Effects of disruption of heat shock genes on susceptibility of Escherichia coli to fluoroquinolones
© Yamaguchi et al; licensee BioMed Central Ltd. 2003
Received: 19 March 2003
Accepted: 12 August 2003
Published: 12 August 2003
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.
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.
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.
FQs are broad-spectrum agents applicable to a range of Gram-positive and Gram-negative infections, and they have good oral absorbability . Because of these advantages, FQs have been widely used against a variety of bacterial infections for about two decades. They target the type II topoisomerases, DNA gyrase and topoisomerase IV, which are essential for controlling the topological state of DNA during replication and transcription .
Bacteria are known to respond to unfavorable conditions, e.g., exposure to toxic chemicals and physical agents, nutrient limitation, or sudden increase in growth temperature, by rapid expression of regulons related to the heat shock, SOS, and oxidative stress responses. DNA damage by UV irradiation, or treatment with naldixic acid, induce both the SOS and heat shock responses [9, 19]. Puromycin has been reported to induce first the SOS and secondly the heat shock responses; hydrogen peroxide induces the oxidative stress and SOS responses; and CdCl2 strongly induces all three stress responses. Protein induction by these responses is widely believed to be important for the organism's survival under hostile conditions. Analogously, the proteins induced when bacterial cells are exposed to antibiotics may affect the susceptibility of the organisms to these agents.
The aim of present study was to identify the bacterial responses affecting the bactericidal action of FQs. We analyzed the proteins induced in Escherichia coli by exposure to FQs, then examined the susceptibilities to these agents of E. coli strains with mutations in the genes encoding these proteins.
Results and Discussion
Analysis of protein synthesis in E. coli exposed to levofloxacin
In E. coli, the expression of heat shock genes is positively regulated at the transcriptional level by the heat shock specific sigma subunit of RNA polymerase, σ32. In addition, the heat shock response is negatively autoregulated by the DnaK chaperone machine that interferes with the efficient binding of σ32 to the RNA polymerase core, turning off the response [2, 4, 16]. Heat-induced aggregation of proteins induces the expression of the heat shock regulon through the titration of the DnaK chaperone by the aggregates [4, 16]. Therefore, in these experiments, the induction of the E. coli heat shock regulon could have been triggered by protein aggregates accumulated after incubation with LVFX.
Antibiotic susceptibility of E. coli strains with mutations in heat shock genes
The heat shock proteins comprise chaperones such as the DnaK/DnaJ/GrpE, GroEL/GroES and IbpA/IbpB systems, and ATP-dependent proteases such as Lon, ClpXP and HslVU. Whereas the chaperones are abundantly synthesized, the levels of the proteases are relatively low even under inducing conditions. In these experiments, proteases were not present in sufficient amounts to identify by mass spectrometry in LVFX-treated cells. However, all the heat shock genes in one regulon under the control of σ32 would be simultaneously induced in cells exposed to LVFX.
Chaperones and proteases are essential for de novo folding and quality control of proteins, acting by preventing protein aggregation and by refolding or degrading misfolded proteins. Therefore, the increased susceptibility of the chaperone and lon mutants can partly be explained by the disruption of the quality control system in E. coli cytosol.
Effect of sulA-disruption on the hypersusceptibility of the Δlon mutant to fluoroquinolones
Effect of the lon mutation in fluoroquinolone-resistant mutants
Bacterial quinolone resistance is usually due to mutations in the genes for targets of these agents: DNA gyrase encoded by gyrA and gyrB, and topoisomerase IV encoded by parC and parE. In several species of Enterobacteriaceae, decreased susceptibility or resistance to FQs is associated with specific point mutations in gyrA [21, 22]. An additional mutation in parC results in greater resistance [10, 20]. We examined whether the Δlon mutation affects the resistance to LVFX in E. coli gyrA and parC mutants.
Effect of recA mutation on susceptibility of E. coli to fluoroquinolone
The present results show that the DnaK and GroEL chaperones have moderate effects on the antimicrobial activity of FQs. These chaperones might contribute to quinolone resistance because they sequester the aggregates that accumulate in cells exposed to FQs. Lon protease markedly affects the bactericidal action of FQs, as indicated by the hypersusceptibility of the lon mutant. This increased susceptibility is corroborated by the effects of subsequent disruption of the gene encoding SulA. SulA protein is induced by the SOS response and degraded by Lon. Collectively, the results suggest that the hypersusceptibility of the lon mutant to FQs could be due to abnormal accumulation of SulA, which is depeleted by Lon, and the continuous induction of the SOS response.
