Modulation of antimicrobial efflux pumps of the major facilitator superfamily in <em>Staphylococcus aureus</em>

Staphylococcus aureus has developed resistance to antimicrobials since its first use. The S. aureus major facilitator superfamily (MFS) efflux pump Tet(K) contributes to resistance to tetracyclines. The efflux pump diminishes antibiotic accumulation, and biofilm hampers the diffusion of antibiotics. None of the currently known compounds have been approved as efflux pump inhibitors (EPIs) for clinical use. In the current study, we screened clinically approved drugs for possible Tet(K) efflux pump inhibition. In silico docking followed by in vitro checkerboard assays, we identified five azoles (the fungal ergosterol synthesis inhibitors) showing the putative EPI-like potential with a fractional inhibitory concentration index of ≤0.5, indicating synergism. The functionality of the azoles was confirmed using ethidium bromide (EtBr) accumulation and efflux inhibition assays. In time-kill kinetics, the combination treatment with butoconazole engendered a marked increase in the bactericidal capacity of tetracycline. When assessing the off-target effects of the azoles, we observed no disruption of bacterial membrane permeability and polarization. Finally, the combination of azoles with tetracycline led to a significant eradication of preformed mature biofilms. This study is the primary representation of azoles that can be repurposed as putative Tet(K) EPIs and to reduce biofilm formation at clinically relevant concentrations. IMPORTANCE Staphylococcus aureus use efflux pumps to transport antibiotics out of the cell and thus increase the dosage at which they endure antibiotics. Also, efflux pumps play a role in biofilm formation by the excretion of extracellular matrix molecules. One way to combat these pathogens may be to reduce the activity of efflux pumps and thereby increase pathogen sensitivity to existing antibiotics. We describe the in silico-based screen of clinically approved drugs that identified antifungal azoles inhibiting Tet(K); a pump belongs to the Major Facilitator Superfamily and shows that these compounds bind to and block the activity of the Tet(K) pump. Azoles enhanced the susceptibility of tetracycline against S. aureus and its methicillin-resistant strains. The combination of azoles with tetracycline led to a significant reduction in preformed biofilms. Repurposing of approved drugs may help solve the classical toxicity issues related to efflux pump inhibitors.

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Structural basis of inhibition of a transporter from Staphylococcus aureus, NorC, through a single-domain camelid antibody

Communications Biology ◽

10.1038/s42003-021-02357-x ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Sushant Kumar ◽

Arunabh Athreya ◽

Ashutosh Gulati ◽

Rahul Mony Nair ◽

Ithayaraja Mahendran ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Pathogenic Bacteria ◽

Major Facilitator Superfamily ◽

Single Domain ◽

Fluoroquinolone Resistance ◽

Structural Basis ◽

Antibody Complex ◽

Complementarity Determining Regions ◽

Major Facilitator ◽

Alternating Access

AbstractTransporters play vital roles in acquiring antimicrobial resistance among pathogenic bacteria. In this study, we report the X-ray structure of NorC, a 14-transmembrane major facilitator superfamily member that is implicated in fluoroquinolone resistance in drug-resistant Staphylococcus aureus strains, at a resolution of 3.6 Å. The NorC structure was determined in complex with a single-domain camelid antibody that interacts at the extracellular face of the transporter and stabilizes it in an outward-open conformation. The complementarity determining regions of the antibody enter and block solvent access to the interior of the vestibule, thereby inhibiting alternating-access. NorC specifically interacts with an organic cation, tetraphenylphosphonium, although it does not demonstrate an ability to transport it. The interaction is compromised in the presence of NorC-antibody complex, consequently establishing a strategy to detect and block NorC and related transporters through the use of single-domain camelid antibodies.

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Two Distinct Major Facilitator Superfamily Drug Efflux Pumps Mediate Chloramphenicol Resistance in Streptomyces coelicolor

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00853-09 ◽

2009 ◽

Vol 53 (11) ◽

pp. 4673-4677 ◽

Cited By ~ 24

Author(s):

James J. Vecchione ◽

Blair Alexander ◽

Jason K. Sello

Keyword(s):

Efflux Pump ◽

Streptomyces Coelicolor ◽

Efflux Pumps ◽

Major Facilitator Superfamily ◽

Close Relative ◽

Gram Positive ◽

Antibacterial Drugs ◽

Chloramphenicol Resistance ◽

Drug Activity ◽

Major Facilitator

ABSTRACT Chloramphenicol, florfenicol, and thiamphenicol are used as antibacterial drugs in clinical and veterinary medicine. Two efflux pumps of the major facilitator superfamily encoded by the cmlR1 and cmlR2 genes mediate resistance to these antibiotics in Streptomyces coelicolor, a close relative of Mycobacterium tuberculosis. The transcription of both genes was observed by reverse transcription-PCR. Disruption of cmlR1 decreased the chloramphenicol MIC 1.6-fold, while disruption of cmlR2 lowered the MIC 16-fold. The chloramphenicol MIC of wild-type S. coelicolor decreased fourfold and eightfold in the presence of reserpine and Phe-Arg-β-naphthylamide, respectively. These compounds are known to potentiate the activity of some antibacterial drugs via efflux pump inhibition. While reserpine is known to potentiate drug activity against gram-positive bacteria, this is the first time that Phe-Arg-β-naphthylamide has been shown to potentiate drug activity against a gram-positive bacterium.

