Role of peroxiredoxin of the AhpC/TSA family in antioxidant defense mechanisms of Francisella tularensis

Abstract

Francisella tularensis is a Gram-negative, facultative intracellular pathogen and the causative agent of a lethal human disease known as tularemia. Due to its extremely high virulence and potential to be used as a bioterror agent, F. tularensis is classified by the CDC as a Category A Select Agent. As an intracellular pathogen, F. tularensis during its intracellular residence encounters a number of oxidative and nitrosative stresses. The roles of the primary antioxidant enzymes SodB, SodC and KatG in oxidative stress resistance and virulence of F. tularensis live vaccine strain (LVS) have been characterized in previous studies. However, very fragmentary information is available regarding the role of peroxiredoxin of the AhpC/TSA family (annotated as AhpC) of F. tularensis SchuS4; whereas the role of AhpC of F. tularensis LVS in tularemia pathogenesis is not known. This study was undertaken to exhaustively investigate the role of AhpC in oxidative stress resistance of F. tularensis LVS and SchuS4. We report that AhpC of F. tularensis LVS confers resistance against a wide range of reactive oxygen and nitrogen species, and serves as a virulence factor. In highly virulent F. tularensis SchuS4 strain, AhpC serves as a key antioxidant enzyme and contributes to its robust oxidative and nitrosative stress resistance, and intramacrophage survival. We also demonstrate that there is functional redundancy among primary antioxidant enzymes AhpC, SodC, and KatG of F. tularensis SchuS4. Collectively, this study highlights the differences in antioxidant defense mechanisms of F. tularensis LVS and SchuS4.

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.