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How Do ABC‐F Proteins Mediate Antibiotic Resistance?

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How Do ABC‐F Proteins Mediate Antibiotic Resistance? BSAC ARM 2016 Resolving a 25‐year old controversy: How do ABC‐F proteins mediate antibiotic resistance? Liam Sharkey O’Neill group, FBS, University of Leeds. The ARE ABC-F proteins The mechanism underlying ARE ABC-F protein mediated resistance has been the subject of debate 50S targeted antibiotic ?? Vga(A) Active efflux Vga(A) Ribosomal protection Binding of 50S and translational inhibition Evidence for efflux Decreased accumulation of erythromycin in the presence of msr(A) WT + Streptogramin B WT + erm(C) + msr(A) Ross JI, Eady EA, Cove JH, Cunliffe WJ, Baumberg S, Reynolds E, Ross JI, Cove JH. 2003. Cantón R et al. 2005. Wootton JC. 1990. Int J Antimicrob Agents 22:228–236. J Antimicrob Chemother 55:489‐495 Mol Microbiol 4:1207–1214. Indirect evidence for protection I: ABC-F proteins are not archetypal transporters Class: DNA repair Function: Export Translational control Import Antibiotic resistance Indirect evidence for protection II: Binding sites of antibiotics within the 50S subunit PTC PTC Exit Tunnel Exit Tunnel ARE ABC-F resistance phenotypes correlate with overlapping antibiotic binding sites within the 50S subunit PTC: A‐site Phenicols and oxazolidinones OptrA PTC PTC: A and P ‐site Streptogramin As, lincosamides and pleuromutilins Vga‐type Lsa‐type Exit Tunnel Exit tunnel: Streptogramin Bs, macrolides Msr‐type Searching for ARE ABC-F mediated cross-resistance to additional 50S targeted antibiotics MIC (µg/ml) against S. aureus RN4220 strain Antibacterial Control + msr(A) + lsa(A) + vga(A) compound Virginiamycin M 1 1 64 64 Lincomycin 0.25 0.125 8 2 Retapamulin 0.03125 0.03125 4 0.125 Erythromycin 0.5 128 0.5 0.5 Leucomycin A1 0.25 0.25 0.25 1 Carbomycin 0.5 0.5 1 2 Tylosin 0.5 0.5 0.5 0.5 Spiramycin1111 A possible explanation for Lsa(A) / Vga(A) mediated macrolide resistance P‐Site Tylosin Carbomycin A‐Site Spiramycin Leucomycin A1 Streptogramin A Carbomycin Tylosin Erythromycin Erythromycin Testing the ribosomal protection hypothesis: In vitro transcription/translation (T/T) assay Production and purification of two ARE ABC-F proteins: Preparation of S. aureus S30 extract Lsa(A) and Vga(A) pC19sa luc Lsa(A) Vga(A) ARE ABC‐F Light Antibiotic + Luciferin Luciferase Transcription Translation RNA Addition of Vga(A) rescues in vitro translation from antibiotic inhibition 100 50 1 2 3456 7 8 0 VGM = Virginiamycin M FA = Fusidic acid L K R Sharkey et al. 2016 mBio 7 no. 2 e01975‐15 Addition of Lsa(A) rescues in vitro translation from antibiotic inhibition Virginiamycin M Lincomycin L K R Sharkey et al. 2016 mBio 7 no. 2 e01975‐15 Recapitulation of phenotypes observed in whole cells within the T/T assay (I) Neither Lsa(A) nor Vga(A) protect an E. coli T-T assay + Lsa(A) + Vga(A) vga(A) does not elicit resistance to Streptogramin As when expressed in E. coli Jacquet E et al. 2008. J Biol Chem 283(37): 25332‐25339. L K R Sharkey et al. 2016 VGM = Virginiamycin M mBio 7 no. 2 e01975‐15 Recapitulation of phenotypes observed in whole cells within the T/T assay (II) ATPase activity is essential for Vga(A) function Jacquet E et al. 2008. J Biol Chem 283(37): 25332‐25339. L K R Sharkey et al. 2016 VGM = Virginiamycin M mBio 7 no. 2 e01975‐15 Recapitulation of phenotypes observed in whole cells within the T/T assay (III) Changes within the Vga(A) linker domain alter resistance phenotype Vga(A) K219T Lenart J et al. 2015. Antimicrob Agents Chemother 59(2): 1360‐1364. L K R Sharkey et al. 2016 LNC = Lincomycin mBio 7 no. 2 e01975‐15 RibosomeRibosome binding binding assay assay with radiolabelled with 3H-lincomycin lincomycin Drug not bound Drug bound to ribosome ARE ABC‐F 3H‐ 3H‐ LNC 30S LNC 30S 30S 3H‐ LNC 50S 50S 50S Filter Lsa(A) causes dissociation of 3H-Lincomycin from staphylococcal ribosomes A A & B) Ribosomes pre‐incubated with Lsa(A) before drug added C) Ribosomes pre‐incubated with drug before Lsa(A) added L. K. R. Sharkey et al. 2016 mBio 7 no. 2 e01975‐15 A model for ARE ABC-F mediated resistance: Comparison to EttA Site of linker extension Drug binding sites . Figure adapted from Lenart J et al. 2015. E‐site P‐site A‐site Antimicrob Agents Chemother 59(2): 1360‐1364 Boel G et al. 2014. Nat Struct Mol Biol. 21(2): 143‐151. & Starosta AL et al. 2014. Chen B et al. 2014. Nat Struct Mol Biol. 21(2): 152‐159. FEMS microbiol rev 38(6): 1172‐1201. A model for ARE ABC-F mediated resistance: Comparison to Tetracycline RPPs Gao YG et al. 2009. Science 326(5953): 694‐699 Dönhöfer A et al. 2012. PNAS 16;109(42):16900‐5 Figure adapted from: Starosta AL et al. 2014; FEMS Microbiol Rev 38:1172‐1201 Conclusions • Two phylogenetically distinct ARE ABC‐F proteins 50S targeted antibiotic protect translation in a cell‐free staphylococcal S30 ?? extract Vga(A) • In vitro T/T results mirror in vivo phenotypes • Lsa(A) causes dissociation of lincomycin fromActive the efflux staphylococcal ribosome Vga(A) • Vga(A) and Lsa(A) confer resistance through ribosomal protection Ribosomal protection Binding of 50S • The ARE ABC‐F family confer resistance through and translational ribosomal protection inhibition Acknowledgements Dr Alex O’Neill and Dr Thomas Edwards The O’Neill lab past and present.
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