Roles of reactive oxygen species-degrading enzymes of Francisella tularensis SCHU S4

Abstract

Francisella tularensis is a facultative intracellular bacterium utilizing macrophages as its primary intracellular habitat and is therefore highly capable of resisting the effects of reactive oxygen species (ROS), potent mediators of the bactericidal activity of macrophages. We investigated the roles of enzymes presumed to be important for protection against ROS. Four mutants of the highly virulent SCHU S4 strain with deletions of the genes encoding catalase (katG), glutathione peroxidase (gpx), a DyP-type peroxidase (FTT0086), or double deletion of FTT0086 and katG showed much increased susceptibility to hydrogen peroxide (H2O2) and slightly increased susceptibility to paraquat but not to peroxynitrite (ONOO(-)) and displayed intact intramacrophage replication. Nevertheless, mice infected with the double deletion mutant showed significantly longer survival than SCHU S4-infected mice. Unlike the aforementioned mutants, deletion of the gene coding for alkyl-hydroperoxide reductase subunit C (ahpC) generated a mutant much more susceptible to paraquat and ONOO(-) but not to H2O2. It showed intact replication in J774 cells but impaired replication in bone marrow-derived macrophages and in internal organs of mice. The live vaccine strain, LVS, is more susceptible than virulent strains to ROS-mediated killing and possesses a truncated form of FTT0086. Expression of the SCHU S4 FTT0086 gene rendered LVS more resistant to H2O2, which demonstrates that the SCHU S4 strain possesses additional detoxifying mechanisms. Collectively, the results demonstrate that SCHU S4 ROS-detoxifying enzymes have overlapping functions, and therefore, deletion of one or the other does not critically impair the intracellular replication or virulence, although AhpC appears to have a unique function.

FIG 1

Changes in bacterial numbers of indicated F. tularensis strains after exposure to 4 mM H₂O₂ for 2 h. The bars represent the average decrease in bacterial viability from three replicates, and the error bars represent the standard error of the mean (SEM) (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). Similar results were observed in two additional experiments. The bacterial number at the initiation of the experiment for each strain was approximately 6.0 log₁₀ CFU.

FIG 2

Inhibition of the growth of F. tularensis strains after exposure to paraquat. A disk containing 10 μl of the indicated concentration of paraquat was placed on an agar plate. The bars represent the average of the growth inhibition zone (diameter) from three separate plates. The error bars represent the SEM (*, P < 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

FIG 3

Changes in bacterial numbers of indicated F. tularensis strains after exposure to SIN-1. The bars represent the average change in bacterial viability from three replicates, and the error bars represent the SEM (**, P ≤ 0.01; ***, P ≤ 0.001). Similar results were observed in two additional experiments. (A) Exposure to 8.0 mM SIN-1 for 2 h. (B) Exposure to 10 mM SIN-1 for 2 h. The bacterial number at the initiation of the experiment for each strain was approximately 6.0 log₁₀ CFU.

FIG 4

Intracellular replication of the indicated F. tularensis strains in BMDM with or without IFN-γ. Cells were infected at an MOI of 30 (without IFN-γ) or 200 (with IFN-γ), and bacterial numbers were determined after 24 h. ***, P < 0.001.

FIG 5

Survival of C57BL/6 mice after infection with SCHU S4 (squares), the ΔFTT0086 mutant (circles), and the ΔFTT0086 ΔkatG mutant (triangles). Mice were injected intradermally with 10 CFU of the indicated strain. Each group contained 5 mice.

FIG 6

Multiple sequence alignments of FTT0086, FTL1773, and homologous DyP-type peroxidases. The sequence alignments were performed using ClustalW, and ESPript was used to display the protein alignment (36, 37). Conserved residues are indicated in white on black. Similar amino acid residues are in bold. The D134, H203, and R220 residues of DyP are indicated with black stars. The GXXDG motif is boxed. The sequences of TyrA and BtDyP were retrieved from PDB under accession no. 2hag and 2gvk, respectively. BtDyp is the Dyp homologue from Bacteroides thetaiotaomicron and TyrA the homologue from Shewanella oneidensis.

FIG 7

Changes in bacterial numbers of the indicated F. tularensis strains after exposure to 1 mM H₂O₂ for 2 h. The bars represent the average decrease in bacterial viability from three separate tubes, and the error bars represent the SEM (***, P ≤ 0.001). Similar results were observed in two additional experiments. The bacterial number at the initiation of the experiment for each strain was approximately 6.0 log₁₀ CFU.