Dubois Oceane, Zoued Abdelrahim
CIRI, Centre International de Recherche en Infectiologie, CNRS UMR5308, Inserm U1111, Université Claude Bernard Lyon 1, ENS de Lyon, Lyon, France.
Methods Mol Biol. 2025;2953:59-69. doi: 10.1007/978-1-0716-4694-6_4.
Proximity-dependent protein biotinylation is a transformative approach for studying bacterial surface proteomes during infection. By engineering a bacterial autotransporter to expose APEX2 on the bacterial surface, this protocol allows for the precise labeling and characterization of bacterial surface proteins, as well as host-derived bacterial-binding proteins (HBBPs) that interact with the pathogen during infection. The methodology integrates bacterial culturing, biotinylation, immunofluorescence labeling, and protein analysis to provide a comprehensive workflow for identifying key players in host-pathogen interactions. Insights gained from this approach can advance our understanding of bacterial virulence strategies and uncover potential therapeutic targets. Although the focus is on autotransporter and APEX2-based proximity labeling, the method can be adapted to other proximity labeling systems or tailored to specific bacterial species or infection models. By expanding the range of tools available to study microbial pathogenesis, surface-exposed proximity labeling opens new avenues for developing innovative strategies to combat infectious diseases.
邻近依赖性蛋白质生物素化是研究感染过程中细菌表面蛋白质组的一种变革性方法。通过对细菌自转运蛋白进行工程改造，使其在细菌表面暴露APEX2，该方案能够精确标记和表征细菌表面蛋白，以及在感染过程中与病原体相互作用的宿主来源的细菌结合蛋白（HBBPs）。该方法整合了细菌培养、生物素化、免疫荧光标记和蛋白质分析，为识别宿主-病原体相互作用中的关键参与者提供了一个全面的工作流程。通过这种方法获得的见解可以增进我们对细菌毒力策略的理解，并揭示潜在的治疗靶点。尽管重点是基于自转运蛋白和APEX2的邻近标记，但该方法可以适用于其他邻近标记系统，或针对特定细菌物种或感染模型进行调整。通过扩大研究微生物发病机制可用的工具范围，表面暴露邻近标记为开发对抗传染病的创新策略开辟了新途径。
