Abstract

Bacteria, such as Escherichia coli, use multidrug efflux pumps to export toxic substrates through their cell membranes. Upon formation of an efflux pump, the aperture of its outer membrane protein TolC opens and thereby enables the extrusion of substrate molecules. The specialty of TolC is its ability to dock to different transporters, making it a highly versatile export protein. Within this study, the transition between two conformations of TolC that are both available as crystal structures was investigated using all-atom molecular dynamics simulations. To create a partially open conformation from a closed one, the stability of the periplasmic aperture was weakened by a double point mutation at the constricting ring, which removes some salt bridges and hydrogen bonds. These mutants, which showed partial opening in previous experiments, did not spontaneously open during a 20-ns equilibration at physiological values of the KCl solution. Detailed analysis of the constricting ring revealed that the cations of the solvent were able to constitute ionic bonds in place of the removed salt bridges, which inhibited the opening of the aperture in simulations. To remove the ions from these binding positions within the available simulation time, an extra force was applied onto the ions. To keep the effect of this additional force rather flexible, it was applied in form of an artificial external electric field perpendicular to the membrane. Depending on the field direction and the ion concentration, these simulations led to a partial opening. In experiments, this energy barrier for the ions can be overcome by thermal fluctuations on a longer timescale.

Summary

Multidrug efflux pumps adversely affect both the clinical effectiveness of existing antibiotics and the discovery process to find new ones. In this study, we reconstituted and characterized by surface plasmon resonance the assembly of AcrAB-TolC, the archetypal multidrug efflux pump from Escherichia coli. We report that the periplasmic AcrA and the outer membrane channel TolC assemble high-affinity complexes with AcrB transporter independently from each other. Antibiotic novobiocin and MC-207,110 inhibitor bind to the immobilized AcrB but do not affect interactions between components of the complex. In contrast, DARPin inhibits interactions between AcrA and AcrB. Mutational opening of TolC channel decreases stability of interactions and promotes disassembly of the complex. The conformation of the membrane proximal domain of AcrA is critical for the formation of AcrAB-TolC and could be targeted for the development of new inhibitors.

Highlights

? The tri-partite AcrAB-TolC complex is assembled between AcrA and TolC bound to AcrB ? Mutational opening of TolC channel reduces its affinity to both AcrA and AcrB ? DARPin inhibitor disrupts the complex formation between dimeric AcrA and AcrB ? Conformation of the membrane proximal domain of AcrA is critical for the formation of AcrAB-TolC complex

Background

The TolC outer membrane channel is a key component of several multidrug resistance (MDR) efflux pumps driven by H⁺ transport in Escherichia coli. While tolC expression is under the regulation of the EvgA-Gad acid resistance regulon, the role of TolC in growth at low pH and extreme-acid survival is unknown.

Methods and Principal Findings

TolC was required for extreme-acid survival (pH 2) of strain W3110 grown aerobically to stationary phase. A tolC deletion decreased extreme-acid survival (acid resistance) of aerated pH 7.0-grown cells by 10⁵-fold and of pH 5.5-grown cells by 10-fold. The requirement was specific for acid resistance since a tolC defect had no effect on aerobic survival in extreme base (pH 10). TolC was required for expression of glutamate decarboxylase (GadA, GadB), a key component of glutamate-dependent acid resistance (Gad). TolC was also required for maximal exponential growth of E. coli K-12 W3110, in LBK medium buffered at pH 4.5–6.0, but not at pH 6.5–8.5. The TolC growth requirement in moderate acid was independent of Gad. TolC-associated pump components EmrB and MdtB contributed to survival in extreme acid (pH 2), but were not required for growth at pH 5. A mutant lacking the known TolC-associated efflux pumps (acrB, acrD, emrB, emrY, macB, mdtC, mdtF, acrEF) showed no growth defect at acidic pH and a relatively small decrease in extreme-acid survival when pre-grown at pH 5.5.

Conclusions

TolC and proton-driven MDR efflux pump components EmrB and MdtB contribute to E. coli survival in extreme acid and TolC is required for maximal growth rates below pH 6.5. The TolC enhancement of extreme-acid survival includes Gad induction, but TolC-dependent growth rates below pH 6.5 do not involve Gad. That MDR resistance can enhance growth and survival in acid is an important consideration for enteric organisms passing through the acidic stomach.