Restricted Sequence Variation in Streptococcus pyogenes Penicillin Binding Proteins

β-Lactam antibiotics are the first-line therapeutic option for Streptococcus pyogenes infections. Despite the global high prevalence of S. pyogenes infections and widespread use of β-lactams worldwide, reports of resistance to β-lactam antibiotics, such as penicillin, have been incredibly rare. Recently, β-lactam resistance, as defined by clinical breakpoints, was detected in two clinical S. pyogenes isolates with accompanying mutations in the active site of the penicillin binding protein PBP2x, raising concerns that β-lactam resistance will become more widespread. We screened a global database of S. pyogenes genome sequences to investigate the frequency of PBP mutations, identifying that PBP mutations are uncommon relative to those of Streptococcus pneumoniae. These findings support clinical observations that β-lactam resistance is rare in S. pyogenes and suggest that there are considerable constraints on S. pyogenes PBP sequence variation.

(PBP) PBP2x missense mutation (T553K) at the transpeptidase active site, which was associated with an 8-fold and 3-fold increased MIC to ampicillin and cefotaxime, respectively, compared to levels for closely related isolates without the PBP2x mutation. In contrast to S. pyogenes, reduced susceptibility to ␤-lactams has been widely reported in S. pneumoniae and is strongly associated with sequence variation in PBPs (3,4).
Using GAS genome sequences from global sources, we sought to determine the prevalence of substitutions across the transpeptidase domains of the GAS PBPs (PBP2x, PBP1a, PBP2a, and PBP1b) compared with domains of S. pneumoniae (which shares PBP2x and PBP1a).
Given the similarity between PBP2x of S. pneumoniae and S. pyogenes (73.4% similarity) (Table S1), we mapped the conservation of residues from the alignment of 9,667 S. pyogenes PBP2x sequences onto the crystal structure of S. pneumoniae PBP2x (Fig. 1)  sequence variants of the S. pyogenes PBP2x sequence, with no frameshifts or premature stop codons (Text S1 and Table S3). We found no instances of the T553K substitution in the PBP2x KSGT motif, as reported in the recent S. pyogenes ␤-lactam-resistant isolates (2). Only four S. pyogenes isolate sequences (0.04%) had substitutions within the transpeptidase active-site motifs of PBP2x ( Fig. 1 and Table 1), corresponding to STMK to SAMK and STMK to STIK. These changes may not have a phenotypic effect on penicillin susceptibility, as STIK recently has been reported in a penicillin-susceptible isolate (GASAR0057) (16). Another 84 (0.9%) of the 9,667 genomes contained mutations at one of four amino acid positions associated with increased tolerance to subclinical ␤-lactam MIC identified through a recent population genomics study of emm1, emm28, and emm89 S. pyogenes (Table S3) (19). Furthermore, no amino acid substitutions were found in the active-site motifs of S. pyogenes PBP1a. In comparison, using population data from Li et al. (4), S. pneumoniae had active-site motif variants in 639/2,520 (25.3%) isolates for PBP2x and 445/2,520 (17.7%) for PBP1a (Table 1). A large proportion of S. pneumoniae substitutions mapped to areas near the active site (Fig. S2).
For S. pneumoniae, the number of substitutions across the whole transpeptidase domain of PBPs has been associated with penicillin resistance. Li et al. (4) found that penicillin MICs increased as the total number of divergent (defined as Ͼ10% amino acids different) transpeptidase domains of PBP2x, PBP1a, and PBP2b increased from 0 to 3. For S. pyogenes we used the most common amino acid sequences of PBP2x and PBP1a as our reference and, for S. pneumoniae, a previously defined wild type as the reference (4). There were considerably fewer PBP2x and PBP1a transpeptidase domains with multiple substitutions for S. pyogenes than for S. pneumoniae (Fig. 2). No S. pyogenes strains had sufficient mutations to reach the 10% threshold. For S. pneumoniae, 18.3% (462 of 2,520 strains) and 19.2% (485 of 2,520 strains) contained divergent PBP2x and PBP1a transpeptidase domains, respectively (Fig. 2). This pattern of greater conservation of S. pyogenes PBPs was also observed for PBP1b and PBP2a in S. pyogenes compared to PBP2b in S. pneumoniae (Fig. S3).

