Perley-Robertson G Evan, Yadav Anuj K, Winogrodzki Judith L, Stubbs Keith A, Mark Brian L, Vocadlo David J
Department of Chemistry, Simon Fraser University , Burnaby, British Columbia V5A 1S6, Canada.
Department of Microbiology, University of Manitoba , Winnipeg, Manitoba R3T 2N2, Canada.
ACS Chem Biol. 2016 Sep 16;11(9):2626-35. doi: 10.1021/acschembio.6b00552. Epub 2016 Aug 5.
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.
可诱导的AmpC β-内酰胺酶可使多种β-内酰胺抗生素失活，并在许多革兰氏阴性菌中产生抗生素耐药性。β-内酰胺抗生素引起的肽聚糖循环紊乱会导致GlcNAc-1,6-脱水MurNAc-肽的积累，这些肽由AmpG转运到细胞质中，在那里它们被加工成AmpC诱导剂。人们对AmpG转运蛋白了解甚少；然而，它们的缺失可恢复对β-内酰胺抗生素的敏感性，这突出表明AmpG是一种潜在的耐药性减弱治疗靶点。我们制备了一种GlcNAc-1,6-脱水MurNAc-荧光团缀合物，并使用活的大肠杆菌原生质球定量分析其被AmpG转运的情况以及竞争性底物对该过程的抑制作用。此外，我们使用这种转运测定法来评估来自铜绿假单胞菌的两种AmpG同源物的功能，结果表明铜绿假单胞菌的AmpG（Pa-AmpG）而非AmpP（Pa-AmpP）可转运这种探针底物。我们通过对Pa-AmpG和Pa-AmpP进行AmpC诱导测定来证实这些结果。这种荧光AmpG探针和基于原生质球的转运测定法将有助于更好地理解肽聚糖循环以及来自主要转运蛋白超家族的通透酶，并可能有助于鉴定对抗AmpC介导的β-内酰胺抗生素耐药性的AmpG拮抗剂。
