A penicillin resistant Streptococcus pneumoniae in the making : characterizing resistance development and cell fitness after acquiring low-affinity penicillin-binding proteins and a mosaic MurM

Streptococcus pneumoniae is an important human pathogen, causing about 100 millions of cases of disease and 1 – 2 million deaths every year. The rapid evolution and spread of multiresistant strains threaten current antibiotic treatments of pneumococcal infections. A continued exploration of the cellular processes in S. pneumoniae is essential for the development of novel therapeutic treatments against this pathogen. β-lactam resistance in S. pneumoniae is mediated through alterations in the targets of these antibiotics: the penicillin-binding proteins (PBPs). PBPs are important enzymes in the cell division machinery, catalysing the final steps in the biosynthesis of the cell wall. Resistant pneumococci have PBPs with a reduced affinity for these antibiotics. How different lowaffinity PBPs contribute to increased β-lactam resistance was studied in this work by introducing low-affinity versions of PBP2x, PBP1a and PBP2b from the highly penicillin resistant S. oralis Uo5 into the sensitive S. pneumoniae R6 strain. The results showed that that the transfer of low-affinity PBPs from S. oralis Uo5 to S. pneumoniae R6 resulted in an increased level of resistance against different β-lactam antibiotics, but not to the level of the donor strain. The results also showed that low-affinity PBPs most likely are transferred sequential in a specific order into sensitive strains. Transformation of the R6 strain with combinations of the low-affinity pbp2x/1a/2b genes demonstrated that pbp2x was always transferred first. Subsequent addition of either the low-affinity pbp1a or pbp2b genes did not appear to be critical, although pbp1a appeared to be preferred before pbp2b. Introduction of all three low-affinity PBPs in the R6 strain provided the highest level of resistance to β-lactams. Cells expressing low-affinity PBPs often have a cell wall enriched in branched stem peptides. HPLC analyses of the stem peptide composition of a selection of the R6 strain expressing different combinations of low-affinity PBPs revealed that the expression of a low-affinity PBP2b is most likely responsible for this phenotype. However, it was not possible to deduce whether this was affected by the presence of a low-affinity PBP2x and/or PBP1a. In addition to low-affinity PBPs, mutated versions of MurM, an enzyme involved in the synthesis of branched stem peptides in the cell wall, is an important resistance-determining factor in S. pneumoniae. The transfer of mosaic MurM versions to sensitive strains of S. pneumoniae under laboratory conditions does, however, appear to have a toxic effect on the penicillin-sensitive cells, and it has been hypothesized that they are dependent on the presence of low-affinity PBPs. In this work it was shown that transfer of the mosaic MurM from S. oralis Uo5 to the sensitive R6 strain depends on the activity of MurN, which attaches the second L-Ala residue in the branched stem peptides. Surprisingly, the transfer of a mosaic MurM into the R6 strain expressing low-affinity PBPs did not result in increased resistance against β-lactams. Overexpression experiments showed that MurM had a severe toxic effect on the cells in a ΔmurN background