6 results on '"Wuen Ee Foong"'
Search Results
2. Allosteric drug transport mechanism of multidrug transporter AcrB
- Author
-
Heng-Keat Tam, Wuen Ee Foong, Christine Oswald, Andrea Herrmann, Hui Zeng, and Klaas M. Pos
- Subjects
Science - Abstract
Gram-negative bacteria can display intrinsic antibiotic resistance due to the action of tripartite efflux pumps, which include a H+/drug antiporter component. Here, the authors present a structure-function analysis of antiporter AcrB in intermediate states of the transport cycle, showing novel drug-binding sites and transport pathways.
- Published
- 2021
- Full Text
- View/download PDF
3. Binding of Tetracyclines to Acinetobacter baumannii TetR Involves Two Arginines as Specificity Determinants
- Author
-
Manuela Sumyk, Stephanie Himpich, Wuen Ee Foong, Andrea Herrmann, Klaas M. Pos, and Heng-Keat Tam
- Subjects
transcription repressor ,antibiotic resistance ,Acinetobacter baumannii ,TetR family ,tetracycline transporter ,tetracycline ,Microbiology ,QR1-502 - Abstract
Acinetobacter baumannii is an important nosocomial pathogen that requires thoughtful consideration in the antibiotic prescription strategy due to its multidrug resistant phenotype. Tetracycline antibiotics have recently been re-administered as part of the combination antimicrobial regimens to treat infections caused by A. baumannii. We show that the TetA(G) efflux pump of A. baumannii AYE confers resistance to a variety of tetracyclines including the clinically important antibiotics doxycycline and minocycline, but not to tigecycline. Expression of tetA(G) gene is regulated by the TetR repressor of A. baumannii AYE (AbTetR). Thermal shift binding experiments revealed that AbTetR preferentially binds tetracyclines which carry a O-5H moiety in ring B, whereas tetracyclines with a 7-dimethylamino moiety in ring D are less well-recognized by AbTetR. Confoundingly, tigecycline binds to AbTetR even though it is not transported by TetA(G) efflux pump. Structural analysis of the minocycline-bound AbTetR-Gln116Ala variant suggested that the non-conserved Arg135 interacts with the ring D of minocycline by cation-π interaction, while the invariant Arg104 engages in H-bonding with the O-11H of minocycline. Interestingly, the Arg135Ala variant exhibited a binding preference for tetracyclines with an unmodified ring D. In contrast, the Arg104Ala variant preferred to bind tetracyclines which carry a O-6H moiety in ring C except for tigecycline. We propose that Arg104 and Arg135, which are embedded at the entrance of the AbTetR binding pocket, play important roles in the recognition of tetracyclines, and act as a barrier to prevent the release of tetracycline from its binding pocket upon AbTetR activation. The binding data and crystal structures obtained in this study might provide further insight for the development of new tetracycline antibiotics to evade the specific efflux resistance mechanism deployed by A. baumannii.
- Published
- 2021
- Full Text
- View/download PDF
4. Tigecycline efflux in Acinetobacter baumannii is mediated by TetA in synergy with RND-type efflux transporters
- Author
-
Jochen Wilhelm, Klaas M. Pos, Wuen Ee Foong, and H.K. Tam
- Subjects
Acinetobacter baumannii ,Microbiology (medical) ,Tetracycline ,Microbial Sensitivity Tests ,Tigecycline ,medicine.disease_cause ,Microbiology ,Bacterial Proteins ,Drug Resistance, Multiple, Bacterial ,Escherichia coli ,polycyclic compounds ,medicine ,Pharmacology (medical) ,Pharmacology ,biology ,Membrane transport protein ,Chemistry ,Minocycline ,biochemical phenomena, metabolism, and nutrition ,biology.organism_classification ,Major facilitator superfamily ,Anti-Bacterial Agents ,Infectious Diseases ,biology.protein ,Efflux ,Cell Division ,medicine.drug - Abstract
Objectives To investigate the role of Major Facilitator Superfamily (MFS)-type transporters from Acinetobacter baumannii AYE in tigecycline efflux. Methods Two putative tetracycline transporter genes of A. baumannii AYE (tetA and tetG) were heterologously expressed in Escherichia coli and drug susceptibility assays were conducted with tigecycline and three other tetracycline derivatives. The importance of TetA in tigecycline transport in A. baumannii was determined by complementation of tetA in WT and Resistance Nodulation cell Division (RND) gene knockout strains of A. baumannii ATCC 19606. Gene expression of the MFS-type tetA gene and RND efflux pump genes adeB, adeG and adeJ in A. baumannii AYE in the presence of tigecycline was analysed by quantitative real-time RT–PCR. Results Overproduction of TetA or TetG conferred resistance to doxycycline, minocycline and tetracycline in E. coli. Cells expressing tetA, but not those expressing tetG, conferred resistance to tigecycline, implying that TetA is a determinant for tigecycline transport. A. baumannii WT and RND-knockout strains complemented with plasmid-encoded tetA are significantly less susceptible to tigecycline compared with non-complemented strains. Efflux pump genes tetA and adeG are up-regulated in A. baumannii AYE in the presence of subinhibitory tigecycline concentrations. Conclusions TetA plays an important role in tigecycline efflux of A. baumannii by removing the drug from cytoplasm to periplasm and, subsequently, the RND-type transporters AdeABC and AdeIJK extrude tigecycline across the outer membrane. When challenged with tigecycline, tetA is up-regulated in A. baumannii AYE. Synergy between TetA and the RND-type transporters AdeABC and/or AdeIJK appears necessary for A. baumannii to confer higher tigecycline resistance via drug efflux.
