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Clinically relevant mutant DNA gyrase alters supercoiling, changes the transcriptome, and confers multidrug resistance.

Webber MA, Ricci V, Whitehead R, Patel M, Fookes M, Ivens A, Piddock LJ - MBio (2013)

Bottom Line: Decreased susceptibility to multiple antibiotics seen with a GyrA Asp87Gly mutant was not a result of increased efflux activity or reduced reactive-oxygen production.These data show that a frequently observed and clinically relevant substitution within GyrA results in altered expression of numerous genes, including those important in bacterial survival of stress, suggesting that GyrA mutants may have a selective advantage under specific conditions.This work suggests that fluoroquinolone resistance mutations may be beneficial under a range of conditions.

View Article: PubMed Central - PubMed

Affiliation: Antimicrobial Agents Research Group, School of Immunity and Infection, Institute of Microbiology and Infection, The University of Birmingham, Edgbaston, Birmingham, United Kingdom.

ABSTRACT

Unlabelled: Bacterial DNA is maintained in a supercoiled state controlled by the action of topoisomerases. Alterations in supercoiling affect fundamental cellular processes, including transcription. Here, we show that substitution at position 87 of GyrA of Salmonella influences sensitivity to antibiotics, including nonquinolone drugs, alters global supercoiling, and results in an altered transcriptome with increased expression of stress response pathways. Decreased susceptibility to multiple antibiotics seen with a GyrA Asp87Gly mutant was not a result of increased efflux activity or reduced reactive-oxygen production. These data show that a frequently observed and clinically relevant substitution within GyrA results in altered expression of numerous genes, including those important in bacterial survival of stress, suggesting that GyrA mutants may have a selective advantage under specific conditions. Our findings help contextualize the high rate of quinolone resistance in pathogenic strains of bacteria and may partly explain why such mutant strains are evolutionarily successful.

Importance: Fluoroquinolones are a powerful group of antibiotics that target bacterial enzymes involved in helping bacteria maintain the conformation of their chromosome. Mutations in the target enzymes allow bacteria to become resistant to these antibiotics, and fluoroquinolone resistance is common. We show here that these mutations also provide protection against a broad range of other antimicrobials by triggering a defensive stress response in the cell. This work suggests that fluoroquinolone resistance mutations may be beneficial under a range of conditions.

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Related in: MedlinePlus

Supercoiling activity and expression of gyrA and topA by SL1344, L821, and L825. (A) Separation of pBR322 isolated from each strain on a representative 0.9% agarose gel containing 25 mg/liter chloroquine. Red arrows indicate altered supercoiling; black arrows indicate direction of electrophoresis. (B) Densitometry plots of the lanes in the gel from panel A. (C) Expression of gyrA and topA, measured by comparative RT-PCR. Error bars indicate standard deviations, and asterisks indicate values that are significantly different from the value for SL1344.
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fig3: Supercoiling activity and expression of gyrA and topA by SL1344, L821, and L825. (A) Separation of pBR322 isolated from each strain on a representative 0.9% agarose gel containing 25 mg/liter chloroquine. Red arrows indicate altered supercoiling; black arrows indicate direction of electrophoresis. (B) Densitometry plots of the lanes in the gel from panel A. (C) Expression of gyrA and topA, measured by comparative RT-PCR. Error bars indicate standard deviations, and asterisks indicate values that are significantly different from the value for SL1344.

Mentions: The level of supercoiling of plasmid pBR322 within SL1344, L821 (GyrA Ser83Phe), and L825 (GyrA Asp87Gly) was determined and found to differ in the two mutants (Fig. 3). Compared to SL1344, both mutants showed a shift in the topoisomer band patterns, indicating a reduction in the degree of negative supercoiling of DNA. This reduction was more marked for L825 (13% reduction in activity) than for L821 (5% reduction in activity) (Fig. 3A and B). These results indicate that mutation within gyrA reduces gyrase activity and as a result lessens the degree of supercoiling of DNA within the cell (as found previously with E. coli [34]). Comparative reverse transcription-PCR (RT-PCR) (Fig. 3C) of topA and gyrA showed a significant increase in gyrA expression in both L821 (~2-fold) and L825 (~4-fold) and significant decreases in topA expression in both mutants (~1.25- and 2.5-fold, respectively) compared to SL1344. These data are consistent with a response to altered supercoiling (34). The larger shift in the ratio of gyrA to topA expression seen in L825 than in L821 suggests a greater functional consequence of the Asp87Gly substitution in this mutant (Fig. 3).


