Role of global regulators and nucleotide metabolism in antibiotic tolerance in Escherichia coli - PubMed (original) (raw)
Role of global regulators and nucleotide metabolism in antibiotic tolerance in Escherichia coli
Sonja Hansen et al. Antimicrob Agents Chemother. 2008 Aug.
Abstract
Bacterial populations produce a small number of persister cells that exhibit multidrug tolerance. Persister cells are largely responsible for the antibiotic recalcitrance of biofilm infections. The mechanism of persister cell formation largely remains unknown due to the challenges in identifying persister genes. We screened an ordered comprehensive library of 3,985 Escherichia coli knockout strains to identify mutants with altered antibiotic tolerance. Stationary-state cultures in 96-well plates were exposed to ofloxacin at a concentration which allows only tolerant persister cells to survive. The persister cell level of each culture was determined. A total of 150 mutants with decreased persistence were identified in the initial screen, and subsequent validation confirmed that neither the growth rate nor the ofloxacin MIC was affected for 10 of them. The genes affected in these strains were dnaJ and dnaK (chaperones), apaH (diadenosine tetraphosphatase), surA (peptidyl-prolyl cis-trans isomerase), fis and hns (global regulators), hnr (response regulator of RpoS), dksA (transcriptional regulator of rRNA transcription), ygfA (5-formyl-tetrahydrofolate cyclo-ligase), and yigB (flavin mononucleotide [FMN] phosphatase). The prominent presence of global regulators among these strains pointed to the likely redundancy of persister cell formation mechanisms: the elimination of a regulator controlling several redundant persister genes would be expected to produce a phenotype. This observation is consistent with previous findings for a possible role of redundant genes such as toxin/antitoxin modules in persister cell formation. ygfA and yigB were of special interest. The mammalian homolog of YgfA (methenyltetrahydrofolate synthetase) catalyzes the conversion of 5-formyl-tetrahydrofolate (THF) into the rapidly degraded 5,10-methenyl-THF, depleting the folate pool. The YigB protein is a phosphatase of FMN which would deplete the pool of this cofactor. Stochastic overexpression of these genes could lead to dormancy and, hence, tolerance by depleting the folate and FMN pools, respectively. Consistent with this scenario, the overexpression of both genes produced increased tolerance to ofloxacin.
Figures
FIG. 1.
Screen for mutants with low levels of persistence. (A) Frequency of amdinocillin-resistant mutants in E. coli K-12 BW25113. Stationary-phase culture in LB medium was serially diluted in twofold steps and plated onto LB agar supplemented with 4 μg/ml amdinocillin. Samples from each dilution were also removed for determination of the colony counts. The frequency was 5 × 10−5 on the basis of the initial cell counts and the number of amdinocillin-resistant mutants arising on LB agar supplemented with amdinocillin. (B) Survival of E. coli BW25113 stationary-phase cells challenged with ofloxacin. Stationary-phase cultures grown in MOPS minimal medium were treated with ofloxacin at the indicated concentrations for 6 h and plated for determination of the colony counts. The experiment was performed in triplicate, and error bars represent standard deviations. (C) Survival of strains from the Keio collection. Mutant strains cultured in MOPS minimal medium were treated with ofloxacin for 6 h, and samples were plated on LB agar supplemented with 4 μg/ml amdinocillin and 20 mM MgSO4. Two hits are circled.
FIG. 2.
Survival of mutants affected in putative persister genes. Cells were grown to stationary phase in MOPS minimal medium in 96-well plates (final concentration, ∼1× 109 CFU/ml) and treated with 5 μg/ml ofloxacin for 6 h. Samples were diluted and spot plated on LB agar. The experiment was performed in triplicate, and error bars represent standard deviations. WT, wild type.
FIG. 3.
Time-dependent killing studies of mutants whose persister genes were affected. One-milliliter cultures were grown to stationary phase in MOPS minimal medium (final concentration, ∼1 × 109 CFU/ml) and treated with 10 μg/ml ofloxacin for the indicated times. Samples were diluted and spot plated on LB agar. The experiment was performed in triplicate, and error bars represent standard deviations. WT, wild type.
FIG. 4.
Effects of Δ_ygfA_ and Δ_ssrS_ mutations on persister cell formation. E. coli BW25113 wild type (WT), a Δ_ygfA_ mutant (ygfA), strain KLE910 (ssrS), and strain KLE911 (ssrS ygfA) were grown to stationary phase in MOPS minimal medium and treated with 10 μg/ml ofloxacin for 6 h. Samples were diluted and spot plated on LB agar. The experiment was performed in triplicate, and error bars represent standard deviations.
FIG. 5.
Survival of mutants whose putative persister genes were affected in exponential phase. (A) One-milliliter cultures were grown to exponential phase in MOPS minimal medium and treated with 5 μg/ml ofloxacin for 3 h. (B) Time-dependent killing studies of mutants whose persister genes were affected. Mutants with a prominent low-level-persistence phenotype in log phase were exposed to 5 μg/ml ofloxacin for the indicated times. Samples were diluted and spot plated on LB agar. The experiment was performed in triplicate, and error bars represent standard deviations. WT, wild type.
FIG. 6.
Survival of mutants with low levels of persistence exposed to different classes of antibiotics. One-milliliter cultures were grown to exponential phase in MOPS minimal medium and challenged for 3 h with 1 μg/ml ciprofloxacin (A), 10 μg/ml streptomycin (B), 100 μg/ml ampicillin (C), and 25 μg/ml benzalkonium chloride (D). Samples were diluted and spot plated on LB agar. The experiment was performed in triplicate, and error bars represent standard deviations. WT, wild type.
FIG. 7.
Effects of ygfA and yigB overexpression on persister cell formation. E. coli BW25113 cells transformed with the pALS21 control (empty vector), pALS22 (yigB), and pALS23 (ygfA) were grown in MOPS minimal medium in the presence of 0.1 mM IPTG to stationary phase. The cultures were treated with ofloxacin for the indicated times. Samples were diluted and spot plated on LB agar. Closed squares, wild type; open squares, KLE921(pALS21); open diamonds, KLE922 (pALS22); closed diamonds, KLE923 (pALS23). The experiment was performed in triplicate, and error bars represent standard deviations.
FIG. 8.
Effects of mutations in global regulators IHF and HU on persister formation. (A and B) Cultures of E. coli BW25113 wild type and mutant strains were grown to stationary phase and treated with ofloxacin for 6 h. Samples were diluted and spot plated on LB agar. (A) HU mutants Δ_hupA_ (hupA) Δ_hupB_ (hupB), and KLE912 hupA hupB (hupAB); (B) IHF mutants Δ_ihfA_ (ihfA) Δ_ihfB_ (ihfB), and KLE913 ihfA ihfB (ihfAB). (C) Time-dependent killing studies of HU and IHF mutants. The cultures were grown to stationary phase in MOPS minimal medium and treated with 10 μg/ml for the indicated times. Samples were diluted and spot plated on LB agar. Closed squares, wild type; closed triangles, KLE912 hupA hupB; open squares, KLE913 ihfA ihfB. All experiments were performed in triplicate, and error bars represent standard deviations.
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