Table 1.
List of bacterial strains and plasmids used in this study.
Table 2.
List of primer sequences used in this study.
Fig 1.
Genomic organization and growth characteristics of the ΔahpC mutant of F. tularensis LVS.
(A) Genomic organization of the ahpC gene. (B) Growth curves of F. tularensis LVS, the ΔahpC mutant and the transcomplemented strains (ΔahpC + pahpC). Equal numbers of bacteria were suspended in Mueller-Hinton broth, and the optical densities (OD600) were recorded every 4 hours.
Fig 2.
The ΔahpC mutant of F. tularensis LVS exhibits enhanced sensitivities towards superoxide generating compounds.
The sensitivities of the wild type F. tularensis (Ft) LVS, the ΔahpC mutant, and the transcomplemented strain (ΔahpC+pahpC) as determined by disc diffusion and spot assays against superoxide-generating compounds menadione (A and D), pyrogallol (B and E) and paraquat (C and F). For disc diffusion assays, the results are expressed as a zone of inhibition in millimeters obtained using the indicated concentrations of the compounds and are expressed as Mean ± S.D. of triplicate samples. In spot assays, the Francisella strains were exposed to serially diluted menadione, pyrogallol and paraquat for 1 and 3 hours and spotted on MH-chocolate agar plates to determine the bacterial killing. The red arrows indicate enhanced killing of the ΔahpC mutant at the indicated concentrations of the compounds. All the results shown are representative of 3 independent experiments conducted. The p values were determined by one-way ANOVA and a p-value of <0.05 is considered statistically significant. *p<0.05; ***p<0.001.
Fig 3.
The ΔahpC mutant of F. tularensis LVS exhibits enhanced sensitivity towards organic peroxides and H2O2.
The sensitivities of the wild type F. tularensis (Ft) LVS, the ΔahpC mutant, and the transcomplemented strain ΔahpC+pahpC as determined by disc diffusion and spot assays against organic peroxides tert-butyl hydroperoxide (TBH) (A and D), cumene hydroperoxide (CHP) (B and E) and H2O2 (C and F). For disc diffusion assays, the results are expressed as zone of inhibition in millimeters obtained using the indicated concentrations of the compounds and are expressed as Mean ± S.D. of triplicate samples. In spot assays, Francisella strains were exposed to serially diluted TBH, CHP, and H2O2 for 1 and 3 hours and spotted on MH-chocolate agar plates to determine the bacterial killing. The red arrows indicate enhanced killing of the ΔahpC mutant at the indicated concentrations of the compounds. (G) Growth curves of F. tularensis LVS, the ΔahpC mutant and the transcomplemented strain (ΔahpC + pahpC) in the absence or presence of 750μM H2O2. Equal numbers of bacteria were suspended in Mueller-Hinton broth and the optical density (OD600) was recorded every 4 hours. All the results shown are representative of 3 independent experiments conducted with identical results. The p values were determined by one-way ANOVA and a p value of <0.05 is considered statistically significant. *p<0.05; ***p<0.001.
Fig 4.
The ΔahpC mutant of F. tularensis LVS exhibits enhanced sensitivity towards RNS.
Spot assays were performed using nitric oxide (NO) donors (A) SNP and (B) Sin-1. Wild type F. tularensis LVS, the ΔahpC mutant and the transcomplemented strains (ΔahpC + pahpC) were exposed to serially diluted compounds for 1 and 3 hours and spotted on MH-chocolate agar plates. The red arrows indicate enhanced killing of the ΔahpC mutant at the indicated concentrations of the compounds. The results shown are representative of 3 independent experiments conducted.
Fig 5.
The ΔahpC mutant of F. tularensis LVS does not exhibit intramacrophage growth defect, but is attenuated for virulence in mice.
