Biotinylation as a tool to enhance the uptake of small molecules in Gram-negative bacteria.

Zhenyu Li,Ankit Pandeya,Hendrik W. van Veen,Olaniyi Alegun,Chad Risko,Yinan Wei,Chamikara Karunasena,Ling Yang

doi:10.1371/journal.pone.0260023

Abstract

Antibiotic resistance is a major public health concern. The shrinking selection of effective antibiotics and lack of new development is making the situation worse. Gram-negative bacteria more specifically pose serious threat because of their double layered cell envelope and effective efflux systems, which is a challenge for drugs to penetrate. One promising approach to breach this barrier is the “Trojan horse strategy”. In this technique, an antibiotic molecule is conjugated with a nutrient molecule that helps the antibiotic to enter the cell through dedicated transporters for the nutrient. Here, we explored the approach using biotin conjugation with a florescent molecule Atto565 to determine if biotinylation enhances accumulation. Biotin is an essential vitamin for bacteria and is obtained through either synthesis or uptake from the environment. We found that biotinylation enhanced accumulation of Atto565 in E. coli. However, the enhancement did not seem to be due to uptake through biotin transporters since the presence of free biotin had no observable impact on accumulation. Accumulated compound was mostly in the periplasm, as determined by cell fractionation studies. This was further confirmed through the observation that expression of streptavidin in the periplasm specifically enhanced the accumulation of biotinylated Atto565. This enhancement was not observed when streptavidin was expressed in the cytoplasm indicating no significant distribution of the compound inside the cytoplasm. Using gene knockout strains, plasmid complementation and mutagenesis studies we demonstrated that biotinylation made the compound a better passenger through OmpC, an outer membrane porin. Density functional theory (DFT)-based evaluation of the three-dimensional geometries showed that biotinylation did not directly stabilize the conformation of the compound to make it favorable for the entry through a pore. Further studies including molecular dynamics simulations are necessary to determine the possible mechanisms of enhanced accumulation of the biotinylated Atto565.

Highlights

The ever growing antibiotic resistance in pathogens has become a serious global health threat and warrants a need for development of new therapeutics [1]
To determine whether biotinylation of compounds would enhance their accumulation in gram-negative bacterial cells, we first measured the accumulation of a florescent compound, Atto565, and its biotinylated form, Atto565-biotin, in E. coli
We observed that Atto565-biotin accumulated at a significantly higher level compared to the parent compound Atto565 (Fig 1)

Summary

Introduction

The ever growing antibiotic resistance in pathogens has become a serious global health threat and warrants a need for development of new therapeutics [1]. Among drug-resistant superbugs, gram-negative bacteria cause more serious concerns because of the dwindling pool of effective therapeutics. Their double layered cell envelope is difficult for antibiotics to penetrate. Together with the phospholipid inner membrane, they make a tough barrier against both hydrophilic and hydrophobic antibiotics. Another major hurdle for antibiotics to cross the cellular envelope is the multiple efflux pumps that effectively transport many antibiotics out of cells [2]. There is an ongoing hunt for novel approaches to breach the barrier to increase the penetration and accumulation efficiency of antibiotics in gram-negative bacteria

Methods

Results

Discussion

Conclusion