Dynamics of the CD9 interactome during bacterial infection of epithelial cells by proximity labelling proteomics

Bacterial species utilise different receptors at the cell membrane to adhere to cells. Previously, we demonstrated that interference with CD9, a human tetraspanin, reduces adherence of multiple species of bacteria to cells. CD9 is not a receptor but organises numerous commandeered host proteins at the cell membrane; however, the full interactome has not yet been delineated. Using a CD9 proximity labelling model, a first for CD9, we observed a diverse interactome, with 710 enriched proteins in uninfected cells. Proximal proteins were associated with various cellular processes, including extracellular matrix (ECM)–receptor interactions and tight junctions. Several known bacterial receptors were also detected, including CD44, CD46, and CD147. The interactome was dynamic during infection with two distinct bacterial species, Neisseria meningitidis and Staphylococcus aureus. In total, 12 human proteins were enriched during meningococcal infection, compared to one during staphylococcal infection, demonstrating different host factor requirements during CD9-mediated bacterial adherence. CD44 or CD147 knockdown reduced staphylococcal and meningococcal adherence, respectively, but not vice versa. However, in combination with CD9 interference, no additive effects were observed, demonstrating association of these proteins during infection. We have developed a tool that measures changes within the CD9 interactome, demonstrated CD9 as a universal organiser of bacterial ‘adhesion platforms’, and shown efficacy of a disrupting CD9-derived peptide.

细菌可借助细胞膜上的各类受体实现对宿主细胞的黏附。此前本研究证实，对人类四次跨膜蛋白（tetraspanin）CD9进行干预，可降低多种细菌对宿主细胞的黏附能力。CD9本身并非受体，而是在细胞膜上组织大量被劫持的宿主蛋白；但目前其完整的相互作用组（interactome）尚未被完全解析。本研究首次针对CD9建立邻近标记模型，借此鉴定得到多样化的相互作用组，在未感染的宿主细胞中筛选到710种富集蛋白。这些邻近蛋白参与多种细胞生物学过程，包括细胞外基质（extracellular matrix, ECM）-受体相互作用以及紧密连接调控。同时还检测到多种已知的细菌黏附受体，包括CD44、CD46及CD147。在分别感染两种不同致病菌——脑膜炎奈瑟菌（Neisseria meningitidis）与金黄色葡萄球菌（Staphylococcus aureus）——的过程中，该相互作用组呈现动态变化。总计有12种人类蛋白在脑膜炎奈瑟菌感染阶段发生富集，而金黄色葡萄球菌感染阶段仅1种，这表明CD9介导的细菌黏附过程中，不同致病菌所需的宿主因子存在显著差异。分别敲低CD44或CD147可分别降低金黄色葡萄球菌与脑膜炎奈瑟菌的黏附能力，但反向操作则无此效果。但当将该手段与CD9干预联合使用时，未观察到叠加效应，表明这些蛋白在感染过程中与CD9存在功能关联。本研究开发了一种可检测CD9相互作用组变化的工具，证实CD9是细菌‘黏附平台’的通用组织者，并验证了一种源自CD9的干扰肽的有效性。