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Bacterial Resistance Mechanisms and Inhibitors of Multidrug Efflux Pumps Belonging to the Major Facilitator Superfamily of Solute Transport Systems

Frontiers in Anti-Infective Drug Discovery ◽

10.2174/9781681082912117050006 ◽

2017 ◽

pp. 109-131

Keyword(s):

Solute Transport ◽

Bacterial Resistance ◽

Efflux Pumps ◽

Resistance Mechanisms ◽

Major Facilitator Superfamily ◽

Transport Systems ◽

Multidrug Efflux ◽

Multidrug Efflux Pumps ◽

Major Facilitator

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Functional and Structural Roles of the Major Facilitator Superfamily Bacterial Multidrug Efflux Pumps

Microorganisms ◽

10.3390/microorganisms8020266 ◽

2020 ◽

Vol 8 (2) ◽

pp. 266 ◽

Cited By ~ 5

Author(s):

Sanath Kumar ◽

Manjusha Lekshmi ◽

Ammini Parvathi ◽

Manisha Ojha ◽

Nicholas Wenzel ◽

...

Keyword(s):

Amino Acid ◽

Antimicrobial Agents ◽

Efflux Pumps ◽

Multidrug Resistant ◽

Amino Acid Sequences ◽

Major Facilitator Superfamily ◽

Multidrug Efflux ◽

Ion Translocation ◽

Multidrug Efflux Pumps ◽

Major Facilitator

Pathogenic microorganisms that are multidrug-resistant can pose severe clinical and public health concerns. In particular, bacterial multidrug efflux transporters of the major facilitator superfamily constitute a notable group of drug resistance mechanisms primarily because multidrug-resistant pathogens can become refractory to antimicrobial agents, thus resulting in potentially untreatable bacterial infections. The major facilitator superfamily is composed of thousands of solute transporters that are related in terms of their phylogenetic relationships, primary amino acid sequences, two- and three-dimensional structures, modes of energization (passive and secondary active), and in their mechanisms of solute and ion translocation across the membrane. The major facilitator superfamily is also composed of numerous families and sub-families of homologous transporters that are conserved across all living taxa, from bacteria to humans. Members of this superfamily share several classes of highly conserved amino acid sequence motifs that play essential mechanistic roles during transport. The structural and functional importance of multidrug efflux pumps that belong to the major facilitator family and that are harbored by Gram-negative and -positive bacterial pathogens are considered here.

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The Varied Role of Efflux Pumps of the MFS Family in the Interplay of Bacteria with Animal and Plant Cells

Microorganisms ◽

10.3390/microorganisms7090285 ◽

2019 ◽

Vol 7 (9) ◽

pp. 285 ◽

Cited By ~ 7

Author(s):

Pasqua ◽

Grossi ◽

Zennaro ◽

Fanelli ◽

Micheli ◽

...

Keyword(s):

Regulatory Networks ◽

Efflux Pump ◽

Efflux Pumps ◽

Major Facilitator Superfamily ◽

Host Cells ◽

Multidrug Efflux ◽

Animal Cells ◽

Multidrug Efflux Pumps ◽

Wide Range ◽

Major Facilitator

Efflux pumps represent an important and large group of transporter proteins found in all organisms. The importance of efflux pumps resides in their ability to extrude a wide range of antibiotics, resulting in the emergence of multidrug resistance in many bacteria. Besides antibiotics, multidrug efflux pumps can also extrude a large variety of compounds: Bacterial metabolites, plant-produced compounds, quorum-sensing molecules, and virulence factors. This versatility makes efflux pumps relevant players in interactions not only with other bacteria, but also with plant or animal cells. The multidrug efflux pumps belonging to the major facilitator superfamily (MFS) are widely distributed in microbial genomes and exhibit a large spectrum of substrate specificities. Multidrug MFS efflux pumps are present either as single-component transporters or as tripartite complexes. In this review, we will summarize how the multidrug MFS efflux pumps contribute to the interplay between bacteria and targeted host cells, with emphasis on their role in bacterial virulence, in the colonization of plant and animal host cells and in biofilm formation. We will also address the complexity of these interactions in the light of the underlying regulatory networks required for the effective activation of efflux pump genes.

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Antimicrobial Activity of a Library of Thioxanthones and Their Potential as Efflux Pump Inhibitors

Pharmaceuticals ◽

10.3390/ph14060572 ◽

2021 ◽

Vol 14 (6) ◽

pp. 572

Author(s):

Fernando Durães ◽

Andreia Palmeira ◽

Bárbara Cruz ◽

Joana Freitas-Silva ◽

Nikoletta Szemerédi ◽

...

Keyword(s):

Quorum Sensing ◽

Mammalian Cells ◽

Efflux Pump ◽

Efflux Pumps ◽

Major Facilitator Superfamily ◽

Design And Synthesis ◽

Resistance Nodulation Division ◽

Efflux Pump Inhibitors ◽

Major Facilitator

The overexpression of efflux pumps is one of the causes of multidrug resistance, which leads to the inefficacy of drugs. This plays a pivotal role in antimicrobial resistance, and the most notable pumps are the AcrAB-TolC system (AcrB belongs to the resistance-nodulation-division family) and the NorA, from the major facilitator superfamily. In bacteria, these structures can also favor virulence and adaptation mechanisms, such as quorum-sensing and the formation of biofilm. In this study, the design and synthesis of a library of thioxanthones as potential efflux pump inhibitors are described. The thioxanthone derivatives were investigated for their antibacterial activity and inhibition of efflux pumps, biofilm formation, and quorum-sensing. The compounds were also studied for their potential to interact with P-glycoprotein (P-gp, ABCB1), an efflux pump present in mammalian cells, and for their cytotoxicity in both mouse fibroblasts and human Caco-2 cells. The results concerning the real-time ethidium bromide accumulation may suggest a potential bacterial efflux pump inhibition, which has not yet been reported for thioxanthones. Moreover, in vitro studies in human cells demonstrated a lack of cytotoxicity for concentrations up to 20 µM in Caco-2 cells, with some derivatives also showing potential for P-gp modulation.

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