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.