Biotinylation as a Tool to Enhance Uptake of Compounds in Gram‐negative Bacteria

Abstract

Gram-negative bacteria pose a major challenge in the development of new antibiotics due to the presence of an extra outer membrane apart from the regular phospholipid plasma membrane. Multidrug efflux pumps exacerbate the problem by pumping out many molecules that do penetrate the membrane barrier. Hence, there is an immense need to develop novel pathways across the cellular envelope so inhibitors can reach their intracellular target. Bacterial nutrient uptake pathway has been exploited to enhance antibiotic uptake through the conjugation of the drug with an essential nutrient. This so-called “Trojan horse” strategy has been explored mostly using the iron uptake system. In this study, we investigated the uptake of a compound conjugated with biotin. Biotin is an essential nutrient for bacteria and many species including E. coli have dedicated biotin uptake transporters. Using fluorescent probe Atto565 as a model compound, we found that biotinylation lead to a significant enhancement of accumulation in E. coli. However, this enhanced accumulation was not affected when large excess of free biotin was present, arguing against the involvement of dedicated biotin transporters. Through quantitative analysis of accumulation in the periplasm and cytoplasm, we found that accumulation was mostly in the periplasmic space. This result was further confirmed by the specific promotion of biotinylated Atto565, but not the parent compound Atto565, when streptavidin was expressed in the periplasm. On further studies, we determined that the enhanced accumulation in the periplasm is due to increased penetration through the outer membrane, not a negative impact on compound efflux. Using gene knockout strains and site directed mutagenesis studies, we showed that the enhanced penetration through outer membrane was via OmpC, a general porin channel present in outer membrane for the uptake of wide variety of small molecules. Thus, we concluded that biotinylation promotes the accumulation of Atto565 to cross the outer membrane of E. coli, making it a better passenger through the OmpC porin.