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Researchers developed a new assay called CHAMP to systematically compare how lipid modifications affect the ability of molecules to penetrate mammalian cells versus Gram-negative bacteria like E. coli. They found that these cell types follow opposite rules: while larger, more hydrophobic lipid conjugates enhance penetration into mammalian cells, most lipid modifications actually reduce accumulation in bacterial cells. This work demonstrates that lipidation—a common strategy for improving drug delivery—operates through fundamentally different mechanisms depending on the target cell type.
Why it matters
These findings explain why there are so few effective lipid-based antibiotics against Gram-negative bacteria and suggest that traditional drug optimization strategies focused on increasing hydrophobicity may be counterproductive for targeting these pathogens. This could redirect how researchers design new antibiotics to combat drug-resistant Gram-negative infections.
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⚠️ Preprint – Noch nicht peer-reviewed
Dieser Artikel wurde noch nicht von unabhängigen Experten begutachtet. Die Ergebnisse sind vorläufig und sollten mit Vorsicht interpretiert werden.
While lipidation is a widely observed strategy to promote membrane permeation, whether the factors governing lipid-driven accumulation are shared across the divergent membranes of mammalian and Gram-negative cells 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 accumulation, whereas larger, more hydrophobic conjugates, including medium-chain, cyclized, and heteroatom-containing lipids, are preferentially internalized by mammalian cells. Through targeted endogenous and exogenous modifications, we further resolve how charge, scaffold composition, and individual envelope barriers shape these patterns. Together, these results establish that lipidation is a context-dependent permeation principle that fundamentally diverges between mammalian and diderm envelopes. By showing that hydrophobic modifications routinely hinder Gram-negative cytosolic entry, this work explains the scarcity of lipidated Gram-negative antimicrobials, exposes the limits of lipophilicity-driven optimization, and redefines the physicochemical boundaries for penetrating the diderm envelope.
Source: Escherichia coli and Mammalian Cells Follow Divergent Rules for Lipid-Driven Cytosolic Accumulation