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

Subject Area Medical Microbiology and Mycology, Hygiene, Molecular Infection Biology
Term from 2008 to 2017
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 73111189
 
Final Report Year 2017

Final Report Abstract

Intra- and inter-species gene transfer events that include commensal streptococci are a driving force for the emergence and spread of penicillin resistant Streptococcus pneumoniae (PRSP). This results in mosaic genes involved in the resistance phenotype. In contrast, selection with β-lactams in the laboratory leads to point mutations in relevant genes. Using a unique collection of PRSP, laboratory mutants, and transformants obtained with DNA of high-level β-lactam resistant strains, several new aspects on the mechanism and dynamics of penicillin resistance could be revealed. All high molecular weight PBPs including PBP1b, and MurM, contributed to resistance in transformants obtained with S. mitis B6 DNA. In contrast, PBP2x, 2b and 1a, and surprisingly MurE, a UDP-N-acetylmuramyl tripeptide synthetase, were identified in transformants with S. oralis Uo5 DNA, but PBP2a, PBP1b and MurM were not involved. In piperacillin resistant mutants, the α-GalGlcDAG synthase CpoA is mutated resulting in an altered glycolipid profile; in addition, a two component system and ImdH (inosin-monophosphate dehydrogenase) play a role in some laboratory mutants. PBP2a which is rarely involved in resistance displays a mosaic structure in clinical isolates, whereas in cefotaxime resistant laboratory mutants disruption of pbp2a occurred due to point mutations or internal duplications, confirming fundamental differences between these two settings. The histidine kinase CiaH plays a role in resistance in cefotaxime resistant laboratory mutants and in one high-level PRSP Hu17. Localization studies of the histidine kinase CiaH using gfp-ciaH derivatives revealed that also this protein is at least temporarily located at septal sites. CiaH localization was strongly affected by ciaH232 of S. pneumoniae Hu17 containing a mutation in the external sensor domain, adding another complexity to the process of cell division. Alterations in PBP2x are a prerequisite for the development of high-level PRSP. Comparative analysis of pbp2x from PRSP and commensal streptococci revealed that four pbp2x alleles of penicillin sensitive S. mitis account for most of the diverse sequence blocks in resistant S. pneumoniae, S. pseudopneumoniae, and S. mitis; moreover, S. infantis and S. oralis sequences were observed in rare PRSP. Specific mutational patterns were detected in PBP2x, depending on the parental sequence blocks. Cells containing mosaic PBP genes of the high-level PRSP Hu17 have an altered muropeptide composition in their cell wall, strongly suggesting an altered enzymatic activity of these proteins. High-level penicillin resistance involves a low affinity PBP2b in addition to PBP2x and PBP1a, whereas by itself it confers only small decrease in penicillin susceptibility. One phenotype associated with a low affinity PBP2b, a tolerant response and reduced lysis upon treatment with -lactam antibiotics, was now shown to be not only beneficial during growth in the laboratory, but also in in vivo mouse models, implicating that this resistance determinant is advantageous for the survival in vivo. Mutations distinct from those of clinical isolates occur in PBP2x of two laboratory mutants, C606 and C405, which apparently affect the amount of the protein. The amount of PBP2xC405 could be restored to wild type levels upon deletion of the chaperone/serine protease HtrA which is part of the regulon controlled by the two component system CiaRH. Thus, HtrA targets PBP2xC405 similar to GFP-PBP2x derivatives, but not native PBP2x. Interestingly, mutations observed in two laboratory mutants are located at the surface of the transpeptidase domain and face the C-terminal PASTA domains. Deletions of 3'-terminal sequences affecting the C-terminal PASTA domains of PBP2x documented for the first time that this region is important for β-lactam binding and thus for the enzymatic activity of PBP2x. Moreover, the PASTA domains are crucial for PBP2x localization at the division septum. Positioning of PBP2x at the division site is similar to PBP1a and the serinethreonine kinase StkP, confirming that PBP2x is a key element of the divisome complex.

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