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Molecular properties of bacterial multidrug transporters.

ABSTRACT: One of the mechanisms that bacteria utilize to evade the toxic effects of antibiotics is the active extrusion of structurally unrelated drugs from the cell. Both intrinsic and acquired multidrug transporters play an important role in antibiotic resistance of several pathogens, including Neisseria gonorrhoeae, Mycobacterium tuberculosis, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Vibrio cholerae. Detailed knowledge of the molecular basis of drug recognition and transport by multidrug transport systems is required for the development of new antibiotics that are not extruded or of inhibitors which block the multidrug transporter and allow traditional antibiotics to be effective. This review gives an extensive overview of the currently known multidrug transporters in bacteria. Based on energetics and structural characteristics, the bacterial multidrug transporters can be classified into five distinct families. Functional reconstitution in liposomes of purified multidrug transport proteins from four families revealed that these proteins are capable of mediating the export of structurally unrelated drugs independent of accessory proteins or cytoplasmic components. On the basis of (i) mutations that affect the activity or the substrate specificity of multidrug transporters and (ii) the three-dimensional structure of the drug-binding domain of the regulatory protein BmrR, the substrate-binding site for cationic drugs is predicted to consist of a hydrophobic pocket with a buried negatively charged residue that interacts electrostatically with the positively charged substrate. The aromatic and hydrophobic amino acid residues which form the drug-binding pocket impose restrictions on the shape and size of the substrates. Kinetic analysis of drug transport by multidrug transporters provided evidence that these proteins may contain multiple substrate-binding sites.

SUBMITTER: Putman M

PROVIDER: S-EPMC99009 | biostudies-literature | 2000 Dec

REPOSITORIES: biostudies-literature

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Molecular properties of bacterial multidrug transporters.

Putman M M van Veen H W HW Konings W N WN

Microbiology and molecular biology reviews : MMBR 20001201 4

One of the mechanisms that bacteria utilize to evade the toxic effects of antibiotics is the active extrusion of structurally unrelated drugs from the cell. Both intrinsic and acquired multidrug transporters play an important role in antibiotic resistance of several pathogens, including Neisseria gonorrhoeae, Mycobacterium tuberculosis, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Vibrio cholerae. Detailed knowledge of the molecular basis of drug recognition and t ...[more]

PMID: 11104814

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Project description:Bacteria use multidrug efflux pumps to export drugs and toxic compounds out of the cell. One of the most important efflux pumps in Escherichia coli is the AcrAB-TolC system. Small regulatory RNAs (sRNAs) are known to be major posttranscriptional regulators that can enhance or repress translation by binding to the 5' untranslated region (UTR) of mRNA targets with the help of a chaperone protein, Hfq. In this study, we investigated the expression of acrA, acrB, and tolC translational fusions using 27 Hfq-dependent sRNAs overexpressed from plasmids. No significant sRNA regulation of acrA or acrB was detected. SdsR (also known as RyeB), an abundant and well-conserved stationary-phase sRNA, was found to repress the expression of tolC, the gene encoding the outer membrane protein of many multidrug resistance efflux pumps. This repression was shown to be by direct base pairing occurring upstream from the ribosomal binding site. SdsR overexpression and its regulation of tolC were found to reduce resistance to novobiocin and crystal violet. Our results suggest that additional targets for SdsR exist that contribute to increased antibiotic sensitivity and reduced biofilm formation. In an effort to identify phenotypes associated with single-copy SdsR and its regulation of tolC, the effect of a deletion of sdsR or mutations in tolC that should block SdsR pairing were investigated using a Biolog phenotypic microarray. However, no significant phenotypes were identified. Therefore, SdsR appears to modulate rather than act as a major regulator of its targets.AcrAB-TolC is a major efflux pump present in E. coli and Gram-negative bacteria used to export toxic compounds; the pump confers resistance to many antibiotics of unrelated classes. In this study, we found that SdsR, a small RNA expressed in stationary phase, repressed the expression of tolC, resulting in increased sensitivity to some antibiotics. This extends the findings of previous studies showing that sRNAs contribute to the regulation of many outer membrane proteins; manipulating or enhancing their action might help in sensitizing bacteria to antibiotics.

