Abstract
ABSTRACTThe Streptococcus pneumoniae clone Hungary19A-6 expresses unusually high levels of β-lactam resistance, which is in part due to mutations in the MurM gene, encoding a transferase involved in the synthesis of branched peptidoglycan. Moreover, it contains the allele ciaH232, encoding the histidine kinase CiaH (M. Müller, P. Marx, R. Hakenbeck, and R. Brückner, Microbiology 157:3104–3112, 2011, https://doi.org/10.1099/mic.0.053157-0). High-level penicillin resistance primarily requires the presence of low-affinity (mosaic) penicillin binding protein (PBP) genes, as, for example, in strain Hu17, a closely related member of the Hungary19A-6 lineage. Interestingly, strain Hu15 is β-lactam sensitive due to the absence of mosaic PBPs. This unique situation prompted us to investigate the development of cefotaxime resistance in transformation experiments with genes known to play a role in this phenotype, pbp2x, pbp1a, murM, and ciaH, and penicillin-sensitive recipient strains R6 and Hu15. Characterization of phenotypes, peptidoglycan composition, and CiaR-mediated gene expression revealed several novel aspects of penicillin resistance. The murM gene of strain Hu17 (murMHu17), which is highly similar to murM of Streptococcus mitis, induced morphological changes which were partly reversed by ciaH232. murMHu17 conferred cefotaxime resistance only in the presence of the pbp2x of strain Hu17 (pbp2xHu17). The ciaH232 allele contributed to a remarkable increase in cefotaxime resistance in combination with pbp2xHu17 and pbp1a of strain Hu17 (pbp1aHu17), accompanied by higher levels of expression of CiaR-regulated genes, documenting that ciaH232 responds to PBP1aHu17-mediated changes in cell wall synthesis. Most importantly, the proportion of branched peptides relative to the proportion of linear muropeptides increased in cells containing mosaic PBPs, suggesting an altered enzymatic activity of these proteins.
Highlights
The subsequent introduction of ciaH232 resulted in cells that appeared more normal in size and form (Fig. 7B). These results suggest that the function of the products of both pbp2xHu17 and pbp1aHu17 differs from that of the respective R6 proteins and, that these products affect cell morphology, whereas ciaH232 counteracts this defect
We used the unique situation of penicillin-sensitive strain Hu15, which is of the same sequence type (ST226) as strain Hu17 and which contains both an altered murM and the ciaH232 allele, to show that these two genes were already present before the introduction of altered penicillin binding protein (PBP) genes, i.e., most likely long before the extensive use of -lactam antibiotics triggered the evolution of penicillin-resistant S. pneumoniae (PRSP)
In the early 1990s, the penicillin resistance of pneumococci was considered to be entirely due to altered PBPs [80]
Summary
Introduction ofciaH232 into R6M (R6MC) had no effect on MIC values but remarkably affected cell morphology. R6MC cells were more oval shaped than R6M cells, and the morphology of R6MC cells was similar to that of cells of the parental R6 strain, whereas chain formation was more pronounced in R6MC cells than parental R6 cells (Fig. 6B). R62x and an R6 strain which carried pbp2xHu17 and pbp1aHu17 (R62x1a) individually or in combination to study their impact on resistance in the presence of pbp2xHu17 alone or together with pbp1aHu17. The introduction of murMHu17 into R62x resulted in strain R62xM and conferred increased cefotaxime and oxacillin MICs (Fig. 5). This demonstrates for the first time that pbp2xHu17 alone suffices as a genetic background to reveal that aac.asm.org 8
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