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Compensatory evolution of pbp mutations restores the fitness cost imposed by β-lactam resistance in Streptococcus pneumoniae.

Albarracín Orio AG, Piñas GE, Cortes PR, Cian MB, Echenique J - PLoS Pathog. (2011)

Bottom Line: Thus, these compensatory combinations of pbp mutant alleles resulted in an increase in the level and spectrum of β-lactam resistance.The clinical origin of the pbp mutations suggests that this intergenic compensatory process is involved in the persistence of β-lactam resistance among circulating strains.We propose that this compensatory mechanism is relevant for β-lactam resistance evolution in Streptococcus pneumoniae.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica Clínica - CIBICI (CONICET), Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.

ABSTRACT
The prevalence of antibiotic resistance genes in pathogenic bacteria is a major challenge to treating many infectious diseases. The spread of these genes is driven by the strong selection imposed by the use of antibacterial drugs. However, in the absence of drug selection, antibiotic resistance genes impose a fitness cost, which can be ameliorated by compensatory mutations. In Streptococcus pneumoniae, β-lactam resistance is caused by mutations in three penicillin-binding proteins, PBP1a, PBP2x, and PBP2b, all of which are implicated in cell wall synthesis and the cell division cycle. We found that the fitness cost and cell division defects conferred by pbp2b mutations (as determined by fitness competitive assays in vitro and in vivo and fluorescence microscopy) were fully compensated by the acquisition of pbp2x and pbp1a mutations, apparently by means of an increased stability and a consequent mislocalization of these protein mutants. Thus, these compensatory combinations of pbp mutant alleles resulted in an increase in the level and spectrum of β-lactam resistance. This report describes a direct correlation between antibiotic resistance increase and fitness cost compensation, both caused by the same gene mutations acquired by horizontal transfer. The clinical origin of the pbp mutations suggests that this intergenic compensatory process is involved in the persistence of β-lactam resistance among circulating strains. We propose that this compensatory mechanism is relevant for β-lactam resistance evolution in Streptococcus pneumoniae.

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Stability in vivo of wild-type and mutant PBPs.Strains Cp1015 (Panel A), Cp1015 pbp2b28 (Panel B) and Cp1015 pbp2b28pbp2x28 pbp1a28 (Panel C) expressing HA-tagged PBPs were grown in Todd Hewitt broth with 0.5% Yeast Extract to an OD600 nm of 0.2 prior to the addition of kanamycin (500 µg/ml) to inhibit protein synthesis. At the time intervals indicated, aliquots were withdrawn and analyzed by SDS-PAGE followed by immunoblotting with an anti-HA monoclonal antibody (see Material and Methods).
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ppat-1002000-g005: Stability in vivo of wild-type and mutant PBPs.Strains Cp1015 (Panel A), Cp1015 pbp2b28 (Panel B) and Cp1015 pbp2b28pbp2x28 pbp1a28 (Panel C) expressing HA-tagged PBPs were grown in Todd Hewitt broth with 0.5% Yeast Extract to an OD600 nm of 0.2 prior to the addition of kanamycin (500 µg/ml) to inhibit protein synthesis. At the time intervals indicated, aliquots were withdrawn and analyzed by SDS-PAGE followed by immunoblotting with an anti-HA monoclonal antibody (see Material and Methods).

Mentions: To investigate the putative cause of these effects on cell morphology and fitness, we analyzed the stability of the proteins encoded by these pbp mutated genes using immunoblotting and inhibiting the protein synthesis by the addition of kanamycin. For these assays, we constructed C-terminal HA tagged PBPs to permit the detection of the proteins using an anti-HA monoclonal antibody. The gene constructs were inserted into the chromosome by insertion-duplication as described previously [18], and these genes were expressed as single copies under the control of their native promoters. These pbp-HA mutants showed the same phenotypes as the original pbp mutants, as well as the compensatory effects demonstrated in the double and triple pbp mutants (data not shown). We observed an increase in the half-life (>120 min) for PBP2b28 compared with the wild-type protein (21 min), not only in pbp2b28 (Fig. 5A) but also in the triple pbp mutant (Fig. 5C). Interestingly, we also detected an increased half-life for PBP1a28 (>120 min) and PBP2x28 (77 min) in the triple pbp mutant compared with half-life of PBP1a (31 min) and PBP2x (29 min) displayed in the wild-type strain (Figs. 5B and 5C). Our hypothesis is that these stability changes in the PBP mutants could have been responsible for the fitness/morphological alterations in the pbp2b mutant and the compensatory effects in the triple pbp mutant, and this will be discussed later.


