Multidrug efflux pumps:structure, function and regulation

Infections arising from multidrug-resistant pathogenic bacteria are spreading rapidly throughout the world and threaten to become untreatable. The origins of resistance are numerous and complex, but one underlying factor is the capacity of bacteria to rapidly export drugs through the intrinsic activity of efflux pumps. In this Review, we describe recent advances that have increased our understanding of the structures and molecular mechanisms of multidrug efflux pumps in bacteria. Clinical and laboratory data indicate that efflux pumps function not only in the drug extrusion process but also in virulence and the adaptive responses that contribute to antimicrobial resistance during infection. The emerging picture of the structure, function and regulation of efflux pumps suggests opportunities for countering their activities

AlphaFold2 predicts the inward‐facing conformation of the multidrug transporter LmrP

As part of the CASP competition, the protein structure prediction algorithm AlphaFold2 generated multiple models of the proton/drug antiporter LmrP. Previous distance restraints from double electron-electron resonance (DEER) spectroscopy, a technique which reports distance distributions between spin labels attached to proteins, suggest that one of the lower-ranked models may have captured a conformation that has so far eluded experimental structure determination

Expression of SMA inhibits SMC proliferation and migration but does not affect activation of MAPKs.

<p>(A) Proliferation of GFP-controls or SMCs overexpressing SMA was determined by MTT assays at various time points after stimulation with 10% FBS. Data (OD₅₆₀ measurements) are presented as mean +/- S.D. of 6 independent experiments *P< 0.001. (B) Cell migration in response to 10% FBS was measured by scratch assays. Hydroxyurea at 5 mmol/L was added to prevent proliferation. Data (percent area covered by cells 17 hours after wounding) are presented as mean +/- S.D. of 6 independent experiments. *P<0.001. Representative micrographs for GFP-controls (co) and SMA-SMCs (SMA) are shown at time 0 right after wounding and at the end of the experiment. (C) Serum-starved cells (GFP-controls and SMA-SMCs) were stimulated with 10% FBS and harvested at the time points indicated. Cell extracts were subjected to Western blot analysis and blots probed with antibodies to recognize the phosphorylated forms of p44/p42 ERK, p38 MAPK or p54/p46 JNK. In all cases, HSC70 was used as a loading control. For each phospho-antibody, a representative result from three independent experiments is shown. The band below phospho-p46 JNK is considered a singularly phosphorylated form that does not shift, or a breakdown product.</p

SMA inhibits serum-induced activation of Rac.

<p>Quiescent cells (GFP-controls or SMCs overexpressing SMA) were stimulated with 10% FBS for 1 and 5 min. Cell lysates were prepared and the amount of GTP-bound Rac (A) and Rho (B) determined by ELISA. All assays were performed in triplicates with two independently prepared cell batches for each condition. Data (OD₄₉₀ measurements) are presented as mean +/- S.D. *P<0.001, n.s. = non-significant. Inserts show Western blots for total Rac and Rho protein in extracts of GFP-controls and SMA-SMCs. HSC-70 was used to confirm equal protein loading.</p

Visualization of actin fibers and focal adhesion sites in SMCs overexpressing SMA.

<p>SMCs were transfected with bicistronic lenti virus expressing GFP alone (co, GFP-control), or in addition VSVG-tagged SMA (SMA). (A) Overexpression of SMA was confirmed by Western blot analysis of cell extracts using antibodies against SMA or VSVG. Staining for β-tubulin was included as loading control. Two independent batches of cells were prepared for controls and SMA overexpression. Note the slight up-shift of the SMA band in SMA-SMC-extracts due to the VSVG-tag. (B) GFP-controls or SMA-SMCs were plated onto fibronectin-coated coverslips and stained with phalloidin for F-actin or with antibody for vinculin. A typical micrograph for each condition including a magnification of an area of interest is shown. Bars equal 50 μm.</p

Expression of a constitutively active Rac1 mutant counteracts the inhibitory effects of SMA on cell migration and growth.

<p>Controls or SMCs overexpressing SMA were transfected with adenovirus to express lacZ (Ad-lacZ) or constitutively active V12rac1 (Ad-rac). All assays were performed in triplicates. (A) Proliferation of cells was determined by MTT assays at day 1 and day 5 after stimulation with 10% FBS. Data (OD₅₆₀ measurements) are presented as mean +/- S.D. (B) Cell migration in response to 10% FBS was measured by scratch assays. Hydroxyurea at 5 mmol/L was added to prevent proliferation. Data (percent area covered by cells 17 hours after wounding) are presented as mean +/- S.D.</p

Knock-down of SMA expression increases the activation of Rac.

<p>SMCs were transfected with non-specific, GC-matched siRNA (control) or siRNA targeting SMA (SMA). (A) Expression of SMA was determined by qPCR measurements and normalized to GAPDH expression. Data (mean + S.D.) from 8 independent transfection experiments are shown. Insert: Western blot analysis of SMA protein expression. The blot was reprobed with beta-actin antibody as a loading control. (B, C) Quiescent controls or cells transfected with SMA-siRNA were stimulated with 10% FBS for 1 and 5 min. Cell lysates were prepared and the amount of GTP-bound Rac (B) and RhoA (C) determined by ELISA following the manufacturer’s instructions. Assays were performed in triplicates with two independent cell batches for each condition. Data (OD490 measurements) are presented as mean +/- S.D. *P<0.001, n.s. = non-significant.</p

Probing the structure of the mechanosensitive channel of small conductance in lipid bilayers with pulsed electron-electron double resonance

Funding: EaStCHEM studentship to E.B. The work was funded by BBSRC grant BB/H017917/1 to JNH, OS & IRB, The Leverhulme Foundation (EM-2012-60\2) and equipment from a Wellcome Trust Capital Award.Mechanosensitive channel proteins are important safety valves against osmotic shock in bacteria, and are involved in sensing touch and sound waves in higher organisms. The mechanosensitive channel of small conductance (MscS) has been extensively studied. Pulsed electron-electron double resonance (PELDOR or DEER) of detergent-solubilized protein confirms that as seen in the crystal structure, the outer ring of transmembrane helices do not pack against the pore- forming helices, creating an apparent void. The relevance of this void to the functional form of MscS in the bilayer is the subject of debate. Here, we report PELDOR measurements of MscS reconstituted into two lipid bilayer systems: nanodiscs and bicelles. The distance measurements from multiple mutants derived from the PELDOR data are consistent with the detergent-solution arrangement of the protein. We conclude, therefore, that the relative positioning of the transmembrane helices is preserved in mimics of the cell bilayer, and that the apparent voids are not an artifact of detergent solution but a property of the protein that will have to be accounted for in any molecular mechanism of gating.Publisher PDFPeer reviewe