Aerolysin enables modular, non-genetic functionalization of living cell surfaces

Methods for installing synthetic functions on living cell surfaces provide powerful approaches for imaging, sensing, and manipulating cell behavior, but many require genetic modification of the target cell or chemical modification of the plasma membrane. Here, we repurpose the glycosylphosphatidylinositol-anchored protein (GPI-AP)-binding toxin aerolysin as a modular chassis for non-genetic cell-surface functionalization. We show that a non-cytotoxic, monomeric aerolysin mutant retains high-affinity and GPI-AP-dependent cell binding when genetically fused to diverse protein cargos. Fluorescent protein-aerolysin fusions robustly label multiple cell types and remain predominantly associated with the cell surface for at least 24 h, in contrast to wheat germ agglutinin, which is extensively internalized. Aerolysin can also be equipped with SpyTag/SpyCatcher to enable modular assembly with independently expressed protein cargos. Importantly, aerolysin supports functional rather than solely optical modification of the cell surface: fusion to the proximity-labeling enzyme APEX2 enables extracellular protein biotinylation, while fusion to HUH endonuclease tags enables covalent attachment of synthetic DNA to living cells. Using this latter architecture, we developed a DNA hairpin sensor that converts cell-surface nuclease activity into a fluorescent signal and distinguishes cells with different levels of extracellular nuclease activity. Together, these results establish non-cytotoxic aerolysin as a genetically encoded, soluble adapter for installing proteins, enzymes, and programmable nucleic acids onto living cells without modification of the target-cell genome.