New Aspects of the Interplay between Penicillin Binding Proteins, murM, and the Two-Component System CiaRH of Penicillin-Resistant Streptococcus pneumoniae Serotype 19A Isolates from Hungary

ABSTRACT The 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.

residues are shown; residues identical to those in strain R6 (bold letters) are indicated by dots. The transpeptidase domain is highlighted in grey.
Alignments were prepared using CLUSTALW.   The peak numbers correspond to those previously published (Bui et al., 2012).

DNA manipulations and construction of mutants
All DNA techniques were carried out as described by Sambrook et al. (Sambrook et al., 1989). E.
coli plasmids were isolated using the QIAprep Spin Miniprep kit (Qiagen). The oligonucleotides used in this study are listed in Table S2 and were obtained from Eurofins Genomics. PCR products were amplified using high-fidelity iProof DNA-polymerase (Bio-Rad) according to the manufacturer's instructions. DNA modifying enzymes were purchased from New England Biolabs or Fermentas (Thermo Scientific), and used as described by the manufacturer. Transfer of the genes pbp2x Hu17 , pbp1a Hu17 , ciaH232, and murM Hu17 to S. pneumoniae R6 and Hu15 and derivatives were verified by DNA sequencing. The letters in subscript indicate the presence of Hu17 genes in the order of the transformation steps; for example, R6 2x1aMC was constructed by introducing successively pbp2x Hu17 , pbp1a Hu17 , murM Hu17 and ciaH232.
Strain R6 M was constructed by a two-step process using the Janus cassette (Sung et al., 2001).
First, the Janus cassette was introduced into murM of the streptomycin resistant S. pneumoniae R6 strR carrying the rpsL41 allele (Salles et al., 1992). Introduction of the Janus cassette confers a Kan R Str S phenotype. In a second step, the Janus cassette was replaced by mosaic murM Hu17 gene resulting in strain R6 M . In detail, two fragments of the MurM gene were amplified by PCR using chromosomal R6 DNA and the oligonucleotide pairs PM263/PM238 and PM246/J2. These two fragments were joined by overlapping PCR with the Janus cassette, which had been amplified using oligonucleotides janus_f/janus_r and chromosomal DNA of CCCOmurM::janus strain (Sauerbier et al., 2012). The resulting fragment murM::kan R -rpsL + (2660 bp) was transformed into R6 strR followed by screening for kanamycin-resistant, streptomycin-sensitive colonies. These transformants contained a non-functional murM. One transformant was subjected to a further transformation using the 2522 bp PCR fragment which was amplified using oligonucleotides PM263/J2 and genomic DNA of strain CCCOmurM Hu17 . Streptomycin-resistant transformants were selected and the presence of murM Hu17 was verified by PCR and sequencing. The strains R6 2xM and R6 2x1aM were constructed as described above.
To introduce the allele ciaH232 into strains R6 M , R6 2x , R6 2xM , R6 2x1a and R6 2x1aM , the Janus replacement method was used as described by Müller et al. (Müller et al., 2011). Briefly, a ciaH::kan R -rpsL + fragment was amplified by PCR using primers ciaH_up_ff/ ciaH_down_rr and genomic DNA of strain RKL161 (Müller et al., 2011). The resulting 3057 bp fragment was used to transform streptomycin resistance derivatives harbouring the rpsL41 allele. The Kan R Str S transformants were selected and correct integration was verified by PCR. One transformant served as recipient for the ciaH232 allele, which was amplified by PCR using oligonucleotides ciaH_up_ff/ ciaH_down_rr and genomic DNA of strain RKL243 (Müller et al., 2011). Streptomycin resistant transformants were selected and the presence of ciaH232 verified by sequencing of the ciaRH region.
To ensure that only the PBP2x gene of Hu17 and not flanking regions were transferred into S.  (Fig. 3).
To generate the strains carrying the mosaic PBP1a Hu17 gene, a PCR fragment covering pbp1a Hu17 was amplified using genomic DNA of strain Hu17 as template and the oligonucleotide pair PM205 and PM180. The 4208 bp PCR product was used to transform the strains followed by cefotaxime selection. The cefotaxime concentrations were slightly above the MIC of recipient strain (R6 2x 0.4 μg/ml; Hu15 2x 0.9 μg/ml). 20 and 12 transformants from strains R6 2x and Hu15 2x , respectively, were screened for presence of low-affinity PBP1a gene after analysis of PBP-profiles as described above. One transformant containing a low affinity PBP1a was chosen for DNA sequencing of pbp1a and used for further experiments (R6 2x1a and Hu15 2x1a ; Fig. 4).