Gene transfer and the evolution of penicillin resistance: PBPs and non-PBP genes in laboratory mutants and clinical isolates in Streptococcus pneumoniae

肺炎链球菌β -内酰胺耐药性的发展涉及多个参与者。最重要的是这些抗生素的靶酶，青霉素结合蛋白（pbp）。要达到高电阻水平，至少需要改变3个pbp。在特殊的临床分离株中，MurM也发生改变，在存在PBPs时产生耐药性。PBPs是主要的转肽酶，负责肽聚糖亚基的交联，MurM修饰这些亚基并有助于肽间桥的合成。在耐药的实验室突变体中，双组分系统CiaRH通过组氨酸蛋白激酶CiaH的突变被激活。我们最近发现细胞壁相关的丝氨酸蛋白酶/伴侣蛋白HtrA是Cia调控的一部分，靶向GFP-BP2x衍生物。此外，肺炎球菌中主要糖脂的生物合成受到糖基转移酶CpoA突变的影响。所有这些成分对细胞生长和分裂都很重要，其中一些已被证明定位于间隔位点，如PBP2x， PBP1a， HtrA以及这里所示的CiaH。目前的建议与这些成分在β -内酰胺抗性发展过程中的相互作用有关。一部分涉及在利用高水平耐药共生菌的DNA转化肺炎链球菌过程中发生的突变。在第二个系列实验中，在匈牙利肺炎链球菌19a -6的一个特异性耐药克隆中分析了已知参与耐药表型的基因的作用。在该克隆中，除了3个pbp外，murM和ciaH也发生了改变。最后介绍了利用gfp融合蛋白定位PBP2x的实验，包括利用细胞壁蛋白酶HtrA对PBP2x突变体的质量控制。

Multiple players are involved in the development of beta-lactam resistance in Streptococcus pneumoniae. Most importanta are the target enzymes of these antibiotics, the penicillin-binding proteins (PBPs). Alterations in at least three PBPs are required to achieve high resistance level. In particular clinical isolates MurM is also altered, conferring resistance in the presence of PBPs. PBPs are the main transpeptidases responsible for crosslinkage of the peptidoglycan subunits, and MurM modifies these subunits and contributes to the synthesis of interpeptide bridges. In resistant laboratory mutants, the two component system CiaRH is activated via mutations in the histidine protein kinase CiaH. We have recently shown that the cell wall associated serine protease/chaperone HtrA which is part of the Cia regulon targets GFP-BP2x derivatives. Moreover, the biosynthesis of the major glycolipid in pneumococci is affected by mutations in the glycosyltransferase CpoA. All these components are important for cell growth and division, and several of them have been shown to localize to septal sites such as PBP2x, PBP1a, HtrA and as will be shown here also CiaH.The current proposal is related to the interplay of these components during the development of beta-lactam resistance. One part concerns mutations that occurred during transformation of S. pneumoniae using DNA of high level resistant commensal species. In a second series of experiments, the contribution of genes known to be involved in the resistance phenotype is analyzed in one partFP-icular resistant clone of S. pneumoniae Hungary19A-6. In this clone, murM and ciaH are altered in addition to three PBPs. Finally experiments concerning the localization of PBP2x using GFP-fusion proteins are described, including quality control of PBP2x mutants by the cell wall protease HtrA.