Superoxide dismutase B gene (sodB)-deficient mutants of Francisella tularensis demonstrate hypersensitivity to oxidative stress and attenuated virulence

Abstract

A Francisella tularensis live vaccine strain mutant (sodB(Ft)) with reduced Fe-superoxide dismutase gene expression was generated and found to exhibit decreased sodB activity and increased sensitivity to redox cycling compounds compared to wild-type bacteria. The sodB(Ft) mutant also was significantly attenuated for virulence in mice. Thus, this study has identified sodB as an important F. tularensis virulence factor.

FIG. 1.

Generation and screening of sodB mutants of F. tularensis LVS. (A) Schematic representation of the strategy used for generation and identification of sodB_Ft mutants. Substitution of GTG for ATG and a PstI site introduced upstream of the start codon for screening of sodB_Ft mutants are indicated. Locations of various primers used for the generation of mutants are indicated by arrows. (B) Confirmation of sodB_Ft mutants. A colony PCR was performed using the flanking primers Xba-sodB and Sal-sodB (95^οC for 1 min, 53^οC for 1 min, and 72^οC for 1 min for 30 cycles, followed by a final extension at 72^οC for 5 min); the amplified products were digested with PstI and run on a 1.2% agarose gel. Lanes 1 and 8, molecular mass markers (1-kb DNA ladder; Invitrogen Corporation, Carlsbad, CA). Lanes 2 and 4, PCR products amplified from two sodB_Ft mutant colonies after the first and second recombination events, respectively, and their digestion with PstI (lanes 3 and 5). Lanes 6 and 7, PCR product amplified from the wild-type F. tularensis LVS and its digestion with PstI. Digestion of PCR products amplified from the sodB_Ft mutant colonies with PstI confirmed mutation of the sodB gene.

FIG. 2.

FeSOD activity assay. Crude cell extracts of F. tularensis LVS and sodB_Ft grown on modified chocolate agar plates were prepared by resuspending 10 mg of the bacterial culture in 1.0 ml of lysis solution (50 mM Tris-HCl [pH 7.4], 0.1 mM EDTA, and 0.2 mg lysozyme per ml). The lysates were clarified, and the protein concentration of each lysate was determined with a bicinchoninic protein assay kit (Pierce, Rockford, IL). Protein (15 μg) was loaded on a 10% native polyacrylamide gel electrophoresis gel and run at 150 V for 90 min at 4°C. The gel was stained for SOD activity by the method of Beauchamp and Fridovich (3). (A) Zymogram demonstrating the expression of active FeSOD of F. tularensis LVS following 24, 48, and 72 h of growth on modified chocolate agar plates. (B) FeSOD activity of sodB_Ft mutants. Lane 1, F. tularensis LVS; lane 2, sodB_Ft mutant; lane 3, a fibrosarcoma cell line transfected with MnSOD was used as a positive control. Note the markedly reduced expression of FeSOD in the sodB_Ft mutant.

FIG. 3.

(A) Growth curve of F. tularensis LVS and sodB_Ft. MHB (25 ml) in a 125-ml flask was inoculated with 1 ml of an overnight culture of F. tularensis LVS or sodB_Ft and agitated at 175 rpm and 37°C in a shaking incubator. Aliquots were removed at the times indicated to measure optical density at 600 nm (OD₆₀₀), and 10-fold dilutions were plated on modified chocolate agar for the determination of CFU levels. (B) Effect of sodB mutation on sensitivity to H₂O₂. F. tularensis LVS and sodB_Ft mutants were grown in MHB to an OD of 0.2. The indicated concentrations of H₂O₂ were then added, and the cultures were allowed to grow for 48 h in a shaking incubator. OD readings were taken every 4 h. (C) Aliquots were removed from the cultures grown in the presence of 1 mM of H₂O₂ at the times indicated and plated on modified chocolate agar plates to determine the CFU counts. Percent survival was calculated using the mean of the results obtained with duplicate samples at the timed intervals and compared to data at 0 h. All results are representative of two independent experiments.

FIG. 4.

Effect of sodB mutation on sensitivity to paraquat. (A) The left and right panels represent F. tularensis LVS and sodB_Ft mutant strains, respectively. Briefly, 1 × 10⁹ F. tularensis LVS or sodB_Ft bacteria were plated on modified chocolate agar plates. Sterile filter paper discs were placed on each plate, and 5 μl of 1.25, 2.5, 5.0, and 10 mg/ml concentrations of freshly prepared paraquat was added to individual discs. The plates were incubated for 48 to 72 h at 37°C, and the zones of growth inhibition were observed. Insets show the zone of growth inhibition observed with kanamycin, which was used as an experimental control. (B) Quantitation of the zone of growth inhibition. The zone of growth inhibition around the discs impregnated with a 10 mg/ml concentration of paraquat was quantitated using AlphaEase FC software (Alpha Innotech Inc., San Leandro, CA). Data represent the means ± standard errors of the means for the zone of inhibition determined for each strain (n = 5 plates per strain) and the cumulative results of two independent experiments. Comparisons between the strains were made using a nonparametric Mann-Whitney test (P < 0.0001). (C) F. tularensis LVS and the sodB_Ft mutant strain were grown in MHB to an OD₆₀₀ of 0.2. Paraquat (1 mM) was then added to the cultures, and cultures were allowed to grow at 37°C with constant shaking. Aliquots were removed at the times indicated, diluted 10-fold, and plated on modified chocolate agar plates to determine the number of CFU. Data are representative of two independent experiments.

FIG. 5.

Effect of sodB mutation on virulence in mice. (A) Survival of C57BL/6 (n = 12) and BALB/c (n = 10) mice infected intranasally with 1 × 10⁴ CFU of F. tularensis LVS or sodB_Ft bacteria. Results are expressed as Kaplan-Meier curves and P values determined using a log rank test. (B) Kinetics and growth of F. tularensis LVS and sodB_Ft in C57BL/6 mice. C57BL/6 mice were inoculated intranasally with 5 × 10³ CFU of F. tularensis LVS or sodB_Ft. Three mice were killed at each indicated time point, and homogenates of the lungs, liver, and spleen were plated for the determination of bacterial burden. Results represent the means ± standard errors of CFU counts (n = 3 per time point). **, P < 0.01; ***, P < 0.001 (using the nonparametric Mann-Whitney test). ND, not detected. All results are representative of two independent experiments.