FQs have contributed successfully to the treatment of various bacterial infections because of their broad-spectrum, potent antimicrobial activity and ease of oral administration. However, their widespread use and often misuse, coupled with emerging resistance, have gradually compromised their utility. The present work suggests that agents capable of inhibiting the Lon protease have potential for combination therapy with FQs.
Bacterial strains and plasmids
Escherichia coli strains used in this study
F- araD139 Δ(argF-lac)U169 rpsL150 relA1 flbB5301 deoC1 ptsF25 rbsR
F- lacY leu str supE thi thr tonA
C600 Sm recA
C600 Sm recA1
MC4100 dnaK::cat sidB1
Same as in MC4100 but araD+
BB7222 rpoH::Km zhf::Tn10 suhX1401
MC4100 groEL44 zie::Tn10
MC4100 groES619 zie::Tn10
CS5086 parC::Cm harboring pJK282-parC1
Levofloxacin was kindly provided by Daiichi Pharmaceutical Co. Ltd. [Tokyo, Japan].
Labeling of proteins producing in bacteria exposed to LVFX or heat shock
Bacterial cells grown in M9-medium  to mid-exponential growth phase were inoculated with LVFX and incubated at 30°C. Cells were labeled with 3.7 Mbq/ml of [35S]-Met and Cys (Protein labeling mix, >37Tbq/mmol, Amersham, Buckinghamshire, U.K.) for 1 min, mixed with TCA (final concentration 5%) and chilled on ice for 15 min. After centrifugation at 16000 g for 2 min, the pellets were washed with acetone and resuspended in sample buffer . The labeling of the heat shock proteins was performed as follows: bacterial cells in mid-exponential growth phase were transferred from 30°C to 42°C, incubated for 10 min and then labeled for 5 min at 42°C.
Resolution and quantitative estimation of proteins
The radiolabeled proteins were separated by SDS-polyacrylamide gel electrophoresis as described previously . The radioactivities incorporated into proteins were quantified using a BAS2000A photoimager (Fuji Film Co. [Tokyo, Japan])
Identification of proteins
Proteins were separated by two-dimensional gel electrophoresis and the protein spots of interest were excised from the gels and subjected to matrix-assisted laser desorption ionization-mass spectrometry according to the procedure described previously .
Fractionation of Proteins
Aliquots (10 ml) of cultures were rapidly cooled in an ice-water bath and centrifuged for 10 min at 5000 g to harvest the cells. Pellets were washed with saline and resuspended in 50 μl of buffer A (50 mM Tris-acetate, pH 7.4, 5 mM EDTA, 20 % sucrose, 1 μg/ml lysozyme) and incubated for 30 min on ice. Cell lysis was performed by addition of 450 μl of buffer B [50 mM Tris-acetate, pH 7.4, 5 mM EDTA, protease inhibitor cocktail (SIGMA [St. Louis, Mo.]) and mixing, followed by sonication with a Branson Sonifier 200. Intact cells were removed by centrifugation at 5000 g for 10 min at 4°C. The supernatant was used as a cytoplasmic and periplasmic protein fraction. The pellet containing membrane-associated and -integral proteins was resuspended in 80 μl of buffer B containing 1 M NaCl and centrifuged at 100000 g for 1 h at 4°C. The supernatant, containing membrane-associated proteins, was dialyzed against buffer C (50 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 10 % Glycerol, 0.1 mM DTT).
Bacterial susceptibility to antibiotics was determined by the twofold standard agar dilution method, except that bacterial cultures were diluted from 10-3 to 10-7 in buffered saline containing 0.01 % of gelatine.
YY analyzed the LVFX-related proteins, constructed some of mutants, and performed the susceptibility testing. TT constructed some of mutants and conducted the experiments with them. AT conducted the mass spectrometric analysis of the LVFX-related proteins. MM identified RecA protein by mass spectrometric analysis. TY opened the project here and prepared the submitted manuscript.
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