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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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Related in: MedlinePlus

Septal localization in pbp mutants.Exponentially grown cells were stained with DAPI and fluorescent vancomycin (Fl-Van, which labels nascent peptidoglycan synthesis and strongly marks septal localization) and analysed by using an epifluorescence microscope (see Material and Methods). The pbp2b showed a septal accumulation in rod-like shaped cells, whereas the double (pbp2b pbp2x or pbp2b pbp1a) and the triple pbp mutants restored their septal localization. Representative images are shown from experiments that were repeated independently three times (bar scale, 1 µm).
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ppat-1002000-g004: Septal localization in pbp mutants.Exponentially grown cells were stained with DAPI and fluorescent vancomycin (Fl-Van, which labels nascent peptidoglycan synthesis and strongly marks septal localization) and analysed by using an epifluorescence microscope (see Material and Methods). The pbp2b showed a septal accumulation in rod-like shaped cells, whereas the double (pbp2b pbp2x or pbp2b pbp1a) and the triple pbp mutants restored their septal localization. Representative images are shown from experiments that were repeated independently three times (bar scale, 1 µm).

Mentions: When cells were stained with fluorescein-labeled vancomycin (Van-FL), which localizes to sites of nascent peptidoglycan synthesis and clearly marks the septum location in the wild-type strain [17] (Fig. S16 in Supporting Information S1), an abnormal septum pattern was revealed in rod-shaped cells of the pbp2b28 mutants, suggesting a clear alteration in cell division (Fig. 4). However, this phenomenon was compensated in the double (pbp2b28 pbp2x28 or pbp2b28 pbp1a28) and triple pbp mutants (Fig. 4). The morphological variation was confirmed by flow cytometry analysis, which allowed determining the population distribution of pneumococci by cell size. These assays showed a displacement favoring a larger cell size in the pbp2b28 mutant, and the restoration of normal size in the triple pbp mutant (Fig. 2B). These results suggest that the septal alterations and the cellular enlargement found in the pbp2b28 mutant could have been responsible for its growth retardation.


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)

Septal localization in pbp mutants.Exponentially grown cells were stained with DAPI and fluorescent vancomycin (Fl-Van, which labels nascent peptidoglycan synthesis and strongly marks septal localization) and analysed by using an epifluorescence microscope (see Material and Methods). The pbp2b showed a septal accumulation in rod-like shaped cells, whereas the double (pbp2b pbp2x or pbp2b pbp1a) and the triple pbp mutants restored their septal localization. Representative images are shown from experiments that were repeated independently three times (bar scale, 1 µm).
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002000-g004: Septal localization in pbp mutants.Exponentially grown cells were stained with DAPI and fluorescent vancomycin (Fl-Van, which labels nascent peptidoglycan synthesis and strongly marks septal localization) and analysed by using an epifluorescence microscope (see Material and Methods). The pbp2b showed a septal accumulation in rod-like shaped cells, whereas the double (pbp2b pbp2x or pbp2b pbp1a) and the triple pbp mutants restored their septal localization. Representative images are shown from experiments that were repeated independently three times (bar scale, 1 µm).
Mentions: When cells were stained with fluorescein-labeled vancomycin (Van-FL), which localizes to sites of nascent peptidoglycan synthesis and clearly marks the septum location in the wild-type strain [17] (Fig. S16 in Supporting Information S1), an abnormal septum pattern was revealed in rod-shaped cells of the pbp2b28 mutants, suggesting a clear alteration in cell division (Fig. 4). However, this phenomenon was compensated in the double (pbp2b28 pbp2x28 or pbp2b28 pbp1a28) and triple pbp mutants (Fig. 4). The morphological variation was confirmed by flow cytometry analysis, which allowed determining the population distribution of pneumococci by cell size. These assays showed a displacement favoring a larger cell size in the pbp2b28 mutant, and the restoration of normal size in the triple pbp mutant (Fig. 2B). These results suggest that the septal alterations and the cellular enlargement found in the pbp2b28 mutant could have been responsible for its growth retardation.

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