Association of a thr-371 substitution in a conserved amino acid motif of penicillin-binding protein 1A with penicillin resistance of Streptococcus pneumoniae

Abstract

We determined the nucleotide sequence between 1,903 and 3,097 bp of pbp1a, which encodes the transpeptidase domain of PBP 1A, from clinical isolates of penicillin-resistant Streptococcus pneumoniae (PRSP) serotypes 19 (n = 8), 6 (n = 9), 23 (n = 6), and 14 (n = 2) and two penicillin-susceptible S. pneumoniae (PSSP) isolates. These serotyped PRSP strains were isolated predominantly in Japan from 1993 through 1997. The 25 resistant strains were classified into five groups on the basis of the extent of sequence differences. Strains in groups I (n = 5; serotype 6), II (n = 3; serotype 19), and III (n = 12; different serotypes) had sequences highly homologous to the sequence of pbp1a of PRSP strains from South Africa, Spain, and the United States. The group IV strain (n = 1; serotype 14) had unique deletions from or insertions in the sequences. The sequences of group V strains (n = 4; serotypes 6 and 23) had relatively few differences from the sequences of the PSSP strains. For strains (n = 18) for which the threonine at codon 371 (Thr-371) in a conserved STMK motif of PBP 1A was substituted with an alanine or a serine (in addition to having altered pbp2x and pbp2b genes), penicillin MICs were >/= 1.0 microgram/ml. The PBPs 1A of these strains showed decreased affinities for [3H]benzylpenicillin and slightly faster mobilities on sodium dodecyl sulfate-polyacrylamide gels. In contrast, for strains (n = 4) without a substitution at Thr-371 in PBP 1A but with mutations in both pbp2x and pbp2b, penicillin MICs were 0.125 to 0.25 microgram/ml, and the affinities of their PBPs 1A were similar to that of PSSP PBPs 1A. Furthermore, for the Thr-371-substituted strains (n = 3) with altered pbp2x genes but native pbp2b genes, penicillin MICs were 0.125 to 0.25 microgram/ml. These results suggest that amino acid substitution of Thr-371 contributes to the development of penicillin resistance in PRSP strains with altered pbp2x and pbp2b genes.

FIG. 1

Nucleotide sequences of part of the pbp1a genes from representative PRSP strains. The sequence of the pbp1a gene of PSSP R6 is shown at the top. Numbering is based on published data for strain R6 (19). Only nucleotides differing from those in the R6 sequence are shown. Asterisks indicate the deletion of nucleotides.

FIG. 2

Deduced amino acid sequence of part of PBP 1A of the strains listed in Fig. 1. Only amino acids differing from the R6 amino acid sequence are shown. Boxes indicate conserved amino acid sequences.

FIG. 3

Mosaic structure of pbp1a genes from PRSP. Regions of the sequence coding for conserved amino acid sequences are marked. Shading indicates blocks where the sequences differ from that for the PSSP R6 strain (white area) by 6% or more, i.e., blocks a to e. Groups are as follows: group I, strains 5/H31, 8/Z2, 11/Z20, 12/Z21, and variant 22/HA5; group II, strains 17/Z46, 20/B98, and 27/SHA3; group III, strains 2/H26, 4/H29, 13/Z34, 18/B43, 19/B90, 21/B99, 28/H0, 3/H28, 6/KU126, 15/Z12, 16/Z13, and variant 7/KK133; group IV, strains 14/Z42; group V, strains 23/HSB21, 24/TJ25, 26/TJ29, and variant 9/Z17.

FIG. 4

PBP profiles of S. pneumoniae strains. Whole cells (10⁹ cells per ml) suspended in 50 mM phosphate buffer (pH 7.0) were labeled with [³H]benzylpenicillin for 10 min at 30°C and were then subjected to SDS-polyacrylamide gel electrophoresis and fluorography (25). The results of PCR detection of altered pbp2x and pbp2b genes (+, altered; −, not altered) and substitution of Thr-371 in PBP 1A are listed below the gel. Penicillin MICs are as follows: for 10/Z19, 0.031 μg/ml; for 22/HA5, 0.25 μg/ml; for 23/HSB21, 0.125 μg/ml; for 24/TJ25, 0.25 μg/ml; for 26/TJ29, 0.25 μg/ml; for 19/B80, 0.25 μg/ml; for 15/Z12, 0.125 μg/ml; for 7/KK133, 2.0 μg/ml; for 6/KU126, 2.0 μg/ml; for 13/Z34, 2.0 μg/ml; for 27/SHA3, 4.0 μg/ml; for 12/Z21, 2.0 μg/ml. “2A” is 2A/2X.

FIG. 5

Competition assays of penicillin for PBPs 1A of strains 22/HA5 (•) and 5/H31 (○). Strains 22/HA5 without a substitution at Thr-371 (MIC, 0.25 μg/ml) and 5/H31 with a substitution at Thr-371 (MIC, 2.0 μg/ml) were compared. PBPs were detected with [³H]benzylpenicillin after preincubation of the cells with various concentrations of penicillin. Relative PBP density was calculated against the PBP density for the control sample that was not exposed to penicillin.