Abstract
Among the most significant challenges in antibacterial discovery is achieving delivery of small molecule inhibitors across the bacterial membrane(s) to reach their intracellular targets. It is considered axiomatic that the typical route for antibiotics across the cytoplasmic membrane (CM) involves diffusion directly through the lipid bilayer, though this concept has not translated into actionable information for rationally achieving ingress of inhibitors into bacteria. Here, using antibiotic accumulation measurements in bacteria inactivated for individual or multiple CM transporters, we provide evidence that a diverse range of established antibiotic classes are transported across the CM by hitch-hiking on carrier proteins. The chemical similarity between some antibiotic classes and the native substrate(s) of the importer they share provides a molecular logic for drug transport. Our results indicate that carrier-mediated uptake is a common route of antibiotic entry into bacteria and highlight the value of metabolite mimicry in designing new antibacterial drugs. IMPORTANCE: Increasing antibiotic resistance among pathogenic bacteria is undermining our ability to treat infection, and new antibacterial drugs are urgently needed to address the problem. One of the most significant challenges in antibacterial discovery is achieving delivery of inhibitors across the bacterial membrane(s) to reach their intracellular targets, reflecting the lack of a granular understanding about how antibiotics enter bacteria. Here, we provide evidence that a common route for established antibiotics into bacteria involves uptake by membrane transport proteins. Our findings thereby offer a clear route forward for antibacterial discovery: mimicry of the natural substrates of transporter proteins to achieve carrier-mediated uptake of inhibitors into the bacterial cell.