- Published
- 2020
5. Binding and Transport of Carboxylated Drugs by the Multidrug Transporter AcrB
- Author
-
Andrea Herrmann, H.K. Tam, Klaas M. Pos, Wuen-Ee Foong, Giuliano Malloci, Paolo Ruggerone, Viveka Nand Malviya, and Attilio Vittorio Vargiu
- Subjects
Molecular model ,Protein Conformation ,Microbial Sensitivity Tests ,Molecular Dynamics Simulation ,medicine.disease_cause ,Article ,03 medical and health sciences ,0302 clinical medicine ,Structural Biology ,Escherichia coli ,medicine ,Binding site ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,Binding Sites ,Chemistry ,Escherichia coli Proteins ,Transporter ,Periplasmic space ,Models, Theoretical ,Transmembrane domain ,Membrane protein ,Chromatography, Gel ,Biophysics ,Efflux ,Multidrug Resistance-Associated Proteins ,030217 neurology & neurosurgery - Abstract
AcrAB(Z)-TolC is the main drug efflux transporter complex in Escherichia coli. The extrusion of various toxic compounds depends on several drug binding sites within the trimeric AcrB transporter. Membrane-localized carboxylated substrates, such as fusidic acid and hydrophobic β-lactams, access the pump via a groove between the transmembrane helices TM1 and TM2. In this article, the transport route from the initial TM1/TM2 groove binding site toward the deep binding pocket located in the periplasmic part has been addressed via molecular modeling studies followed by functional and structural characterization of several AcrB variants. We propose that membrane-embedded drugs bind initially to the TM1/TM2 groove, are oriented by the AcrB PN2 subdomain, and are subsequently transported via a PN2/PC1 interface pathway directly toward the deep binding pocket. Our work emphasizes the exploitation of multiple transport pathways by AcrB tuned to substrate physicochemical properties related to the polyspecificity of the pump.
- Published
- 2020
6. The chloramphenicol/H+ antiporter CraA of Acinetobacter baumannii AYE reveals a broad substrate specificity
- Author
-
Jan J Crames, Klaas M. Pos, Beate Averhoff, Wuen Ee Foong, and H.K. Tam
- Subjects
0301 basic medicine ,Microbiology (medical) ,Florfenicol ,Acinetobacter baumannii ,Models, Molecular ,Protein Conformation ,030106 microbiology ,medicine.disease_cause ,Antiporters ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Bacterial Proteins ,Drug Resistance, Bacterial ,medicine ,Pharmacology (medical) ,030212 general & internal medicine ,Amino Acid Sequence ,Cloning, Molecular ,Escherichia coli ,Pharmacology ,Dequalinium ,biology ,Membrane transport protein ,Chloramphenicol ,Biological Transport ,Sequence Analysis, DNA ,biology.organism_classification ,Thiamphenicol ,Molecular biology ,Major facilitator superfamily ,Anti-Bacterial Agents ,Infectious Diseases ,chemistry ,biology.protein ,medicine.drug ,Acinetobacter Infections ,Hydrogen - Abstract
Objectives To identify major facilitator superfamily (MFS)-type chloramphenicol transporters of Acinetobacter baumannii AYE, to characterize its substrate specificity and identify CraA substrate and H+ binding sites. Methods Five ORFs predicted to encode chloramphenicol transporters were heterologously expressed in Escherichia coli and their substrate specificity was determined by drug susceptibility assays on solid agar medium. CraA transport properties were determined via whole cell fluorescence experiments using ethidium and dequalinium. ACMA quenching was used to characterize the H+/drug antiport process in everted membrane vesicles. The function of CraA in A. baumannii was determined by drug susceptibility assay using A. baumannii ATCC 19606 ΔcraA. Results CraA, ABAYE0913 and CmlA5 are functionally active when overproduced in E. coli. ABAYE0913 conferred resistance to florfenicol and benzalkonium, CmlA5 conferred resistance to chloramphenicol and thiamphenicol, and craA expression resulted in resistance to chloramphenicol, thiamphenicol, florfenicol, ethidium, dequalinium, chlorhexidine, benzalkonium, mitomycin C and TPP+. Cell expressing craA_E38A showed no resistance to all tested drugs, implying that Glu-38 is involved in the binding of drugs and/or protons. Functional assays indicated that substitution of Asp-46 to Ala resulted in severe susceptibility to cationic drugs, chloramphenicol and thiamphenicol. In contrast, Glu-338 is important for the recognition of chloramphenicol, florfenicol, chlorhexidine and dequalinium. Conclusions This study suggests that CraA has a broad substrate specificity, similar to that of E. coli MdfA. However, due to the presence of three charged residues in the transmembrane region conferring different susceptibility profiles upon substitution to Ala, we postulate that CraA has a different substrate recognition mode compared with MdfA.
- Published
- 2018
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.