Antibiotic Capture by Bacterial Lipocalins Uncovers an Extracellular Mechanism of Intrinsic Antibiotic Resistance

Miguel A Valvano,Omar M El-Halfawy,Slade A Loutet,Joanna B Goldberg,Rebecca J Ingram,Michael E P Murphy,Javier Klett,Sonsoles Martín-Santamaría

doi:10.1128/mbio.00225-17

Abstract

ABSTRACTThe potential for microbes to overcome antibiotics of different classes before they reach bacterial cells is largely unexplored. Here we show that a soluble bacterial lipocalin produced by Burkholderia cenocepacia upon exposure to sublethal antibiotic concentrations increases resistance to diverse antibiotics in vitro and in vivo. These phenotypes were recapitulated by heterologous expression in B. cenocepacia of lipocalin genes from Pseudomonas aeruginosa, Mycobacterium tuberculosis, and methicillin-resistant Staphylococcus aureus. Purified lipocalin bound different classes of bactericidal antibiotics and contributed to bacterial survival in vivo. Experimental and X-ray crystal structure-guided computational studies revealed that lipocalins counteract antibiotic action by capturing antibiotics in the extracellular space. We also demonstrated that fat-soluble vitamins prevent antibiotic capture by binding bacterial lipocalin with higher affinity than antibiotics. Therefore, bacterial lipocalins contribute to antimicrobial resistance by capturing diverse antibiotics in the extracellular space at the site of infection, which can be counteracted by known vitamins.

Highlights

The potential for microbes to overcome antibiotics of different classes before they reach bacterial cells is largely unexplored
BcnA is a secreted bacterial lipocalin required for full resistance of B. cenocepacia to different classes of antibiotics
We investigated the role of B. cenocepacia BcnA (BCAL3311) and BcnB (BCAL3310) by constructing individual deletion mutants in their corresponding genes and assessing bacterial susceptibility to model bactericidal antibiotics representing different classes, including rifamycins, fluoroquinolones (DNA replication inhibitors), several ␤-lactams, and cationic antimicrobial peptides

Summary

Introduction

The potential for microbes to overcome antibiotics of different classes before they reach bacterial cells is largely unexplored. We show that a soluble bacterial lipocalin produced by Burkholderia cenocepacia upon exposure to sublethal antibiotic concentrations increases resistance to diverse antibiotics in vitro and in vivo. These phenotypes were recapitulated by heterologous expression in B. cenocepacia of lipocalin genes from Pseudomonas aeruginosa, Mycobacterium tuberculosis, and methicillin-resistant Staphylococcus aureus. We discovered a previously unrecognized mode of general bacterial antibiotic resistance operating in the extracellular space, which depends on bacterial protein molecules called lipocalins These molecules are highly conserved in most bacteria and have the ability to capture different classes of antibiotics outside bacterial cells. This finding provides a clinically applicable strategy whereby known vitamins could become antibiotic adjuvants by increasing the concentration of free antibiotics in the proximity of bacterial cells, thereby boosting their microbicidal activity

Methods

Results

Conclusion