A PBP2x from a Clinical Isolate of Streptococcus pneumoniae Exhibits an Alternative Mechanism for Reduction of Susceptibility to β-Lactam Antibiotics

Abstract

The human pathogen Streptococcus pneumoniae is one of the main causative agents of respiratory tract infections. At present, clinical isolates of S. pneumoniae often exhibit decreased susceptibility toward ␤-lactams, a phenomenon linked to multiple mutations within the penicillin-binding proteins (PBPs). PBP2x, one of the six PBPs of S. pneumoniae, is the first target to be modified under antibiotic pressure. By comparing 89 S. pneumoniae PBP2x sequences from clinical and public data bases, we have identified one major group of sequences from drug-sensitive strains as well as two distinct groups from drug-resistant strains. The first group includes proteins that display high similarity to PBP2x from the well characterized resistant strain Sp328. The second group includes sequences in which a signature mutation, Q552E, is found adjacent to the third catalytic motif. In this work, a PBP2x from a representative strain from the latter group (S. pneumoniae 5259) was biochemically and structurally characterized. Phenotypical analyses of transformed pneumococci show that the Q552E substitution is responsible for most of the reduction of strain susceptibility toward ␤-lactams. The crystal structure of 5259-PBP2x reveals a change in polarity and charge distribution around the active site cavity, as well as rearrangement of strand ␤3, emulating structural changes observed for other PBPs that confer drug resistance to Gram-positive pathogens. Interestingly, the active site of 5259-PBP2x is in closed conformation, whereas that of Sp328-PBP2x is open. Consequently, S. pneumoniae has evolved to employ the same protein in two distinct mechanisms of antibiotic resistance.

Identical Penicillin-Binding Domains in Penicillin-Binding Proteins of Streptococcus pneumoniae Clinical Isolates with Different Levels of β-Lactam Resistance

“…Sequence type B1 lacked the T338A substitution but was characterized by the Q552E mutation, which typifies a second family of sequences. Based on the crystal structure of PBP 2x from isolate 5259, it has been proposed that the mechanism of affinity reduction for ␤-lactams in PBP 2x sequences that have the Q552E substitution is distinct from that of sequences with a mutation in position 338 (20,25). The T338A substitution is found in the three sequences of group C, which differed from the R6 PBP 2x penicillin-binding domain by 39 to 41 mutations.…”

An Important Site in PBP2x of Penicillin-Resistant Clinical Isolates of Streptococcus pneumoniae : Mutational Analysis of Thr338

“…3) (38), strongly suggesting the presence of compensatory mutations in the mosaic PBP2x. In this context it would be interesting to compare the structure of PBP2x laboratory mutants with the known structures of mosaic PBP2x (13,50).…”

Evidence from Artificial Septal Targeting and Site-Directed Mutagenesis that Residues in the Extracytoplasmic β Domain of DivIB Mediate Its Interaction with the Divisomal Transpeptidase PBP 2B

“…5C we have used the crystal structure of PBP2x, an ortholog from S. pneumoniae (6,26,27), as a proxy for the structure of PBP 2B. The extracytoplasmic regions of these two transpeptidases are 33% identical, and the sequence alignment extends over all the entire length of PBP2x; thus, the PBP2x structure is likely a good surrogate for that of PBP 2B.…”