Role of class A penicillin-binding proteins in PBP5-mediated β-lactam resistance in Enterococcus faecalis
Role of class A penicillin-binding proteins in PBP5-mediated β-lactam resistance in Enterococcus faecalis
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DOI：
10.1128/jb.186.5.1221-1228.2004
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发表时间：
2004-03-01
影响因子：
3.2
通讯作者：
Arthur, M
中科院分区：
文献类型：
--
作者：
Arbeloa, A;Segal, H;Arthur, M
肽聚糖聚合复合物含有A类和B类的多模块青霉素结合蛋白（PBP），它们分别将保守的c端转肽酶模块与n端糖基转移酶或形态发生模块相结合。在粪肠球菌中，B类PBP5介导对头孢菌素类β -内酰胺类抗生素（如头孢曲松）的内在耐药。为了鉴定PBP5的糖基转移酶伴侣，我们在粪肠杆菌JH2-2的三个A类PBP基因（ponA、pbpF和pbpZ）中引入了缺失组合。在单或双缺失突变体中，只有JH2-2 DeltaponA DeltapbpF对头孢曲松敏感。从粪肠球菌中异源表达pbpF可恢复头孢曲松耐药性，而编码金黄色葡萄球菌单功能糖基转移酶的mgt则不能。因此，在β -内酰胺存在的情况下，对肽聚糖聚合至关重要的PBP5伙伴形成了粪肠杆菌a类PBPs的一个子集，并且观察到与粪肠杆菌同源物的异源互补。pbpF的定点诱变证实，转肽酶模块的催化丝氨酸残基不需要产生耐药性。虽然这三个A类PBP基因的缺失会导致产生时间的增加和肽聚糖交联的减少，但这三个A类PBP基因对生存能力都不是必需的。由于粪肠杆菌染色体不包含任何额外的糖基转移酶相关基因，这些观察结果表明，三突变体中的聚糖链聚合是由一种新型的糖基转移酶进行的。后一种酶不受莫诺霉素的抑制，因为三个A类PBP基因的缺失导致了对这种糖基转移酶抑制剂的高水平抗性。
Peptidoglycan polymerization complexes contain multimodular penicillin-binding proteins (PBP) of classes A and B that associate a conserved C-terminal transpeptidase module to an N-terminal glycosyltransferase or morphogenesis module, respectively. In Enterococcus faecalis, class B PBP5 mediates intrinsic resistance to the cephalosporin class of beta-lactam antibiotics, such as ceftriaxone. To identify the glycosyltransferase partner(s) of PBP5, combinations of deletions were introduced in all three class A PBP genes of E. faecalis JH2-2 (ponA, pbpF, and pbpZ). Among mutants with single or double deletions, only JH2-2 DeltaponA DeltapbpF was susceptible to ceftriaxone. Ceftriaxone resistance was restored by heterologous expression of pbpF from Enterococcus faecium but not by mgt encoding the monofunctional glycosyltransferase of Staphylococcus aureus. Thus, PBP5 partners essential for peptidoglycan polymerization in the presence of beta-lactams formed a subset of the class A PBPs of E. faecalis, and heterospecific complementation was observed with an ortholog from E.faecium. Site-directed mutagenesis of pbpF confirmed that the catalytic serine residue of the transpeptidase module was not required for resistance. None of the three class A PBP genes was essential for viability, although deletion of the three genes led to an increase in the generation time and to a decrease in peptidoglycan cross-linking. As the E. faecalis chromosome does not contain any additional glycosyltransferase-related genes, these observations indicate that glycan chain polymerization in the triple mutant is performed by a novel type of glycosyltransferase. The latter enzyme was not inhibited by moenomycin, since deletion of the three class A PBP genes led to high-level resistance to this glycosyltransferase inhibitor.