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Effects of disruption of heat shock genes on susceptibility of Escherichia coli to fluoroquinolones.

Yamaguchi Y, Tomoyasu T, Takaya A, Morioka M, Yamamoto T - BMC Microbiol. (2003)

Bottom Line: The present results show that the bactericidal action of FQs is moderately affected by the DnaK and GroEL chaperones and strongly affected by the Lon protease.FQs have contributed successfully to the treatment of various bacterial infections, but their widespread use and often misuse, coupled with emerging resistance, have gradually compromised their utility.Our results suggest that agents capable of inhibiting the Lon protease have potential for combination therapy with FQs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, Graduate School of Pharmaceutical Sciences, Chiba University, Japan. Yuko@p.chiba-u.ac.jp

ABSTRACT

Background: It is well known that expression of certain bacterial genes responds rapidly to such stimuli as exposure to toxic chemicals and physical agents. It is generally believed that the proteins encoded in these genes are important for successful survival of the organism under the hostile conditions. Analogously, the proteins induced in bacterial cells exposed to antibiotics are believed to affect the organisms' susceptibility to these agents.

Results: We demonstrated that Escherichia coli cells exposed to levofloxacin (LVFX), a fluoroquinolone (FQ), induce the syntheses of heat shock proteins and RecA. To examine whether the heat shock proteins affect the bactericidal action of FQs, we constructed E. coli strains with mutations in various heat shock genes and tested their susceptibility to FQs. Mutations in dnaK, groEL, and lon increased this susceptibility; the lon mutant exhibited the greatest effects. The increased susceptibility of the lon mutant was corroborated by experiments in which the gene encoding the cell division inhibitor, SulA, was subsequently disrupted. SulA is induced by the SOS response and degraded by the Lon protease. The findings suggest that the hypersusceptibility of the lon mutant to FQs could be due to abnormally high levels of SulA protein resulting from the depletion of Lon and the continuous induction of the SOS response in the presence of FQs.

Conclusion: The present results show that the bactericidal action of FQs is moderately affected by the DnaK and GroEL chaperones and strongly affected by the Lon protease. FQs have contributed successfully to the treatment of various bacterial infections, but their widespread use and often misuse, coupled with emerging resistance, have gradually compromised their utility. Our results suggest that agents capable of inhibiting the Lon protease have potential for combination therapy with FQs.

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Fractionation of proteins synthesized in E. coli exposed to LVFX. Proteins from cultures labeled in the presence of 10 μg/ml LVFX were fractionated as described in Methods and separated by SDS-PAGE (A). The protein samples from untreated cells were run in lanes 1, 3, 5, 7 and 9. The samples from LVFX-treated cells were in lanes 2, 4, 6, 8 and 10. Lanes contain protein fractions as follows: lanes 1 and 2, whole cell lysates; lanes 3 and 4, cytoplasmic and periplasmic protein fractions; lanes 5 and 6, membrane-associated protein fractions. The membrane-associated proteins were solubilized in 1 M NaCl and then dialyzed described in Methods. After centrifugation, the pellets were solubilized in lysis buffer and applied in lanes 7 and 8. The supernatants were applied in lanes 9 and 10. Portions of the protein samples applied in lanes 7 and 8 were also subjected to two-dimensional gel electrophoresis and the results are shown in (B) and (C), respectively.
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Figure 3: Fractionation of proteins synthesized in E. coli exposed to LVFX. Proteins from cultures labeled in the presence of 10 μg/ml LVFX were fractionated as described in Methods and separated by SDS-PAGE (A). The protein samples from untreated cells were run in lanes 1, 3, 5, 7 and 9. The samples from LVFX-treated cells were in lanes 2, 4, 6, 8 and 10. Lanes contain protein fractions as follows: lanes 1 and 2, whole cell lysates; lanes 3 and 4, cytoplasmic and periplasmic protein fractions; lanes 5 and 6, membrane-associated protein fractions. The membrane-associated proteins were solubilized in 1 M NaCl and then dialyzed described in Methods. After centrifugation, the pellets were solubilized in lysis buffer and applied in lanes 7 and 8. The supernatants were applied in lanes 9 and 10. Portions of the protein samples applied in lanes 7 and 8 were also subjected to two-dimensional gel electrophoresis and the results are shown in (B) and (C), respectively.

Mentions: The 40 kDa protein showing the most marked induction in Figure 1 does not appear on the two-dimensional gel (Figure 2). We therefore fractionated the proteins from LVFX treated E. coli cells along with those from control cells. As shown in Figure 3, the 40 kDa protein seems to be largely associated with high salt-soluble proteins (lanes 6 and 8). A portion of the high salt-soluble fraction was separated by two-dimensional gel electrophoresis; the spot corresponding to the LVFX-induced 40 kDa protein was subjected to mass spectrometric analysis and identified as RecA. RecA is usually cytosolic in E. coli and binds to single-stranded regions of damaged DNA [14]. Detection of a large amount of RecA protein in the high salt-soluble fraction suggests that when it is synthesized in abnormally large amounts, it aggregates with various denatured and misfolded proteins.