(A) Raw264.7 macrophages were infected with the F. tularensis (Ft) LVS, the ΔahpC mutant or the transcomplemented strain (ΔahpC+pahpC) at 10 and 100 MOI (n = 3 biological replicates). The cells were lysed after 4 and 24 hrs of infection, serially diluted and plated on MH-chocolate agar plates for enumeration of bacterial CFU. The data are representative of three independent experiments conducted and are expressed as Log10 CFU/mL. (B) C57BL/6 and phox-/- mice (n = 4 mice/group) were infected intranasally with 1x104 CFUs of F. tularensis LVS or the ΔahpC mutant and observed for mortality for a period of 21 days post-infection. The results are expressed as Kaplan-Meier survival curves, and the p values were determined using the Log-rank test. The comparison shown is between the wild type mice infected with Ft LVS and the ΔahpC mutant.
Fig 6.
AhpC of F. tularensis SchuS4 is a major antioxidant enzyme that protects against oxidative stress induced by superoxide generating compounds.
The sensitivities of the wild type F. tularensis (Ft) SchuS4, the ΔahpC, ΔsodC and the ΔkatG mutants of SchuS4 as determined by disc diffusion (A), spot assay (B) and bacterial killing assay (C) against superoxide-generating compound, menadione. The sensitivity of the indicated strains against paraquat (D) and pyrogallol (E) was determined using the indicated concentration of the compounds by disc diffusion assay. For the disc diffusion assays, the results are expressed as a zone of inhibition in millimeters and are expressed as Mean ± S.D. The red arrows in (B) indicate enhanced killing of the SchuS4 ΔahpC mutant at the indicated concentrations of menadione. For bacterial killing assay (C) indicated bacterial strains were exposed to menadione (6.25μg/mL) and the bacterial numbers were enumerated after 1 and 4 hours of exposure. PBS, and ethanol required for suspension of menadione were used as controls. The data shown are representative of 2 independent experiments each conducted with 3 biological replicates and were analyzed by one-way ANOVA. **P<0.01; ***P<0.001.
Fig 7.
AhpC of F. tularensis SchuS4 protects against oxidative stress induced by peroxides.
The sensitivities of the wild type F. tularensis (Ft) SchuS4, the ΔahpC, ΔsodC and the ΔkatG mutants of SchuS4 as determined by disc diffusion assays against tert-butyl hydroperoxide (TBH) (A), cumene hydroperoxide (CHP) (C) and H2O2 (E), and by spot assays against TBH (B) and CHP (D). For the disc diffusion assays, the results are expressed as zone of inhibition in millimeters and are expressed as Mean ± S.D. The red arrows in (B and D) indicate enhanced killing of the ΔahpC mutant at the indicated concentrations of the compounds. The data shown are representative of two independent experiments each conducted with 3 biological replicates and were analyzed by one-way ANOVA, and p values were recorded. *P<0.05; ***P<0.001.
Fig 8.
Exposure to nitric oxide generating compounds results in enhanced killing of the ΔahpC mutant of F. tularensis SchuS4.
Spot assays were performed using (A) sodium nitroprusside (SNP) and (B) Sin-1. Wild type F. tularensis (Ft) SchuS4, the ΔahpC, ΔsodC and the ΔkatG mutants of SchuS4 were exposed to serially diluted compounds for 1 hour and spotted on MH-chocolate agar plates. The red arrows indicate enhanced killing of the ΔahpC mutant at the indicated concentrations of the compounds. The results shown are representative of two independent experiments conducted.
Fig 9.
The ΔahpC mutant of F. tularensis SchuS4 is attenuated for intramacrophage growth.
Raw264.7 macrophages were infected with the wild type F. tularensis (Ft) SchuS4, the ΔahpC, ΔsodC and the ΔkatG mutants of SchuS4 at 100 MOI (n = 3 biological replicates). The cells were lysed after 4 and 24 hrs of infection, serially diluted and plated on MH-chocolate agar plates for enumeration of bacterial CFU. The data are expressed as Log10 CFU/mL. The data shown are representative of two independent experiments each conducted with 3 biological replicates and were analyzed by one-way ANOVA, and p values were recorded. **P<0.01.