Competition between protons and substrate for binding to the major facilitator superfamily multidrug/H+ antiporter MdtM

Electrochemical Gradient ◽

Multidrug Efflux ◽

Protein Fluorescence ◽

Ph Values ◽

Major Facilitator ◽

Intrinsic Protein Fluorescence

Abstract Proton electrochemical gradient-driven multidrug efflux activity of representatives of the major facilitator superfamily (MFS) of secondary active transporters contributes to antimicrobial resistance of pathogenic bacteria. Integral to the mechanism of these transporters is a proposed competition between substrate and protons for the binding site of the protein. The current work investigated the competition between protons and antimicrobial substrate for binding to the Escherichia coli MFS multidrug/H+ antiporter MdtM by measuring the quench of intrinsic protein fluorescence upon titration of substrate tetraphenylphosphonium into a solution of purified MdtM over a range of pH values between pH 8.8 and 5.9. The results, which revealed that protons inhibit binding of substrate to MdtM in a competitive manner, are consistent with those reported in a study on the related MFS multidrug/H+ antiporter MdfA and provide further evidence that competition for binding between substrate and protons is a general feature of secondary multidrug efflux.

Novel Chromosomally Encoded Multidrug Efflux Transporter MdeA in Staphylococcus aureus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.3.909-917.2004 ◽

2004 ◽

Vol 48 (3) ◽

pp. 909-917 ◽

Cited By ~ 88

Author(s):

Jianzhong Huang ◽

Paul W. O'Toole ◽

Wei Shen ◽

Heather Amrine-Madsen ◽

Xinhe Jiang ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Pathogenic Bacteria ◽

Multidrug Efflux ◽

Expression Library ◽

Reading Frame ◽

Transmembrane Segments ◽

Antibiotic Compounds ◽

Major Facilitator ◽

Spontaneous Mutants

ABSTRACT Antibiotic efflux is an important mechanism of resistance in pathogenic bacteria. Here we describe the identification and characterization of a novel chromosomally encoded multidrug resistance efflux protein in Staphylococcus aureus, MdeA (multidrug efflux A). MdeA was identified from screening an S. aureus open reading frame expression library for resistance to antibiotic compounds. When overexpressed, MdeA confers resistance on S. aureus to a range of quaternary ammonium compounds and antibiotics, but not fluoroquinolones. MdeA is a 52-kDa protein with 14 predicted transmembrane segments. It belongs to the major facilitator superfamily and is most closely related, among known efflux proteins, to LmrB of Bacillus subtilis and EmrB of Escherichia coli. Overexpression of mdeA in S. aureus reduced ethidium bromide uptake and enhanced its efflux, which could be inhibited by reserpine and abolished by an uncoupler. The mdeA promoter was identified by primer extension. Spontaneous mutants selected for increased resistance to an MdeA substrate had undergone mutations in the promoter for mdeA, and their mdeA transcription levels were increased by as much as 15-fold. The mdeA gene was present in the genomes of all six strains of S. aureus examined. Uncharacterized homologs of MdeA were present elsewhere in the S. aureus genome, but their overexpression did not mediate resistance to the antibacterials tested. However, MdeA homologs were identified in other bacteria, including Bacillus anthracis, some of which were shown to be functional orthologs of MdeA.

Characterization of SotA and SotB, TwoErwinia chrysanthemi Proteins Which Modify Isopropyl-β-d-Thiogalactopyranoside and Lactose Induction of the Escherichia coli lac Promoter

Journal of Bacteriology ◽

10.1128/jb.182.5.1340-1345.2000 ◽

2000 ◽

Vol 182 (5) ◽

pp. 1340-1345 ◽

Cited By ~ 9

Author(s):

Guy Condemine

Keyword(s):

Escherichia Coli ◽

Physiological Role ◽

Intracellular Concentration ◽

Receptor Protein ◽

Multidrug Efflux ◽

Iptg Induction ◽

Lactose Induction ◽

Transporter Family ◽

ABSTRACT The expression, in Escherichia coli, of variants of theErwinia chrysanthemi secretion genes outB andoutS under the Ptac promoter is toxic to the cells. During attempts to clone E. chrysanthemi genes able to suppress this toxicity, I identified two genes, sotA andsotB, whose products are able to reduce the isopropyl-β-d-thiogalactopyranoside (IPTG) induction of the E. coli lac promoter. SotA and SotB belong to two different families of the major facilitator superfamily. SotA is a member of the sugar efflux transporter family, while SotB belongs to the multidrug efflux family. The results presented here suggest that SotA and SotB are sugar efflux pumps. SotA reduces the intracellular concentration of IPTG, lactose, and arabinose. SotB reduces the concentration of IPTG, lactose, and melibiose. Expression ofsotA and sotB is not regulated by their substrates, but sotA is activated by the cyclic AMP receptor protein (CRP), while sotB is repressed by CRP. Lactose is weakly toxic for E. chrysanthemi. This toxicity is increased in a sotB mutant which cannot efficiently efflux lactose. This first evidence for a physiological role of sugar efflux proteins suggests that their function could be to reduce the intracellular concentration of toxic sugars or sugar metabolites.

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 ◽

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.

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 ◽

Transport Systems ◽

Multidrug Efflux ◽

Multidrug Efflux Pumps ◽

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 ◽

Multidrug Efflux ◽

Ion Translocation ◽

Multidrug Efflux Pumps ◽

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.

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 ◽

Host Cells ◽

Multidrug Efflux ◽

Animal Cells ◽

Multidrug Efflux Pumps ◽

Wide Range ◽

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.

Lysophospholipid Flipping across the Escherichia coli Inner Membrane Catalyzed by a Transporter (LplT) Belonging to the Major Facilitator Superfamily

Journal of Biological Chemistry ◽

10.1074/jbc.m414368200 ◽

2005 ◽

Vol 280 (12) ◽

pp. 12028-12034 ◽

Cited By ~ 40

Author(s):

Edgar M. Harvat ◽

Yong-Mei Zhang ◽

Can V. Tran ◽

Zhongge Zhang ◽

Matthew W. Frank ◽

...

Keyword(s):

Escherichia Coli ◽

Inner Membrane ◽

No Single Irreplaceable Acidic Residues in the Escherichia coli Secondary Multidrug Transporter MdfA

Journal of Bacteriology ◽

10.1128/jb.00422-06 ◽

2006 ◽

Vol 188 (15) ◽

pp. 5635-5639 ◽

Cited By ~ 20

Author(s):

Nadejda Sigal ◽

Shahar Molshanski-Mor ◽

Eitan Bibi

Keyword(s):

Escherichia Coli ◽

Critical Role ◽

Energy Coupling ◽

Multidrug Transporter ◽

Coupling Mechanism ◽

Acidic Residues ◽

Major Facilitator ◽

Mfs Transporters ◽

Solute Transporters

ABSTRACT The largest family of solute transporters (major facilitator superfamily [MFS]) includes proton-motive-force-driven secondary transporters. Several characterized MFS transporters utilize essential acidic residues that play a critical role in the energy-coupling mechanism during transport. Surprisingly, we show here that no single acidic residue plays an irreplaceable role in the Escherichia coli secondary multidrug transporter MdfA.