Clinically relevant mutant DNA gyrase alters supercoiling, changes the transcriptome, and confers multidrug resistance.

Webber MA, Ricci V, Whitehead R, Patel M, Fookes M, Ivens A, Piddock LJ - MBio (2013)

Supercoiling activity and expression of gyrA and topA by SL1344, L821, and L825. (A) Separation of pBR322 isolated from each strain on a representative 0.9% agarose gel containing 25 mg/liter chloroquine. Red arrows indicate altered supercoiling; black arrows indicate direction of electrophoresis. (B) Densitometry plots of the lanes in the gel from panel A. (C) Expression of gyrA and topA, measured by comparative RT-PCR. Error bars indicate standard deviations, and asterisks indicate values that are significantly different from the value for SL1344.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3735185&req=5

fig3: Supercoiling activity and expression of gyrA and topA by SL1344, L821, and L825. (A) Separation of pBR322 isolated from each strain on a representative 0.9% agarose gel containing 25 mg/liter chloroquine. Red arrows indicate altered supercoiling; black arrows indicate direction of electrophoresis. (B) Densitometry plots of the lanes in the gel from panel A. (C) Expression of gyrA and topA, measured by comparative RT-PCR. Error bars indicate standard deviations, and asterisks indicate values that are significantly different from the value for SL1344.
Mentions: The level of supercoiling of plasmid pBR322 within SL1344, L821 (GyrA Ser83Phe), and L825 (GyrA Asp87Gly) was determined and found to differ in the two mutants (Fig. 3). Compared to SL1344, both mutants showed a shift in the topoisomer band patterns, indicating a reduction in the degree of negative supercoiling of DNA. This reduction was more marked for L825 (13% reduction in activity) than for L821 (5% reduction in activity) (Fig. 3A and B). These results indicate that mutation within gyrA reduces gyrase activity and as a result lessens the degree of supercoiling of DNA within the cell (as found previously with E. coli [34]). Comparative reverse transcription-PCR (RT-PCR) (Fig. 3C) of topA and gyrA showed a significant increase in gyrA expression in both L821 (~2-fold) and L825 (~4-fold) and significant decreases in topA expression in both mutants (~1.25- and 2.5-fold, respectively) compared to SL1344. These data are consistent with a response to altered supercoiling (34). The larger shift in the ratio of gyrA to topA expression seen in L825 than in L821 suggests a greater functional consequence of the Asp87Gly substitution in this mutant (Fig. 3).

Bottom Line: Decreased susceptibility to multiple antibiotics seen with a GyrA Asp87Gly mutant was not a result of increased efflux activity or reduced reactive-oxygen production.These data show that a frequently observed and clinically relevant substitution within GyrA results in altered expression of numerous genes, including those important in bacterial survival of stress, suggesting that GyrA mutants may have a selective advantage under specific conditions.This work suggests that fluoroquinolone resistance mutations may be beneficial under a range of conditions.

View Article: PubMed Central - PubMed

Affiliation: Antimicrobial Agents Research Group, School of Immunity and Infection, Institute of Microbiology and Infection, The University of Birmingham, Edgbaston, Birmingham, United Kingdom.

ABSTRACT

Unlabelled: Bacterial DNA is maintained in a supercoiled state controlled by the action of topoisomerases. Alterations in supercoiling affect fundamental cellular processes, including transcription. Here, we show that substitution at position 87 of GyrA of Salmonella influences sensitivity to antibiotics, including nonquinolone drugs, alters global supercoiling, and results in an altered transcriptome with increased expression of stress response pathways. Decreased susceptibility to multiple antibiotics seen with a GyrA Asp87Gly mutant was not a result of increased efflux activity or reduced reactive-oxygen production. These data show that a frequently observed and clinically relevant substitution within GyrA results in altered expression of numerous genes, including those important in bacterial survival of stress, suggesting that GyrA mutants may have a selective advantage under specific conditions. Our findings help contextualize the high rate of quinolone resistance in pathogenic strains of bacteria and may partly explain why such mutant strains are evolutionarily successful.

Importance: Fluoroquinolones are a powerful group of antibiotics that target bacterial enzymes involved in helping bacteria maintain the conformation of their chromosome. Mutations in the target enzymes allow bacteria to become resistant to these antibiotics, and fluoroquinolone resistance is common. We show here that these mutations also provide protection against a broad range of other antimicrobials by triggering a defensive stress response in the cell. This work suggests that fluoroquinolone resistance mutations may be beneficial under a range of conditions.

Show MeSH
Related in: MedlinePlus