Reynolds Peter E, Ambur O Herman, Casadewall Barbara, Courvalin Patrice
Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK1.
Unité des Agents Antibactériens, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris, Cedex 15, France2.
Microbiology (Reading). 2001 Sep;147(Pt 9):2571-2578. doi: 10.1099/00221287-147-9-2571.
VanD-type Enterococcus faecium BM4339 is constitutively resistant to vancomycin and to low levels of teicoplanin. This strain produces peptidoglycan precursors terminating in D-lactate but, unlike VanA- and VanB-type strains, E. faecium BM4339 has a mutated ddl ligase gene and cannot synthesize D-Ala-D-Ala. Consequently, although it possesses vanX(D) and vanY(D) genes, it should not require an active VanX-type DD-dipeptidase or a VanY-type DD-carboxypeptidase for resistance. The vanY(D) gene contains the signatures of a penicillin-binding protein (PBP) and is believed to encode a penicillin-sensitive DD-carboxypeptidase. The enzyme activity was found to be membrane-bound and inhibited by low concentrations of benzylpenicillin in membrane preparations and in intact bacteria, indicating that the active site was present on the outside surface of the membrane. The 38 kDa protein was revealed as a PBP present in more copies per cell than conventional PBPs and all the protein was accessible to benzylpenicillin added externally, confirming the localization of the active site. A glycopeptide-susceptible strain of E. faecium lacked this PBP, and the membrane-bound DD-carboxypeptidase activity was less than 5% of that of E. faecium BM4339. Although the active site of VanY(D) was external to the membrane, UDP-MurNAc-tetrapeptide was produced internally, probably from UDP-MurNAc-pentadepsipeptide. The presence of benzylpenicillin at low concentrations in the growth medium substantially reduced the amount of tetrapeptide produced, indicating that inhibition of VanY(D) by benzylpenicillin influenced production of peptidoglycan precursors internally. A model to explain these contrasting observations is proposed.
VanD型屎肠球菌BM4339对万古霉素和低水平替考拉宁具有组成型抗性。该菌株产生以D-乳酸为末端的肽聚糖前体，但与VanA和VanB型菌株不同，屎肠球菌BM4339的ddl连接酶基因发生了突变，无法合成D-Ala-D-Ala。因此，尽管它具有vanX(D)和vanY(D)基因，但它的抗性可能不需要活性的VanX型DD-二肽酶或VanY型DD-羧肽酶。vanY(D)基因具有青霉素结合蛋白（PBP）的特征，被认为编码一种对青霉素敏感的DD-羧肽酶。在膜制剂和完整细菌中发现该酶活性是膜结合的，并受到低浓度苄青霉素的抑制，这表明活性位点存在于膜的外表面。38 kDa的蛋白质被鉴定为一种PBP，每个细胞中的拷贝数比传统PBP更多，并且所有蛋白质都能被外部添加的苄青霉素所接触，这证实了活性位点的定位。一株对糖肽敏感的屎肠球菌缺乏这种PBP，其膜结合的DD-羧肽酶活性不到屎肠球菌BM4339的5%。尽管VanY(D)的活性位点在膜外，但UDP-MurNAc-四肽可能是在内部由UDP-MurNAc-五肽脱酰肽产生的。生长培养基中低浓度苄青霉素的存在显著减少了四肽的产生量，这表明苄青霉素对VanY(D)的抑制影响了内部肽聚糖前体的产生。本文提出了一个模型来解释这些相互矛盾的观察结果。
