Structure-Dependent Effects of Lipid Conjugation on Cytosolic Accumulation in Escherichia coli and Mammalian Cells

Lipidation is a widely used strategy to promote membrane permeation, yet whether the factors governing lipid-driven accumulation are shared across mammalian and Gram-negative envelopes remains unresolved. Here, we apply the Chloroalkane Azide-based Membrane Penetration (CHAMP) assay to a systematically designed library of lipid conjugates in both HeLa and E. coli cells. CHAMP, developed by our group, pairs a minimally disruptive azide tag with a cytosolically anchored HaloTag to quantify cytosolic accumulation directly. The two systems show divergent trends: most lipid modifications reduce E. coli cytosolic accumulation, whereas larger, more hydrophobic conjugates, including medium-chain, cyclized, and heteroatom-containing lipids, are preferentially internalized by mammalian cells. By systematically editing the compound scaffold and, separately, perturbing individual layers of the bacterial envelope, we resolve how charge, scaffold composition, and specific envelope barriers shape these patterns. Together, these results indicate that lipidation is a context-dependent permeation strategy rather than a general one. By showing that hydrophobic modifications frequently hinder cytosolic entry into E. coli, this work offers a permeability-based hypothesis for the scarcity of lipidated antibacterials with cytosolic targets, and delineates where lipophilicity-driven optimization is likely to fail in the diderm envelope.