DISCUSSION
Expanding on recent findings (19), we found no evidence that mutations are present in the ␤-lactam binding site KSGTAQ motif of PBP2x among 9,667 genetically and geographically diverse S. pyogenes genome sequences. While sporadic mutations were observed in PBP proteins, only four isolates contained mutations in the transpeptidase active sites of PBP2x and PBP1a. A further 84 strains (Ͻ1%) carried PBP2x amino acid variations recently associated with an increased tolerance to subclinical penicillin MIC (19). Although the report of two S. pyogenes isolates with clinical ␤-lactam resistance associated with pbp2x mutations is concerning (2), our findings provide reassurance that PBP mutations leading to clinical resistance are extremely limited, and perhaps unique, occurrences at this stage. Similar observations have been reported within closely related beta-hemolytic streptococci such as Streptococcus agalactiae and Streptococcus dysgalactiae subspecies equisimilis, where PBP mutations conferring reduced penicillin susceptibility or resistance were observed (18,20), but without conclusive evidence of clonal expansion through current population-based surveillance investigations (6,20). We found a high degree of conservation of GAS PBP2x and PBP1a at transpeptidase active sites and across the broader transpeptidase domains. In comparison, PBP2x and PBP1a for S. pneumoniae were far less conserved, suggesting that there are strong evolutionary constraints in these domains for S. pyogenes that is not the case for S. pneumoniae. This may be due to several factors, including the lack of structural plasticity possible in PBP proteins of GAS (S. pyogenes lacks a PBP2b homolog), different ␤-lactam-resistant communities within the environmental niches occupied, lower natural transformation efficiency of GAS relative to that of S. pneumoniae, and a necessity for other chromosomal compensatory mutations to facilitate the maintenance of clinically relevant PBP mutations, as has been suggested for group B streptococci (2). Studies of penicillin-resistant S. pyogenes generated through mutagenesis (21) or serial passage in penicillin-containing medium (22) demonstrated that mutants with raised penicillin MICs appeared to have alterations in PBPs with reduced penicillin affinity (21). Notably, mutants grow more slowly, have aberrant colony morphology compared to that of wild-type strains (21), and are avirulent, with a decrease in M protein production (22). These laboratory experiments, together with the absence of naturally occurring isolates with greater than five amino acid substitutions in PBP2x or PBP1a, strongly suggest that changes to the PBPs are associated with a significant fitness cost. However, as subclinical low-level ␤-lactam resistance theoretically could confer biological advantages to S. pyogenes carriage, maintaining vigilance through population-based S. pyogenes surveillance for PBP variants is encouraged (19).
To investigate the inferred crystal structure location of S. pyogenes PBP2x mutations relative to that of the S. pneumoniae orthologue, S. pyogenes PBP2x sequence variations were plotted onto the S. pneumoniae PBP2x crystal structure bound to oxacillin (PDB entry 5OIZ) (29). Sequence conservation, as determined by the frequency (for S. pyogenes) and percentage (for S. pneumoniae) of variant amino acids compared to the consensus, was rendered onto the PBP2x crystal structure using UCSF Chimera (30).
We defined the PBP2x and PBP1a transpeptidase regions as those used in an assessment of 2,520 invasive S. pneumoniae isolates by Li et al. (4) and determined and plotted the number of pairwise amino acid differences within these regions using Distances Matrix in Geneious Prime (28) and ggplot2 in R version 3.6.1 (31). Similarly, we also assessed the conservation of PBP1b and PBP2a proteins for the 9,667 S. pyogenes genomes and the transpeptidase region of PBP2b for S. pneumoniae.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. TEXT S1, TXT file, 0.   The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.