EmrA1 membrane fusion protein of Francisella tularensis LVS is required for resistance to oxidative stress, intramacrophage survival and virulence in mice

Abstract

Francisella tularensis is a category A biodefence agent that causes a fatal human disease known as tularaemia. The pathogenicity of F. tularensis depends on its ability to persist inside host immune cells primarily by resisting an attack from host-generated reactive oxygen and nitrogen species (ROS/RNS). Based on the ability of F. tularensis to resist high ROS/RNS levels, we have hypothesized that additional unknown factors act in conjunction with known antioxidant defences to render ROS resistance. By screening a transposon insertion library of F. tularensis LVS in the presence of hydrogen peroxide, we have identified an oxidant-sensitive mutant in putative EmrA1 (FTL_0687) secretion protein. The results demonstrate that the emrA1 mutant is highly sensitive to oxidants and several antimicrobial agents, and exhibits diminished intramacrophage growth that can be restored to wild-type F. tularensis LVS levels by either transcomplementation, inhibition of ROS generation or infection in NADPH oxidase deficient (gp91Phox(-/-)) macrophages. The emrA1 mutant is attenuated for virulence, which is restored by infection in gp91Phox(-/-) mice. Further, EmrA1 contributes to oxidative stress resistance by affecting secretion of Francisella antioxidant enzymes SodB and KatG. This study exposes unique links between transporter activity and the antioxidant defence mechanisms of F. tularensis.

Fig. 1. Identification and characterization of H₂O₂ sensitive mutants of F. tularensis LVS

(A) H₂O₂ sensitive mutants identified by screening of transposon mutant library of F. tularensis LVS in the presence of 250μM of H₂O_2. (B) The H₂O₂ sensitive mutants were grown in large culture volumes (25 ml) in the absence or presence of 250μM H₂O₂. The cultures were grown for 24 hrs and OD₆₀₀ readings were recorded every 4 hrs. (C) Disc diffusion assay with superoxide generating compound pyrogallol (Concentration = 50μg/disc). The data are cumulative of two independent experiments. The data were analyzed by ANOVA with a Tukey-Kramer post-test, and a cut-off p value of 0.05 or less was considered significant. *, p<0.05; **, p<0.01. (D) MHS- cells were infected with the F. tularensis (Ft) LVS, or the H₂O₂ sensitive mutants at 100 MOI (n=4 biological replicates). The cells were lysed at 24 hrs and plated on MH-chocolate agar plates for enumeration of bacterial CFU. The data are representative of three independent experiments conducted with identical results and were analyzed by ANOVA with a Tukey-Kramer post-test, and a cut-off p value of 0.05 or less was considered significant. Comparisons are shown with Ft LVS. **, p<0.01

Fig. 2. Genomic organization of the emrA1 gene locus of F. tularensis LVS

(A) The genomic organization of the emrA1 gene locus. Arrows on the top indicate the location of primers used for RT-PCR while the small arrows indicate location of primers used for qRT-PCR. The inverted triangle depicts the location of transposon insertion in the emrA1 gene. (B) Unique organization of components of the Emr multidrug efflux pump of F. tularensis. Genes enclosed in dashed box indicate that all the three components of the Emr multidrug efflux pump are transcribed at the same location. (C) RT-PCR analysis of the intergenic regions between FTL_0689 and FTL_0688 (primers A+B in Fig. 1A); FTL_0688 and FTL_0687 (C+D), and FTL_0687 and FTL_0686c (E+F). RNA (no RT) and DNA were kept as negative and positive controls, respectively. (D) Alignment of the conserved RLS motif in EmrA1 of F. tularensis LVS, SchuS4 and other gram-negative bacterial pathogens. The numbers indicate amino acid positions. The arrows indicate the position of conserved RLS motifs.

Fig. 3. The emrA1 mutant of F. tularensis is highly sensitive to oxidative stress

(A) F. tularensis (Ft) LVS, emrA1 and emrA1+ pemrA1 transcomplemented strains were grown in MH-broth containing 1000 μM of H₂O₂. The cultures were grown for 24 hrs and OD₆₀₀ readings were recorded every 4 hrs. (B) The bacterial killing assay. The bacterial cultures were exposed to 1000 μM of H₂O_2, 1 mM Pyrogallol or 1mM Paraquat for 1 and 3 hrs (n=3 biological replicates). The cultures were diluted 10-fold and plated on MH-chocolate agar plates for bacterial enumeration. The results are expressed as Log₁₀ CFU/ml. The data are representative of three independent experiments conducted with identical results. The data were analyzed by ANOVA with a Tukey-Kramer post-test, and a cut-off p value of 0.05 or less was considered significant. Comparisons are shown with Ft LVS. **, p<0.01; ***, p<0.001. (D) Disc diffusion assay with superoxide generating compound pyrogallol (Concentration = 50μg/disc).

