背景:宿主-微生物相互作用对人类健康和全球生态系统非常重要,因此,阐明复杂的宿主-微生物相互作用和相关的蛋白质表达促使需要开发灵敏和准确的生化技术。当前的蛋白质组学技术从宿主或微生物的角度揭示信息,但不提供相应合作伙伴的数据。此外,同时研究反映宿主和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.这提出了开发用于宿主-微生物相互作用的双物种蛋白质组学方法的需要。
目的:我们的目标是建立一种正向+反向稳定同位素标记细胞培养中的氨基酸(SILAC)蛋白质组学方法,以同时标记和定量宿主和微生物的新表达蛋白,而无需物理分离。用于研究宿主-微生物直接相互作用的机制。
方法:使用秀丽隐杆线虫-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.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。
结果:根据我们的蛋白质组学结果,我们研究了病原体铁清除剂和宿主铁摄取蛋白之间的铁竞争,其中铜绿假单胞菌上调了pyoverdine合成蛋白(PvdA)(5.2313的倍数变化)和分泌的pyoverdine,和C.elegans表达铁蛋白(FTN-2)(3.4057的倍数变化)。利用Galangin实现了铁竞争的针对性干预,一种生姜衍生的植物化学物质,可抑制铜绿假单胞菌中的焦菌素产生和生物膜形成。高良姜-环丙沙星组合疗法可以消除鱼类伤口感染模型中的铜绿假单胞菌生物膜,使动物得以生存。
结论:我们的工作提供了一种新颖的基于SILAC的蛋白质组学方法,该方法可以同时评估宿主和微生物蛋白质组,未来在高等宿主生物和其他微生物物种中的应用。它还提供了对宿主-微生物相互作用机制的见解,为抗感染治疗提供新的策略。
BACKGROUND: 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.
OBJECTIVE: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.