title: A Fluorescent Transport Assay Enables Studying AmpG Permeases Involved in Peptidoglycan Recycling and Antibiotic Resistance.
abstract: Inducible AmpC β-lactamases deactivate a broad-spectrum of β-lactam antibiotics and afford antibiotic resistance in many Gram-negative bacteria. The disturbance of peptidoglycan recycling caused by β-lactam antibiotics leads to accumulation of GlcNAc-1,6-anhydroMurNAc-peptides, which are transported by AmpG to the cytoplasm where they are processed into AmpC inducers. AmpG transporters are poorly understood; however, their loss restores susceptibility toward β-lactam antibiotics, highlighting AmpG as a potential target for resistance-attenuating therapeutics. We prepare a GlcNAc-1,6-anhydroMurNAc-fluorophore conjugate and, using live E. coli spheroplasts, quantitatively analyze its transport by AmpG and inhibition of this process by a competing substrate. Further, we use this transport assay to evaluate the function of two AmpG homologues from Pseudomonas aeruginosa and show that P. aeruginosa AmpG (Pa-AmpG) but not AmpP (Pa-AmpP) transports this probe substrate. We corroborate these results by AmpC induction assays with Pa-AmpG and Pa-AmpP. This fluorescent AmpG probe and spheroplast-based transport assay will enable improved understanding of PG recycling and of permeases from the major facilitator superfamily of transport proteins and may aid in identification of AmpG antagonists that combat AmpC-mediated resistance toward β-lactam antibiotics.
authors.affiliation: Department of Chemistry, Simon Fraser University , Burnaby, British Columbia V5A 1S6, Canada.
authors.forename: G Evan
authors.initials: GE
authors.lastname: Perley-Robertson
authors.affiliation: Department of Chemistry, Simon Fraser University , Burnaby, British Columbia V5A 1S6, Canada.
authors.forename: Anuj K
authors.initials: AK
authors.lastname: Yadav
authors.affiliation: Department of Microbiology, University of Manitoba , Winnipeg, Manitoba R3T 2N2, Canada.
authors.forename: Judith L
authors.initials: JL
authors.lastname: Winogrodzki
authors.affiliation: School of Chemistry and Biochemistry, University of Western Australia , Crawley, Western Australia 6009, Australia.
authors.forename: Keith A
authors.initials: KA
authors.lastname: Stubbs
authors.affiliation: Department of Microbiology, University of Manitoba , Winnipeg, Manitoba R3T 2N2, Canada.
authors.forename: Brian L
authors.initials: BL
authors.lastname: Mark
authors.affiliation: Department of Chemistry, Simon Fraser University , Burnaby, British Columbia V5A 1S6, Canada.
authors.forename: David J
authors.initials: DJ
authors.lastname: Vocadlo
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D001692:Biological Transport; D024881:Drug Resistance, Bacterial; D005456:Fluorescent Dyes / Q000378:metabolism*; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000378:metabolism*; D010457:Peptidoglycan / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology*
chemical_list: C084284:AmpG protein, Bacteria; D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D026901:Membrane Transport Proteins; D010457:Peptidoglycan