A Transporter Interactome Is Essential for the Acquisition of Antimicrobial Resistance to Antibiotics

<div><p>Awareness of the problem of antimicrobial resistance (AMR) has escalated and drug-resistant infections are named among the most urgent problems facing clinicians today. Our experiments here identify a transporter interactome and portray its essential function in acquisition of antimicrobial resistance. By exposing <i>E</i>. <i>coli</i> cells to consecutive increasing concentrations of the fluoroquinolone norfloxacin we generated in the laboratory highly resistant strains that carry multiple mutations, most of them identical to those identified in clinical isolates. With this experimental paradigm, we show that the MDTs function in a coordinated mode to provide an essential first-line defense mechanism, preventing the drug reaching lethal concentrations, until a number of stable efficient alterations occur that allow survival. Single-component efflux transporters remove the toxic compounds from the cytoplasm to the periplasmic space where TolC-dependent transporters expel them from the cell. We postulate a close interaction between the two types of transporters to prevent rapid leak of the hydrophobic substrates back into the cell. The findings change the prevalent concept that in Gram-negative bacteria a single multidrug transporter, AcrAB-TolC type, is responsible for the resistance. The concept of a functional interactome, the process of identification of its members, the elucidation of the nature of the interactions and its role in cell physiology will change the existing paradigms in the field. We anticipate that our work will have an impact on the present strategy searching for inhibitors of AcrAB-TolC as adjuvants of existing antibiotics and provide novel targets for this urgent undertaking.</p></div

Sequence of events leading to high-level resistance.

<p>In this sequence, the role of the MDTs is to allow fixation of mutations that otherwise would be lost in the next increase in concentration.</p

The role of the transporters in maintenance of high-level resistance.

<p>A. Solid phenotype in the presence of 400 μM norfloxacin reveals a small but distinct difference in the resistance of Δ<i>acrB</i>-EV18 compared to WT-EV18. The suppliers are not required for maintenance of the high-level resistance of WT-EV18 to norfloxacin. B. Solid phenotype in the presence of 30 μM norfloxacin. Nil strains of <i>acrB</i> generated after 5, 10 and 18 days of evolution revealed that it plays an important role in maintenance of high-level resistance at every stage. However, after a short exposure to high norfloxacin (50 μM), resistance increases again. Nil strains were generated by deletion of <i>acrB</i> (WT-EV18-Δ<i>acrB</i>) and of the three suppliers (WT-EV18-Δ3) as described under Experimental Procedures.</p

Expression of TolC dependent transporters increases during evolution in the Δ<i>acrB</i> strain.

<p>The levels of RNA transcripts of the genes <i>acrB</i>, <i>acrD acrF</i>, <i>mdtF</i> and <i>macB</i> were determined in WT-EV18 and Δ<i>acrB</i>-EV18.</p

Experimental design for the evolution of high-level resistance to norfloxacin.

<p><b>A.</b> Cells were grown overnight in the presence of 0.05 μM norfloxacin and cultures that reached at least A₆₀₀ = 1 were further diluted to LB-KP_i containing twice the norfloxacin concentration. The process was continued with a daily two-fold increase in the norfloxacin concentration for 18 days till the concentration (400 μM) could no longer be increased because of solubility problems. Throughout the evolution process, all strains were stored, tested daily using PCR to verify the stability of all knockouts and IC₅₀ values were determined. <b>B top</b>: Time course of the experiment with wild type (red), Δ<i>acrB</i> (blue) and Δ3 (Δ<i>mdfA</i>, Δ<i>mdtM</i> and Δ<i>emrE</i>, green); the mutations appearing during the experiment in the wild type strain are highlighted; <b>B bottom</b>: IC₅₀ values for each strain determined daily. The IC₅₀ values for days 14–18 are not shown because inhibition of growth of WT and Δ<i>acrB</i> strains was too low for accurate determination and they are, at least, 400 μM. <b>C:</b> Mutations identified in the genome of each one of the strains at the end of the evolution process.</p