Compensatory evolution of pbp mutations restores the fitness cost imposed by β-lactam resistance in Streptococcus pneumoniae.

Albarracín Orio AG, Piñas GE, Cortes PR, Cian MB, Echenique J - PLoS Pathog. (2011)

Stability in vivo of wild-type and mutant PBPs.Strains Cp1015 (Panel A), Cp1015 pbp2b28 (Panel B) and Cp1015 pbp2b28pbp2x28 pbp1a28 (Panel C) expressing HA-tagged PBPs were grown in Todd Hewitt broth with 0.5% Yeast Extract to an OD600 nm of 0.2 prior to the addition of kanamycin (500 µg/ml) to inhibit protein synthesis. At the time intervals indicated, aliquots were withdrawn and analyzed by SDS-PAGE followed by immunoblotting with an anti-HA monoclonal antibody (see Material and Methods).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040684&req=5

ppat-1002000-g005: Stability in vivo of wild-type and mutant PBPs.Strains Cp1015 (Panel A), Cp1015 pbp2b28 (Panel B) and Cp1015 pbp2b28pbp2x28 pbp1a28 (Panel C) expressing HA-tagged PBPs were grown in Todd Hewitt broth with 0.5% Yeast Extract to an OD600 nm of 0.2 prior to the addition of kanamycin (500 µg/ml) to inhibit protein synthesis. At the time intervals indicated, aliquots were withdrawn and analyzed by SDS-PAGE followed by immunoblotting with an anti-HA monoclonal antibody (see Material and Methods).
Mentions: To investigate the putative cause of these effects on cell morphology and fitness, we analyzed the stability of the proteins encoded by these pbp mutated genes using immunoblotting and inhibiting the protein synthesis by the addition of kanamycin. For these assays, we constructed C-terminal HA tagged PBPs to permit the detection of the proteins using an anti-HA monoclonal antibody. The gene constructs were inserted into the chromosome by insertion-duplication as described previously [18], and these genes were expressed as single copies under the control of their native promoters. These pbp-HA mutants showed the same phenotypes as the original pbp mutants, as well as the compensatory effects demonstrated in the double and triple pbp mutants (data not shown). We observed an increase in the half-life (>120 min) for PBP2b28 compared with the wild-type protein (21 min), not only in pbp2b28 (Fig. 5A) but also in the triple pbp mutant (Fig. 5C). Interestingly, we also detected an increased half-life for PBP1a28 (>120 min) and PBP2x28 (77 min) in the triple pbp mutant compared with half-life of PBP1a (31 min) and PBP2x (29 min) displayed in the wild-type strain (Figs. 5B and 5C). Our hypothesis is that these stability changes in the PBP mutants could have been responsible for the fitness/morphological alterations in the pbp2b mutant and the compensatory effects in the triple pbp mutant, and this will be discussed later.

Bottom Line: Thus, these compensatory combinations of pbp mutant alleles resulted in an increase in the level and spectrum of β-lactam resistance.The clinical origin of the pbp mutations suggests that this intergenic compensatory process is involved in the persistence of β-lactam resistance among circulating strains.We propose that this compensatory mechanism is relevant for β-lactam resistance evolution in Streptococcus pneumoniae.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica Clínica - CIBICI (CONICET), Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.

ABSTRACT
The prevalence of antibiotic resistance genes in pathogenic bacteria is a major challenge to treating many infectious diseases. The spread of these genes is driven by the strong selection imposed by the use of antibacterial drugs. However, in the absence of drug selection, antibiotic resistance genes impose a fitness cost, which can be ameliorated by compensatory mutations. In Streptococcus pneumoniae, β-lactam resistance is caused by mutations in three penicillin-binding proteins, PBP1a, PBP2x, and PBP2b, all of which are implicated in cell wall synthesis and the cell division cycle. We found that the fitness cost and cell division defects conferred by pbp2b mutations (as determined by fitness competitive assays in vitro and in vivo and fluorescence microscopy) were fully compensated by the acquisition of pbp2x and pbp1a mutations, apparently by means of an increased stability and a consequent mislocalization of these protein mutants. Thus, these compensatory combinations of pbp mutant alleles resulted in an increase in the level and spectrum of β-lactam resistance. This report describes a direct correlation between antibiotic resistance increase and fitness cost compensation, both caused by the same gene mutations acquired by horizontal transfer. The clinical origin of the pbp mutations suggests that this intergenic compensatory process is involved in the persistence of β-lactam resistance among circulating strains. We propose that this compensatory mechanism is relevant for β-lactam resistance evolution in Streptococcus pneumoniae.

Show MeSH
Related in: MedlinePlus