How do ABC‐F proteins mediate antibiotic resistance?
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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.
Transcript of 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 SharkeyO’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)
Vga(A)
Active efflux
Binding of 50S and translational
inhibition
Ribosomalprotection
? ?
Evidence for efflux
Decreased accumulation of erythromycin in the presence of msr(A)
Ross JI, Eady EA, Cove JH, Cunliffe WJ, Baumberg S, Wootton JC. 1990. Mol Microbiol 4:1207–1214.
WT
+ msr(A)
+ Streptogramin B
+ erm(C)
WT
Reynolds E, Ross JI, Cove JH. 2003. Int J Antimicrob Agents 22:228–236.
Cantón R et al. 2005.J Antimicrob Chemother 55:489‐495
Indirect evidence for protection I:ABC-F proteins are not archetypal transporters
Class:
Export ImportFunction:DNA repair
Translational controlAntibiotic resistance
Indirect evidence for protection II:
Binding sites of antibiotics within the 50S subunit
Exit Tunnel
PTCPTC
Exit Tunnel
ARE ABC-F resistance phenotypes correlate with
overlapping antibiotic binding sites within the 50S subunit
Exit Tunnel
PTC
PTC: A‐sitePhenicols and oxazolidinones
OptrA
PTC: A and P ‐siteStreptogramin As, lincosamides and pleuromutilins
Vga‐type
Lsa‐type
Exit tunnel:Streptogramin Bs, macrolides
Msr‐type
Searching for ARE ABC-F mediated cross-resistance to
additional 50S targeted antibiotics
Antibacterial
compound
MIC (µg/ml) against S. aureus RN4220 strain
Control + msr(A) + lsa(A) + vga(A)
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
Spiramycin 1 1 1 1
A possible explanation for Lsa(A) / Vga(A) mediated macrolide resistance
Tylosin
Spiramycin
Erythromycin
Carbomycin
Leucomycin A1
A‐Site
P‐Site
Streptogramin A
Carbomycin
Erythromycin
Tylosin
Testing the ribosomal protection hypothesis:In vitro transcription/translation (T/T) assay
Preparation of S. aureus S30 extractProduction and purification of two ARE ABC-F proteins:Lsa(A) and Vga(A)
Lsa(A) Vga(A)pC19sa luc
Light
RNA
Luciferase
+ Luciferin
Transcription
Translation
Antibiotic
ARE ABC‐F
0
50
100
3 4 5 6 7 81 2
Addition of Vga(A) rescues in vitro translationfrom antibiotic inhibition
VGM = Virginiamycin MFA = Fusidic acid L K R Sharkey et al. 2016
mBio 7 no. 2 e01975‐15
Addition of Lsa(A) rescues in vitro translationfrom antibiotic inhibition
Virginiamycin M Lincomycin
L K R Sharkey et al. 2016mBio 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)
VGM = Virginiamycin M
Jacquet E et al. 2008.J Biol Chem 283(37): 25332‐25339.
vga(A) does not elicit resistance to Streptogramin As when expressed in E. coli
L K R Sharkey et al. 2016mBio 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.
VGM = Virginiamycin M L K R Sharkey et al. 2016mBio 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
L K R Sharkey et al. 2016mBio 7 no. 2 e01975‐15
K219T
LNC = Lincomycin
Vga(A)
Lenart J et al. 2015. Antimicrob Agents Chemother 59(2): 1360‐1364.
Ribosome binding assay with radiolabelled lincomycin
30S
50S
3H‐LNC 30S
50S
3H‐LNC
30S
50S
3H‐LNC
Drug not bound Drug bound to ribosome
Ribosome binding assay with 3H-lincomycin
Filter
ARE ABC‐F
Lsa(A) causes dissociation of 3H-Lincomycin fromstaphylococcal ribosomes
L. K. R. Sharkey et al. 2016mBio 7 no. 2 e01975‐15
A & B) Ribosomes pre‐incubated with Lsa(A) before drug added C) Ribosomes pre‐incubated with drug before Lsa(A) added
A
A model for ARE ABC-F mediated resistance:Comparison to EttA
Figure adapted from Lenart J et al. 2015. Antimicrob Agents Chemother 59(2): 1360‐1364& Starosta AL et al. 2014. FEMS microbiol rev 38(6): 1172‐1201.
Site of linker extension Drug binding sites
E‐site P‐site A‐site.
Boel G et al. 2014. Nat Struct Mol Biol. 21(2): 143‐151.
Chen B et al. 2014. Nat Struct Mol Biol. 21(2): 152‐159.
A model for ARE ABC-F mediated resistance:Comparison to Tetracycline RPPs
Dönhöfer A et al. 2012. PNAS 16;109(42):16900‐5Gao YG et al. 2009. Science 326(5953): 694‐699
Figure adapted from:Starosta AL et al. 2014; FEMS Microbiol Rev 38:1172‐1201
Conclusions
50S targeted antibiotic
Vga(A)
Vga(A)
Active efflux
Binding of 50S and translational
inhibition
Ribosomalprotection
? ?
• Two phylogenetically distinct ARE ABC‐F proteins protect translation in a cell‐free staphylococcal S30 extract
• In vitro T/T results mirror in vivo phenotypes
• Lsa(A) causes dissociation of lincomycin from the staphylococcal ribosome
• Vga(A) and Lsa(A) confer resistance through ribosomal protection
• The ARE ABC‐F family confer resistance through ribosomal protection
Acknowledgements
Dr Alex O’Neill and Dr Thomas Edwards
The O’Neill lab past and present