Fig. 4. The emrA1 mutant of F. tularensis is deficient for intramacrophage survival and its killing by macrophages is ROS-dependent

MHS- cells (A, B and C) or primary BMDMs derived from wild type C57BL/6 (D) or gp91Phox^−/− mice (E) were infected with the F. tularensis (Ft) LVS, the emrA1 mutant or the transcomplemented strain (emrA1 + pemrA1) at 100 MOI (n=4 biological replicates). The cells were lysed at 4 and 24 hrs and plated on MH-chocolate agar plates for enumeration of bacterial CFU. The MH-S cells were treated with ROS inhibitors apocynin (B) or N- Acetyl Cysteine (NAC) (C) prior to infection. The data are representative of three independent experiments conducted with identical results. The data are expressed as Log₁₀ CFU/ml. The data were analyzed by ANOVA with a Tukey-Kramer post-test, and a cut-off p value of 0.05 or less was considered significant. Comparisons are shown with Ft LVS. **, p<0.01.

Fig. 5. EmrA1 mutant of F. tularensis is attenuated for virulence and its virulence is restored in gp91Phox^−/− mice

Survival of C57BL/6 mice (n = 5) infected intranasally with 1×10⁵ (A) and 1×10⁶ CFU (B) of F. tularensis LVS or the emrA1 mutant and observed for mortality. C57BL/6 wild type mice or gp91Phox^−/− mice (n=3) were infected intranasally with 1×10⁵ (C) and 1×10⁶ CFU (D) of the emrA1 mutant. The data from a single experiment are shown. The mice were observed for the indicated periods for morbidity and mortality. The results are expressed as Kaplan-Meier survival curves and the P values were determined using Log-rank test.

Fig. 6. EmrA1 mutant fails to secrete antioxidant enzymes SodB and KatG

The cultures of F. tularensis (Ft) LVS, the emrA1 mutant or the transcomplemented strain (emrA1+ pemrA1) were grown in MH- (A) or BHI-broth (B). The culture filtrates or the lysates of the bacterial pellet were analyzed at the indicated times by western blot analysis using anti-SodB and anti-KatG antibodies. (C) Aliquots of the bacterial strain grown in MHB or BHI were collected at the indicated time points, diluted 10-fold and plated on MH-chocolate agar plates to enumerate bacterial numbers (n=3). (D) The western blots of the MH-B or BHI-grown culture filtrates probed with anti-SodB antibodies were stripped and re-probed with antibodies against 50S ribosomal protein Rp1L at the indicated times (top panel). Agarose gel electrophoresis of the genomic DNA isolated from culture filtrates from the BHI-grown bacteria at indicated time points (lower panel). (E) Determination of catalase activity of secreted KatG by amplex red assay. The amount of H₂O₂ consumed by culture filtrates of Ft LVS, the emrA1 mutant and the transcomplemented strain collected at the indicated times. The data are representative of at least 3–5 independent experiments conducted with identical results. The data were analyzed by ANOVA with a Tukey-Kramer post-test, and a cut-off p value of 0.05 or less was considered significant. Comparisons are shown with Ft LVS. ***, p<0.001.

Fig. 7. EmrA1 contributes to the secretion of KatG and extracellular KatG is essential to overcome H₂O₂ toxicity

(A) Growth curves of F. tularensis (Ft) LVS and the sodB, sodBC and ΔkatG mutant in the absence or presence of 250μM of H₂O_2. The cultures were grown for 24 hrs and OD₆₀₀ readings were recorded every 4 hrs. Growth curves of F. tularensis (Ft) LVS and the emrA1 mutant (B) and ΔkatG mutant (C) in the absence or presence of 250μM of H₂O₂ and with or without the addition of recombinant F. tularensis KatG (rKatG). The cultures were grown for 24 hrs and OD₆₀₀ readings were recorded every 4 hrs. (D) F. tularensis LVS (LVS) and the emrA1 mutant were grown in MH-broth for 4 and 8 hrs. The bacteria were pelleted and the supernatants were collected and filtered through a 0.22μ filter to remove the bacteria. The F. tularensis LVS pellets were then either resuspended in its own culture supernatants or those from the emrA1 mutant. Similarly, the emrA1 pellets were either resuspended in its own culture supernatants or those from F. tularensis LVS. The resuspended bacteria were either left untreated or exposed to 500μM of H₂O₂ for 1 hr. The bacteria were diluted 10-fold and plated on MH-chocolate-agar plates to enumerate the viable bacteria. The data are representative of 3 independent experiments conducted with identical results. The data were analyzed by ANOVA with a Tukey-Kramer post-test, and a cut-off p value of 0.05 or less was considered significant. Comparisons are shown with Ft LVS. *, p<0.05, **, p<0.01.

Fig. 8. Secretion of SodB and katG is independent of TolC and Sec-dependent secretion pathway

(A) Growth curves of F. tularensis (Ft) LVS and the ΔtolC mutant in the absence or presence of 500μM of H₂O₂. (B) The culture filtrates or the lysates of the bacterial pellet of F. tularensis (Ft) LVS or the ΔtolC mutant grown in BHI-broth were analyzed at the indicated times by western blot analysis using anti-SodB and anti-KatG antibodies. The data are representative of at least 2–3 independent experiments conducted with identical results. (C) Wild type F. tularensis (Ft) LVS and the secA and secE mutants of F. tularensis LVS were grown in BHI-broth for 4, 8 and 12 hrs. The culture filtrates or the lysates of the bacterial pellet were analyzed at the indicated times by western blot analysis using anti-SodB and anti-KatG antibodies. The data are representative of 2 independent experiments conducted with identical results.