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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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Protein localization in wild-type and pbp mutants by fluorescence microscopy.A) Localization of FtsZ. Microphotographs indicate the most common patterns of FtsZ localization through the cell cycle for wt and pbp mutants. Polyclonal antibody against FtsZ was used for its detection by immunofluorescence microscopy. In the Cp1015 (wt) strain, FtsZ is localized at the equatorial ring and septum, In Cp 1015 pbp2b28, the patterns suggest FtsZ delocalization and cell cycle disruption. The triple pbp mutant showed a wild-type FtsZ placement. B) Localization of PBP2b28-GFP. Microphotographs indicate the most common patterns found for PBP2b localization through the cell cycle in the wt and pbp2b28 mutant. Representative images are shown from experiments that were repeated independently three times (bar scale, 1 µm).
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ppat-1002000-g006: Protein localization in wild-type and pbp mutants by fluorescence microscopy.A) Localization of FtsZ. Microphotographs indicate the most common patterns of FtsZ localization through the cell cycle for wt and pbp mutants. Polyclonal antibody against FtsZ was used for its detection by immunofluorescence microscopy. In the Cp1015 (wt) strain, FtsZ is localized at the equatorial ring and septum, In Cp 1015 pbp2b28, the patterns suggest FtsZ delocalization and cell cycle disruption. The triple pbp mutant showed a wild-type FtsZ placement. B) Localization of PBP2b28-GFP. Microphotographs indicate the most common patterns found for PBP2b localization through the cell cycle in the wt and pbp2b28 mutant. Representative images are shown from experiments that were repeated independently three times (bar scale, 1 µm).

Mentions: We also investigated the putative cause of a morphological change in the pbp2b28 mutant, and particularly in the cell division process. It was previously proposed that the life cycle of bacterial cells consists of repeated controlled enlargement, septum formation, and cell division [19]. In this scenario, FtsZ is an essential protein, which was postulated as the force generator that drives the cell division process, since the correct localization of all proteins involved in this mechanism is dependent on FtsZ [19], [20]. To evaluate the impact of pbp mutations on cell division, we determined the FtsZ localization by immunofluorescence microscopy, using a polyclonal antibody against FtsZ. Before septum formation, the FtsZ division ring has been described to be localized at the mid cell, as we also observed for the wild-type strain (Fig. 6A). However, an atypical FtsZ placement was found in pbp2b cells, with an apparent helical structure rather than the mid-cell localization found in wild-type cells. In the pbp2b28 pbp2x28 mutants, although we observed a coccoid morphology, the FtsZ localization was still altered. In contrast, a total FtsZ placement restoration was found in the triple pbp mutants (Fig. 6A). The higher stability of PBP2b28 led us to speculate that this increased protein level could also cause a delocalization of this protein mutant. Therefore, we constructed the PBP2b-GFP and PBP2b28-GFP fusions, expressed ectopically from a multicopy plasmid, in order to study their localization in S. pneumoniae cells. We observed that PBP2b-GFP was localized equatorially in the wild-type strain as described previously [16]. However, PBP2b28-GFP revealed an atypical helical distribution, similar to FtsZ in the pbp2b28 mutant (Fig. 6B), suggesting that PBP2b could interact with FtsZ. To investigate this, we analyzed this putative interaction by using a bacterial two-hybrid system (Bacteriomatch II, Stratagene), confirming this hypothesis by the detection of a positive interaction between PBP2b28 (or PBP2b) and FtsZ, but not between PBP2b28 (or PBP2b) and PBP2x. This served as a control of specificity in addition to the positive and negative controls included in this system (Fig. 7).


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)

Protein localization in wild-type and pbp mutants by fluorescence microscopy.A) Localization of FtsZ. Microphotographs indicate the most common patterns of FtsZ localization through the cell cycle for wt and pbp mutants. Polyclonal antibody against FtsZ was used for its detection by immunofluorescence microscopy. In the Cp1015 (wt) strain, FtsZ is localized at the equatorial ring and septum, In Cp 1015 pbp2b28, the patterns suggest FtsZ delocalization and cell cycle disruption. The triple pbp mutant showed a wild-type FtsZ placement. B) Localization of PBP2b28-GFP. Microphotographs indicate the most common patterns found for PBP2b localization through the cell cycle in the wt and pbp2b28 mutant. Representative images are shown from experiments that were repeated independently three times (bar scale, 1 µm).
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Related In: Results  -  Collection

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

ppat-1002000-g006: Protein localization in wild-type and pbp mutants by fluorescence microscopy.A) Localization of FtsZ. Microphotographs indicate the most common patterns of FtsZ localization through the cell cycle for wt and pbp mutants. Polyclonal antibody against FtsZ was used for its detection by immunofluorescence microscopy. In the Cp1015 (wt) strain, FtsZ is localized at the equatorial ring and septum, In Cp 1015 pbp2b28, the patterns suggest FtsZ delocalization and cell cycle disruption. The triple pbp mutant showed a wild-type FtsZ placement. B) Localization of PBP2b28-GFP. Microphotographs indicate the most common patterns found for PBP2b localization through the cell cycle in the wt and pbp2b28 mutant. Representative images are shown from experiments that were repeated independently three times (bar scale, 1 µm).
Mentions: We also investigated the putative cause of a morphological change in the pbp2b28 mutant, and particularly in the cell division process. It was previously proposed that the life cycle of bacterial cells consists of repeated controlled enlargement, septum formation, and cell division [19]. In this scenario, FtsZ is an essential protein, which was postulated as the force generator that drives the cell division process, since the correct localization of all proteins involved in this mechanism is dependent on FtsZ [19], [20]. To evaluate the impact of pbp mutations on cell division, we determined the FtsZ localization by immunofluorescence microscopy, using a polyclonal antibody against FtsZ. Before septum formation, the FtsZ division ring has been described to be localized at the mid cell, as we also observed for the wild-type strain (Fig. 6A). However, an atypical FtsZ placement was found in pbp2b cells, with an apparent helical structure rather than the mid-cell localization found in wild-type cells. In the pbp2b28 pbp2x28 mutants, although we observed a coccoid morphology, the FtsZ localization was still altered. In contrast, a total FtsZ placement restoration was found in the triple pbp mutants (Fig. 6A). The higher stability of PBP2b28 led us to speculate that this increased protein level could also cause a delocalization of this protein mutant. Therefore, we constructed the PBP2b-GFP and PBP2b28-GFP fusions, expressed ectopically from a multicopy plasmid, in order to study their localization in S. pneumoniae cells. We observed that PBP2b-GFP was localized equatorially in the wild-type strain as described previously [16]. However, PBP2b28-GFP revealed an atypical helical distribution, similar to FtsZ in the pbp2b28 mutant (Fig. 6B), suggesting that PBP2b could interact with FtsZ. To investigate this, we analyzed this putative interaction by using a bacterial two-hybrid system (Bacteriomatch II, Stratagene), confirming this hypothesis by the detection of a positive interaction between PBP2b28 (or PBP2b) and FtsZ, but not between PBP2b28 (or PBP2b) and PBP2x. This served as a control of specificity in addition to the positive and negative controls included in this system (Fig. 7).

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