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

肺炎链球菌对β-内酰胺类抗生素耐药的形成涉及多个因素。最重要的是这些抗生素的靶酶，青霉素结合蛋白(PBPs)。要达到高阻力水平，至少需要改变三个多溴联苯。特别是，临床分离株的杂音也发生了改变，在多溴联苯存在的情况下产生了耐药性。PBPs是负责交联肽多糖亚基的主要转肽酶，MURM修饰这些亚基并促进肽间桥的合成。在抗药性的实验室突变体中，双组分系统CiaRH通过组氨酸蛋白激酶CiaH的突变而激活。我们最近发现，细胞壁相关丝氨酸蛋白酶/伴侣HtrA是CIA调节子的一部分，以GFP-BP2x衍生物为靶标。此外，肺炎球菌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的生长和分裂都是重要的，其中几个已经被证明定位于隔膜部位，如PBP2x，PBP1a，HtrA和as也将在这里显示CiaH。目前的建议与这些成分在β-内酰胺耐药形成过程中的相互作用有关。其中一部分涉及使用高水平抗性共生物种的DNA转化肺炎链球菌过程中发生的突变。在第二系列实验中，分析了肺炎链球菌Hungary19A-6的部分耐药克隆中已知的与耐药表型有关的基因的贡献。在这个克隆中，除了三个PbP外，Murm和ciaH也发生了改变。最后描述了利用GFP融合蛋白定位PBP2x的实验，包括通过细胞壁蛋白酶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.