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