Project description:BackgroundThe multidrug resistance (MDR) transporters, P-glycoprotein (P-gp, encoded by ABCB1) and breast cancer resistance protein (BCRP/ABCG2) contribute to the blood-brain barrier (BBB), protecting the brain from drug exposure. The impact of infection on MDR in the developing human BBB remains to be determined. We hypothesized that exposure to bacterial and viral pathogen-associated molecular patterns (PAMPs) modify MDR expression and activity in human fetal brain endothelial cells (hfBECs) isolated from early and mid-gestation brain microvessels.MethodsWe modelled infection (4 h and 24 h) using the bacterial PAMP, lipopolysaccharide (LPS; a toll-like receptor [TLR]-4 ligand) or the viral PAMPs, polyinosinic polycytidylic acid (Poly I:C; TLR-3 ligand) and single-stranded RNA (ssRNA; TLR-7/8 ligand). mRNA expression was assessed by qPCR, whereas protein expression was assessed by Western blot or immunofluorescence. P-gp and BCRP activity was evaluated by Calcein-AM and Chlorin-6 assays.ResultsTLRs-3,4 and 8 were expressed by the isolated hfBECs. Infection mimics induced specific pro-inflammatory responses as well as changes in P-gp/ABCB1 or BCRP/ABCG2 expression (P < 0.05). LPS and ssRNA significantly decreased P-gp activity at 4 and 24 h in early and mid-gestation (P < 0.03-P < 0.001), but significantly increased BCRP activity in hfBECs in a dose-dependent pattern (P < 0.05-P < 0.002). In contrast, Poly-IC significantly decreased P-gp activity after 4 h in early (P < 0.01) and mid gestation (P < 0.04), but not 24 h, and had no overall effect on BCRP activity, though BCRP activity was increased with the highest dose at 24 h in mid-gestation (P < 0.05).ConclusionsInfectious PAMPs significantly modify the expression and function of MDR transporters in hfBECs, though effects are PAMP-, time- and dose-specific. In conclusion, bacterial and viral infections during pregnancy likely have profound effects on exposure of the fetal brain to physiological and pharmacological substrates of P-gp and BCRP, potentially leading to altered trajectories of fetal brain development.

Project description:BACKGROUND:Much of the structure-based mechanistic understandings of the function of SLC6A neurotransmitter transporters emerged from the study of their bacterial LeuT-fold homologs. It has become evident, however, that structural differences such as the long N- and C-termini of the eukaryotic neurotransmitter transporters are involved in an expanded set of functional properties to the eukaryotic transporters. These functional properties are not shared by the bacterial homologs, which lack the structural elements that appeared later in evolution. However, mechanistic insights into some of the measured functional properties of the eukaryotic transporters that have been suggested to involve these structural elements are sparse or merely descriptive. RESULTS:To learn how the structural elements added in evolution enable mechanisms of the eukaryotic transporters in ways not shared with their bacterial LeuT-like homologs, we focused on the human dopamine transporter (hDAT) as a prototype. We present the results of a study employing large-scale molecular dynamics simulations and comparative Markov state model analysis of experimentally determined properties of the wild-type and mutant hDAT constructs. These offer a quantitative outline of mechanisms in which a rich spectrum of interactions of the hDAT N-terminus and C-terminus contribute to the regulation of transporter function (e.g., by phosphorylation) and/or to entirely new phenotypes (e.g., reverse uptake (efflux)) that were added in evolution. CONCLUSIONS:The findings are consistent with the proposal that the size of eukaryotic neurotransmitter transporter termini increased during evolution to enable more functions (e.g., efflux) not shared with the bacterial homologs. The mechanistic explanations for the experimental findings about the modulation of function in DAT, the serotonin transporter, and other eukaryotic transporters reveal separate roles for the distal and proximal segments of the much larger N-terminus in eukaryotic transporters compared to the bacterial ones. The involvement of the proximal and distal segments - such as the role of the proximal segment in sustaining transport in phosphatidylinositol 4,5-bisphosphate-depleted membranes and of the distal segment in modulating efflux - may represent an evolutionary adaptation required for the function of eukaryotic transporters expressed in various cell types of the same organism that differ in the lipid composition and protein complement of their membrane environment.

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Molecular properties of bacterial multidrug transporters.

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Molecular properties of bacterial multidrug transporters.

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