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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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Susceptibility of E. coli Δlon and ΔsulA double mutant to LVFX. (A), (B), (C) and (D) show the results of spot testing with agar plates containing 0 μg/ml, 0.0125 μg/ml, 0.025 μg/ml and 0.05 μg/ml LVFX, respectively.
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Figure 5: Susceptibility of E. coli Δlon and ΔsulA double mutant to LVFX. (A), (B), (C) and (D) show the results of spot testing with agar plates containing 0 μg/ml, 0.0125 μg/ml, 0.025 μg/ml and 0.05 μg/ml LVFX, respectively.

Mentions: The Lon protease degrades SulA, which inhibits separation by binding to FtsZ, a key cell division protein [1]. SulA is a SOS response protein induced by DNA damage after UV-irradiation and quinolone-treatment [23]. We examined the possibility that the hypersensitivity of the Δlon mutant is due to the accumulation of SulA protein. The sulA gene was disrupted in both wild-type and Δlon-mutant cells and then the resultant double mutants were compared for susceptibility to LVFX. As shown in Figure 5, the hypersusceptibility of Δlon was suppressed by the subsequent disruption of sulA, suggesting that the increased sensitivity is due to the inhibition of cell division by abnormal accumulation of SulA. It therefore appears that in Δlon, depletion of the Lon protease and continuous induction of the SOS response in the presence of LVFX result in abnormally high levels of the cell division inhibitor SulA. Complementation analysis also indicates that continuous induction of SulA by FQs contributes to quinolone killing. This hypothesis is supported by the finding that the ΔsulA mutant is less sensitive to LVFX than the wild type (Figure 5). Moreover, disruption of lon did not affect the susceptibility of bacteria to antibiotics other than quinolones, such as ampicillin, cephalothin, streptomycin, polymixin-B or rifampicin (our unpublished data).


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)

Susceptibility of E. coli Δlon and ΔsulA double mutant to LVFX. (A), (B), (C) and (D) show the results of spot testing with agar plates containing 0 μg/ml, 0.0125 μg/ml, 0.025 μg/ml and 0.05 μg/ml LVFX, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Susceptibility of E. coli Δlon and ΔsulA double mutant to LVFX. (A), (B), (C) and (D) show the results of spot testing with agar plates containing 0 μg/ml, 0.0125 μg/ml, 0.025 μg/ml and 0.05 μg/ml LVFX, respectively.
Mentions: The Lon protease degrades SulA, which inhibits separation by binding to FtsZ, a key cell division protein [1]. SulA is a SOS response protein induced by DNA damage after UV-irradiation and quinolone-treatment [23]. We examined the possibility that the hypersensitivity of the Δlon mutant is due to the accumulation of SulA protein. The sulA gene was disrupted in both wild-type and Δlon-mutant cells and then the resultant double mutants were compared for susceptibility to LVFX. As shown in Figure 5, the hypersusceptibility of Δlon was suppressed by the subsequent disruption of sulA, suggesting that the increased sensitivity is due to the inhibition of cell division by abnormal accumulation of SulA. It therefore appears that in Δlon, depletion of the Lon protease and continuous induction of the SOS response in the presence of LVFX result in abnormally high levels of the cell division inhibitor SulA. Complementation analysis also indicates that continuous induction of SulA by FQs contributes to quinolone killing. This hypothesis is supported by the finding that the ΔsulA mutant is less sensitive to LVFX than the wild type (Figure 5). Moreover, disruption of lon did not affect the susceptibility of bacteria to antibiotics other than quinolones, such as ampicillin, cephalothin, streptomycin, polymixin-B or rifampicin (our unpublished data).

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