Drastic changes in the peptidoglycan composition of penicillin resistant laboratory mutants ofStreptococcus pneumoniae

Abstract

The penicillin MIC of 2 Streptococcus pneumoniae clinical isolates was increased 100-fold (from 0.02 to 2.0 micromilligrams) and 20-fold (from 0.5 to 10.0 micromilligrams) through gradual exposure of the bacteria to increasing concentrations of penicillin in the laboratory. In both mutants the affinity of all four high molecular mass penicillin binding proteins (PBPs) for penicillin was drastically reduced accompanied by major changes in the composition of peptidoglycan as resolved by HPLC. The ratio of crosslinked to monomeric peptides became virtually inverted in the resistant cell walls with monomers representing two-thirds of the muropeptide species. The proportion of the crosslinked tri-tetra dimer, a major component of the cell wall of the original isolates, decreased to one-third or one-sixth of its normal representation, while the amounts of tripeptide monomers with an alanyl-serine substitution on the lysine epsilon amino group increased by close to a factor of two. The growth rates of both mutants decreased by a factor of approximately two, as compared to the original bacteria.

Biochemical characterization of penicillin-resistant and -sensitive penicillin-binding protein 2x transpeptidase activities of Streptococcus pneumoniae and mechanistic implications in bacterial resistance to beta-lactam antibiotics

“…On the other hand, the cell wall peptidoglycan of penicillinresistant clinical isolates contains mainly abnormal branchedstem peptides (16,40,42,43). These studies suggest that the abnormal branched-stem peptides might be the preferred substrates for penicillin-resistant PBPs in vivo (16,40,42,43). We do indeed show that R-PBP2x differed drastically from SPBP2x kinetically when it was assayed with the thioester S2d as a substrate.…”

“…Studies have shown that the cell wall peptidoglycan composition of penicillin-resistant clinical isolates of S. pneumoniae differs significantly from that of penicillin-sensitive strains (16,40,42,43). The cell wall peptidoglycan of penicillin-sensitive strains contains a majority of monomeric and oligomeric forms of linear-stem peptides (16,40,42,43). On the other hand, the cell wall peptidoglycan of penicillinresistant clinical isolates contains mainly abnormal branchedstem peptides (16,40,42,43).…”

“…However, the underlying biochemical basis of resistance is not well understood. Studies have shown that the cell wall peptidoglycan composition of penicillin-resistant clinical isolates of S. pneumoniae differs significantly from that of penicillin-sensitive strains (16,40,42,43). The cell wall peptidoglycan of penicillin-sensitive strains contains a majority of monomeric and oligomeric forms of linear-stem peptides (16,40,42,43).…”

“…First, penicillin-resistant clinical isolates of S. pneumoniae are often faster growers compared with the penicillin-resistant mutants generated in the laboratory (16,27,28,43). Second, the degree of cross-linking in the peptidoglycan of penicillin-resistant clinical isolates of S. pneumoniae was shown to be similar to that of penicillin-sensitive strains, but a drastic reduction in the cross-linking of the peptidoglycan was observed for penicillin-resistant laboratory mutants (16,40,42,43). Third, penicillin-resistant mutants generated in the laboratory were usually associated with a low level rather than a high level of resistance to the drug (21,22,27,28).…”

“…A substantial amount of evidence shows that the cell wall composition of S. pneumoniae penicillin-resistant isolates differs drastically from that of penicillin-sensitive strains (16,40,42,43). On the basis of this observation, it was proposed that an alteration in the substrate specificity of penicillin-resistant PBPs might have resulted in the heterogeneity of cell wall composition (16,40,42,43).…”