How Does Berberine Hydrochloride Exporter Work?

IMPORTANCE In this work, we evaluated the role of berberine (BBR) as an inhibitor of the MFS efflux pump MdfA from E. coli. We demonstrated that low levels of BBR significantly increased intracellular ciprofloxacin concentrations and restored antibiotic susceptibility of the reporter strain. Molecular dynamics simulations revealed the effect of BBR on the conformational transition of MdfA. Our data suggested that driving forces for MdfA’s conformational transition were affected by BBR and provided evidence for BBR’s extended application as an effective inhibitor of MdfA.

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Infections by Gram-negative pathogens are usually difficult to manage due to the drug export by efflux pumps. With the evolution and horizontal transfer of efflux pumps, there is an urgent need to discover safe and effective efflux pump inhibitors. Here, we found that the natural compound berberine (BBR), a traditional medicine for intestinal infection, is an inhibitor against the major facilitator superfamily (MFS) efflux pump MdfA in Escherichia coli. The impact of BBR on MdfA was evaluated in a recombinant E. coli reporter strain. We demonstrated that low levels of BBR significantly increased intracellular ciprofloxacin concentrations and restored antibiotic susceptibility of the reporter strain. At the same time, we conducted molecular dynamics simulations to investigate the mechanisms of BBR’s effect on MdfA. Our data indicated that BBR can aggregate to the periplasmic and cytoplasmic sides of MdfA in both of its inward and outward conformations. Protein rigidities were affected to different degrees. More importantly, two major driving forces for the conformational transition, salt bridges and hydrophilic interactions with water, were changed by BBR’s aggregation to MdfA, which affected its conformational transition. In summary, our data provide evidence for the extended application of BBR as an efflux pump inhibitor at a clinically meaningful level. We also reveal the mechanisms and provide insights into BBR’s effect on the reciprocal motion of MdfA.

INTRODUCTION

Infectious diseases are considered to be the primary leading causes of death around the world, and nearly 10% of them are caused by bacterial infections (1). However, overuse and misuse of antibiotics has resulted in an explosion of antibiotic resistance, which now is a public health issue of greatest importance (2). Among the factors that contribute to the survival of bacterial pathogens in the presence of antibiotics, efflux pumps play a crucial role by transporting a broad range of substrates out of the cytoplasm (3). Indeed, some previously effective antibiotics used for treating Gram-negative pathogen infections, such as azithromycin, streptomycin, and tetracycline, have become less effective because of efflux pumps (4).

Until now, six families of proton-driven efflux pumps have been identified in Gram-negative pathogens, namely, the resistance-nodulation-cell division (RND) family, multidrug and toxic compound extrusion (MATE) family, major facilitator superfamily (MFS), small multidrug resistance (SMR) family, proteobacterial antimicrobial compound efflux (PACE) family, and p-aminobenzoyl-glutamate transporter (AbgT) family (5,–7). Among these efflux pumps, the MFS efflux pump family has been deduced to contain the largest and most diverse family among all the efflux pump superfamilies in all kingdoms of life (8). For instance, 25% of prokaryotic transporters belong to the MFS family, and there are more than 100 MFS efflux pumps in the human genome (9, 10). In Gram-negative pathogens, some representative MFS efflux pumps have been investigated, such as MdfA from Escherichia coli, KmrA from Klebsiella pneumoniae, and SmvA from Salmonella enterica (11,–13). Studies of antibiotic susceptibilities indicate that these MFS efflux pumps often confer a narrow range of quinolone resistance, which is an important class of drug in antibiotic treatment of severe infections (14). More seriously, increasing evidence suggests that these efflux pumps are being transferred via mobile genetic elements, posing potential concerns for antibiotic therapy clinically (15, 16). Therefore, discovery efforts for effective and safe inhibitors continue.

The MFS efflux pump is embedded in the inner membrane of Gram-negative pathogens (17). The structure of an MFS efflux pump is composed of two six-helix rigid domains forming a central transmembrane channel (8, 18), and a rocker-switch model has been proposed to describe the export process by switching between inward- and outward-facing conformations ( ), which are believed to be the excited and ground states, respectively (19). Based on the mechanism of reciprocal movement, two kinds of inhibitors have been investigated to reduce drug export. First, ionophores such as carbonyl cyanide m-chlorophenyl hydrazone (CCCP) are used to disrupt the proton motive force and thereby inactivate all importers and exporters, including MFS efflux pumps (20). However, these inhibitors are usually toxic to humans. The other kind of inhibitor is MFS specific, such as verapamil and omeprazole, while the high concentrations at which efflux pumps are inhibited (usually over 50 μg/mL) also limit their applications (21).

Plant-derived secondary metabolites are believed to be the main source of natural efflux pump inhibitors (22). Among the identified isoquinoline alkaloid compounds, berberine (BBR) is a promising one due to its safety for humans. Indeed, BBR has been widely used for the treatment of intestinal infections, as it blocks the adherence of pathogens to the inner surface of the intestine (23). Moreover, the relationship between BBR and efflux pumps has also been illustrated. First, as a fluorescent compound which can be excited at 335 nm, BBR is often applied to monitor the efflux behavior of bacteria (24). Second, BBR has been demonstrated to hijack the MFS efflux pump Mdr1p and restore antibiotic susceptibility of Candida albicans, thus showing great potential as a safe efflux pump inhibitor (25). However, the inhibitory mechanisms behind this action are still unclear. First, among the characterized structures of MFS efflux pumps, only a few of them are in outward-facing conformations (ground state). The lack of different outward-facing conformations of MFS efflux pumps has restricted our understanding of the ground state (26). Moreover, export of substrate is accomplished by a large conformational transition of an efflux pump, and substrates will interact with different regions or motifs during the whole cycle. Therefore, molecular docking is insufficient to describe this dynamic process. To better understand the interactions between BBR and MFS efflux pumps, more investigations should be conducted.

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Measuring antibiotic efflux in Gram-negative pathogens is an important way to estimate the level of antibiotic resistance (27). Bacterial efflux can be evaluated at either the population level or single-cell level (28). Conventional determinations of efflux at the population level can be divided into direct measurement of efflux with ethidium bromide and measurement of intracellular accumulation of an efflux substrate (28, 29). These methods have bee demonstrated to be useful in estimating the effects of an inhibitor on certain efflux pumps (30, 31). With the development of chemical biology and microfluidics equipment, efflux in a single-cell or cell-free system can be applied to detect or visualize efflux with modified fluorescent substances (32,–34). For MFS efflux pumps from Gram-negative pathogens, their roles and substrates have been clearly elucidated. These efflux pumps usually confer a narrow range of antibiotic resistance against quinolones (35). Therefore, measuring bacterial sensitivity and intracellular substrate concentration are supposed to evaluate the efficiency of inhibitor against the overexpressed efflux pump.

Here, we apply a representative MFS efflux pump, MdfA, the two conformations of which have been captured, to study BBR’s inhibitory effect in vitro and in silico, and the commercially available pure BBR compound was used. We constructed a recombinant E. coli strain to test its antibiotic susceptibility and intracellular antibiotic concentrations in the presence of BBR. At the same time, conventional molecular dynamics simulations were carried out to demonstrate the impact of BBR on the conformational transition of MdfA. We aimed to provide new insights into the interactions between BBR and MdfA.

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