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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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Effect of pbp mutations on cell morphology.Cells were prepared for microscopy and stained with DAPI as described in Materials and Methods (bar scale, 5 µm). (A) Merged phase-contrast and DAPI fluorescence image of the pbp mutants showing a bacillar morphology, contrasting with the typical coccoid shape of the CP1015 (wt) strain. (B) Cell shape modifications of the cell populations detected by flow cytometry analysis. Exponentially growing bacterial cells were injected into a flow cytometer and the results were analyzed with the WinMDI software. To compare populations, the data for each strain were plotted on a two-dimensional graph (x-axis, forward scatter; y-axis, side scatter).
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ppat-1002000-g002: Effect of pbp mutations on cell morphology.Cells were prepared for microscopy and stained with DAPI as described in Materials and Methods (bar scale, 5 µm). (A) Merged phase-contrast and DAPI fluorescence image of the pbp mutants showing a bacillar morphology, contrasting with the typical coccoid shape of the CP1015 (wt) strain. (B) Cell shape modifications of the cell populations detected by flow cytometry analysis. Exponentially growing bacterial cells were injected into a flow cytometer and the results were analyzed with the WinMDI software. To compare populations, the data for each strain were plotted on a two-dimensional graph (x-axis, forward scatter; y-axis, side scatter).

Mentions: It is known that PBPs are responsible for peptidoglycan synthesis. These proteins form the main component of the cell wall, and are involved in longitudinal wall growth and the cell division process. Morlot and coworkers [16] have proposed certain functions for PBP2b, PBP2a and PBP1a in S. pneumoniae, but the specific roles of PBPs in the growth and division phases are not yet well understood in bacteria. PBPs are also recognized as the targets for βLs, the most frequently used antimicrobials for treating bacterial infections. Therefore, considering that the interaction between drug resistance and the cell division processes remains unclear [4], we decided to investigate this connection. First, we examined the effects of pbp mutations on S. pneumoniae cell morphology, and then the putative cell division alterations. Microscopic examinations of single pbp1a28 and pbp2x28 mutants in Cp1015 strain did not display any morphological alterations, whereas the pbp2b28 mutant (Fig. 2A) and all 10 strains bearing pbp2b mutations from clinical strains showed a rod shape (data not shown). This atypical phenotype was also found for pbp2b mutants with a different genetic background to that of Cp1015, such as the R6 and D39 laboratory strains (data not shown).


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)

Effect of pbp mutations on cell morphology.Cells were prepared for microscopy and stained with DAPI as described in Materials and Methods (bar scale, 5 µm). (A) Merged phase-contrast and DAPI fluorescence image of the pbp mutants showing a bacillar morphology, contrasting with the typical coccoid shape of the CP1015 (wt) strain. (B) Cell shape modifications of the cell populations detected by flow cytometry analysis. Exponentially growing bacterial cells were injected into a flow cytometer and the results were analyzed with the WinMDI software. To compare populations, the data for each strain were plotted on a two-dimensional graph (x-axis, forward scatter; y-axis, side scatter).
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002000-g002: Effect of pbp mutations on cell morphology.Cells were prepared for microscopy and stained with DAPI as described in Materials and Methods (bar scale, 5 µm). (A) Merged phase-contrast and DAPI fluorescence image of the pbp mutants showing a bacillar morphology, contrasting with the typical coccoid shape of the CP1015 (wt) strain. (B) Cell shape modifications of the cell populations detected by flow cytometry analysis. Exponentially growing bacterial cells were injected into a flow cytometer and the results were analyzed with the WinMDI software. To compare populations, the data for each strain were plotted on a two-dimensional graph (x-axis, forward scatter; y-axis, side scatter).
Mentions: It is known that PBPs are responsible for peptidoglycan synthesis. These proteins form the main component of the cell wall, and are involved in longitudinal wall growth and the cell division process. Morlot and coworkers [16] have proposed certain functions for PBP2b, PBP2a and PBP1a in S. pneumoniae, but the specific roles of PBPs in the growth and division phases are not yet well understood in bacteria. PBPs are also recognized as the targets for βLs, the most frequently used antimicrobials for treating bacterial infections. Therefore, considering that the interaction between drug resistance and the cell division processes remains unclear [4], we decided to investigate this connection. First, we examined the effects of pbp mutations on S. pneumoniae cell morphology, and then the putative cell division alterations. Microscopic examinations of single pbp1a28 and pbp2x28 mutants in Cp1015 strain did not display any morphological alterations, whereas the pbp2b28 mutant (Fig. 2A) and all 10 strains bearing pbp2b mutations from clinical strains showed a rod shape (data not shown). This atypical phenotype was also found for pbp2b mutants with a different genetic background to that of Cp1015, such as the R6 and D39 laboratory strains (data not shown).

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