Effects of disruption of heat shock genes on susceptibility of Escherichia coli to fluoroquinolones.

Yamaguchi Y, Tomoyasu T, Takaya A, Morioka M, Yamamoto T - BMC Microbiol. (2003)

Fractionation of proteins synthesized in E. coli exposed to LVFX. Proteins from cultures labeled in the presence of 10 μg/ml LVFX were fractionated as described in Methods and separated by SDS-PAGE (A). The protein samples from untreated cells were run in lanes 1, 3, 5, 7 and 9. The samples from LVFX-treated cells were in lanes 2, 4, 6, 8 and 10. Lanes contain protein fractions as follows: lanes 1 and 2, whole cell lysates; lanes 3 and 4, cytoplasmic and periplasmic protein fractions; lanes 5 and 6, membrane-associated protein fractions. The membrane-associated proteins were solubilized in 1 M NaCl and then dialyzed described in Methods. After centrifugation, the pellets were solubilized in lysis buffer and applied in lanes 7 and 8. The supernatants were applied in lanes 9 and 10. Portions of the protein samples applied in lanes 7 and 8 were also subjected to two-dimensional gel electrophoresis and the results are shown in (B) and (C), respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC184496&req=5

Figure 3: Fractionation of proteins synthesized in E. coli exposed to LVFX. Proteins from cultures labeled in the presence of 10 μg/ml LVFX were fractionated as described in Methods and separated by SDS-PAGE (A). The protein samples from untreated cells were run in lanes 1, 3, 5, 7 and 9. The samples from LVFX-treated cells were in lanes 2, 4, 6, 8 and 10. Lanes contain protein fractions as follows: lanes 1 and 2, whole cell lysates; lanes 3 and 4, cytoplasmic and periplasmic protein fractions; lanes 5 and 6, membrane-associated protein fractions. The membrane-associated proteins were solubilized in 1 M NaCl and then dialyzed described in Methods. After centrifugation, the pellets were solubilized in lysis buffer and applied in lanes 7 and 8. The supernatants were applied in lanes 9 and 10. Portions of the protein samples applied in lanes 7 and 8 were also subjected to two-dimensional gel electrophoresis and the results are shown in (B) and (C), respectively.
Mentions: The 40 kDa protein showing the most marked induction in Figure 1 does not appear on the two-dimensional gel (Figure 2). We therefore fractionated the proteins from LVFX treated E. coli cells along with those from control cells. As shown in Figure 3, the 40 kDa protein seems to be largely associated with high salt-soluble proteins (lanes 6 and 8). A portion of the high salt-soluble fraction was separated by two-dimensional gel electrophoresis; the spot corresponding to the LVFX-induced 40 kDa protein was subjected to mass spectrometric analysis and identified as RecA. RecA is usually cytosolic in E. coli and binds to single-stranded regions of damaged DNA [14]. Detection of a large amount of RecA protein in the high salt-soluble fraction suggests that when it is synthesized in abnormally large amounts, it aggregates with various denatured and misfolded proteins.

Bottom Line: The present results show that the bactericidal action of FQs is moderately affected by the DnaK and GroEL chaperones and strongly affected by the Lon protease.FQs have contributed successfully to the treatment of various bacterial infections, but their widespread use and often misuse, coupled with emerging resistance, have gradually compromised their utility.Our results suggest that agents capable of inhibiting the Lon protease have potential for combination therapy with FQs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, Graduate School of Pharmaceutical Sciences, Chiba University, Japan. Yuko@p.chiba-u.ac.jp

ABSTRACT

Background: It is well known that expression of certain bacterial genes responds rapidly to such stimuli as exposure to toxic chemicals and physical agents. It is generally believed that the proteins encoded in these genes are important for successful survival of the organism under the hostile conditions. Analogously, the proteins induced in bacterial cells exposed to antibiotics are believed to affect the organisms' susceptibility to these agents.

Results: We demonstrated that Escherichia coli cells exposed to levofloxacin (LVFX), a fluoroquinolone (FQ), induce the syntheses of heat shock proteins and RecA. To examine whether the heat shock proteins affect the bactericidal action of FQs, we constructed E. coli strains with mutations in various heat shock genes and tested their susceptibility to FQs. Mutations in dnaK, groEL, and lon increased this susceptibility; the lon mutant exhibited the greatest effects. The increased susceptibility of the lon mutant was corroborated by experiments in which the gene encoding the cell division inhibitor, SulA, was subsequently disrupted. SulA is induced by the SOS response and degraded by the Lon protease. The findings suggest that the hypersusceptibility of the lon mutant to FQs could be due to abnormally high levels of SulA protein resulting from the depletion of Lon and the continuous induction of the SOS response in the presence of FQs.

Conclusion: The present results show that the bactericidal action of FQs is moderately affected by the DnaK and GroEL chaperones and strongly affected by the Lon protease. FQs have contributed successfully to the treatment of various bacterial infections, but their widespread use and often misuse, coupled with emerging resistance, have gradually compromised their utility. Our results suggest that agents capable of inhibiting the Lon protease have potential for combination therapy with FQs.

Show MeSH
Related in: MedlinePlus