Luan, Hao Piliang, Chua Song Lin
Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Kowloon, Hong Kong Special Administrative Region.
School of Life Science and Technology, ShanghaiTech University, China.
J Adv Res. 2024 Sep 2. doi: 10.1016/j.jare.2024.08.038.
Host-microbe interactions are important to human health and ecosystems globally, so elucidating the complex host-microbe interactions and associated protein expressions drives the need to develop sensitive and accurate biochemical techniques. Current proteomics techniques reveal information from the point of view of either the host or microbe, but do not provide data on the corresponding partner. Moreover, it remains challenging to simultaneously study host-microbe proteomes that reflect the direct competition between host and microbe. This raises the need to develop a dual-species proteomics method for host-microbe interactions.
We aim to establish a forward + reverse Stable Isotope Labeling with Amino acids in Cell culture (SILAC) proteomics approach to simultaneously label and quantify newly-expressed proteins of host and microbe without physical isolation, for investigating mechanisms in direct host-microbe interactions.
Using Caenorhabditis elegans-Pseudomonas aeruginosa infection model as proof-of-concept, we employed SILAC proteomics and molecular pathway analysis to characterize the differentially-expressed microbial and host proteins. We then used molecular docking and chemical characterization to identify chemical inhibitors that intercept host-microbe interactions and eliminate microbial infection.
Based on our proteomics results, we studied the iron competition between pathogen iron scavenger and host iron uptake protein, where P. aeruginosa upregulated pyoverdine synthesis protein (PvdA) (fold-change of 5.2313) and secreted pyoverdine, and C. elegans expressed ferritin (FTN-2) (fold-change of 3.4057). Targeted intervention of iron competition was achieved using Galangin, a ginger-derived phytochemical that inhibited pyoverdine production and biofilm formation in P. aeruginosa. The Galangin-ciprofloxacin combinatorial therapy could eliminate P. aeruginosa biofilms in a fish wound infection model, and enabled animal survival.
Our work provides a novel SILAC-based proteomics method that can simultaneously evaluate host and microbe proteomes, with future applications in higher host organisms and other microbial species. It also provides insights into the mechanisms dictating host-microbe interactions, offering novel strategies for anti-infective therapy.
宿主-微生物相互作用对全球人类健康和生态系统都很重要，因此阐明复杂的宿主-微生物相互作用及相关蛋白质表达推动了开发灵敏且准确的生化技术的需求。当前的蛋白质组学技术从宿主或微生物的角度揭示信息，但未提供关于相应伙伴的数据。此外，同时研究反映宿主与微生物之间直接竞争的宿主-微生物蛋白质组仍然具有挑战性。这就产生了开发一种用于宿主-微生物相互作用的双物种蛋白质组学方法的需求。
我们旨在建立一种正向+反向细胞培养中氨基酸稳定同位素标记（SILAC）蛋白质组学方法，以在不进行物理分离的情况下同时标记和定量宿主和微生物新表达的蛋白质，用于研究宿主-微生物直接相互作用的机制。
以秀丽隐杆线虫-铜绿假单胞菌感染模型作为概念验证，我们采用SILAC蛋白质组学和分子途径分析来表征差异表达的微生物和宿主蛋白质。然后我们使用分子对接和化学表征来鉴定拦截宿主-微生物相互作用并消除微生物感染的化学抑制剂。
基于我们的蛋白质组学结果，我们研究了病原体铁清除剂与宿主铁摄取蛋白之间的铁竞争，其中铜绿假单胞菌上调了绿脓菌素合成蛋白（PvdA）（变化倍数为5.2313）并分泌了绿脓菌素，而秀丽隐杆线虫表达了铁蛋白（FTN-2）（变化倍数为3.4057）。使用高良姜素实现了对铁竞争的靶向干预，高良姜素是一种源自生姜的植物化学物质，可抑制铜绿假单胞菌中绿脓菌素的产生和生物膜形成。高良姜素-环丙沙星联合疗法可在鱼类伤口感染模型中消除铜绿假单胞菌生物膜，并使动物存活。
我们的工作提供了一种基于SILAC的新型蛋白质组学方法，可同时评估宿主和微生物蛋白质组，未来可应用于更高等的宿主生物和其他微生物物种。它还提供了对决定宿主-微生物相互作用机制的见解，为抗感染治疗提供了新策略。
