Peptidoglycan Cross-Linking in Glycopeptide-Resistant Actinomycetales

Abstract

ABSTRACTSynthesis of peptidoglycan precursors ending ind-lactate (d-Lac) is thought to be responsible for glycopeptide resistance in members of the orderActinomycetalesthat produce these drugs and in related soil bacteria. More recently, the peptidoglycan of several members of the orderActinomycetaleswas shown to be cross-linked byl,d-transpeptidases that use tetrapeptide acyl donors devoid of the target of glycopeptides. To evaluate the contribution of these resistance mechanisms, we have determined the peptidoglycan structure ofStreptomyces coelicolorA(3)2, which harbors avanHAXgene cluster for the production of precursors ending ind-Lac, andNonomuraeasp. strain ATCC 39727, which is devoid ofvanHAXand produces the glycopeptide A40296. Vancomycin retained residual activity againstS. coelicolorA(3)2 despite efficient incorporation ofd-Lac into cytoplasmic precursors. This was due to ad,d-transpeptidase-catalyzed reaction that generated a stem pentapeptide recognized by glycopeptides by the exchange ofd-Lac ford-Ala and Gly. The contribution ofl,d-transpeptidases to resistance was limited by the supply of tetrapeptide acyl donors, which are essential for the formation of peptidoglycan cross-links by these enzymes. In the absence of a cytoplasmic metallo-d,d-carboxypeptidase, the tetrapeptide substrate was generated by hydrolysis of the C-terminald-Lac residue of the stem pentadepsipeptide in the periplasm in competition with the exchange reaction catalyzed byd,d-transpeptidases. InNonomuraeasp. strain ATCC 39727, the contribution ofl,d-transpeptidases to glycopeptide resistance was limited by the incomplete conversion of pentapeptides into tetrapeptides despite the production of a cytoplasmic metallo-d,d-carboxypeptidase. Since the level of drug production exceeds the level of resistance, we propose thatl,d-transpeptidases merely act as a tolerance mechanism in this bacterium.