Bacillus anthracis-derived nitric oxide is essential for pathogen virulence and survival in macrophages

Abstract

Phagocytes generate nitric oxide (NO) and other reactive oxygen and nitrogen species in large quantities to combat infecting bacteria. Here, we report the surprising observation that in vivo survival of a notorious pathogen-Bacillus anthracis-critically depends on its own NO-synthase (bNOS) activity. Anthrax spores (Sterne strain) deficient in bNOS lose their virulence in an A/J mouse model of systemic infection and exhibit severely compromised survival when germinating within macrophages. The mechanism underlying bNOS-dependent resistance to macrophage killing relies on NO-mediated activation of bacterial catalase and suppression of the damaging Fenton reaction. Our results demonstrate that pathogenic bacteria use their own NO as a key defense against the immune oxidative burst, thereby establishing bNOS as an essential virulence factor. Thus, bNOS represents an attractive antimicrobial target for treatment of anthrax and other infectious diseases.

Fig. 1.

NO protects B. anthracis against oxidative stress. (A) bNOS-dependent NO production in vivo. Representative fluorescent image of bacteria treated with Cu(II)-based NO-detecting probe (CuFL). B. anthracis Sterne (WT) and Δnos cells were grown in LB medium to OD₆₀₀ ≈0.5 followed by CuFL addition. (B) bNOS-mediated cytoprotection in B. anthracis. WT and Δnos cells were grown aerobically in LB medium to late log phase (OD₆₀₀ ≈0.8–0.9) at 30°C. An aliquot from each culture was diluted with an equal amount of fresh prewarmed LB medium and plated on BHI agar. 3MM disks were saturated with 10 mM H₂O₂ and placed on top of the bacterial lawns. Where indicated, the Brain Heart Infusion (BHI) media contained NOS inhibitors: 1 mM NG-methyl-l-arginine (L-NMA) or 1 mM L-NAME. Δ indicates the increase of the sterile zone. Values shown are the means and SD from four independent experiments. (C) Effect of nos deletion on catalase activity. Cells in the late exponential growth phase were collected one min after dilution with fresh LB medium and lysed immediately, and H₂O₂ degradation (catalase) activity was measured as described in Materials and Methods. Values are the mean ± SE from three experiments. (D) Rapid and specific protection against H₂O₂ toxicity by exogenous NO. Reagents were added as indicated to aerobically grown WT (Sterne) cells at OD₆₀₀ ≈0.5 (in LB medium at 37°C). An aqueous solution of NO (bar 2), NO-donor MAHMA NONOate (bar 4), NaNO₂ (bar 7), or oxidized NO (bar 8) was added 5 sec before challenging cells with 10 mM H₂O₂ for 30 min. NO was oxidized by bubbling air into an aqueous solution of the gas for 2 h. In additional controls, NO was mixed with H₂O₂ before addition to cells (bar 6), and 10 mM H₂O₂ was added after preincubation (5 sec) with 30 μM H₂O₂ (bar 9). The percentage of surviving cells was determined by colony counting and is shown as the mean ± SD from three experiments. (E) Time course of NO-mediated cytoprotection. Culture aliquots were taken after preincubation with NO for the indicated time intervals and challenged with 10 mM H₂O₂ for 30 min. Chloramphenicol (Cm, 200 μg/ml) was added 10 min before NO/H₂O₂ treatment. Data are shown as the mean ± SD from three experiments.

Fig. 2.

bNOS is essential for B. anthracis virulence in mice and survival in macrophages. (A) LD50 of anthrax spores as a function of bNOS activity. The indicated amounts of B. anthracis spores (Sterne or Δnos) were inoculated s.c. into 6- to 7-week-old A/J mice (n = 10 for each group). Infected animals were monitored and moribund animals were euthanized. Shown are the effect of LD100 (Left) and LD50 (Right) doses, previously established for this animal model (17) and adjusted to compensate for the compromised virulence of Δnos (Right). (B) Effect of the nos deletion on anthrax spore germination in macrophages. (Left) Comparison of WT and Δnos B. anthracis survival in J774A.1 macrophages as a function of time. Viable bacteria within macrophages were recovered and colony-forming units were determined 12 h after infection. Data are the mean of six independent experiments. To follow the spore germination status (Right), bacterial colony-forming units from macrophages were determined directly (vegetative cells) or after treatment for 40 min at 65°C (spores) at 40 min (t₀) or 2h (t₂) after phagocytosis. Data are the mean ± SD of three independent experiments (P < 0.05).

Fig. 3.

NO production in B. anthracis infected macrophages. (A) NN accumulation in clarified supernatants of J774A.1 macrophages 2 and 10 h after infection with Sterne or Δnos spores or noninfected control macrophages (MF). Experimental conditions are as in Fig. 2B. Values are the mean ± SD from four experiments. (B) Direct monitoring of NO production in J774A.1 infected or cytokines-treated macrophages (IL1β, 1 nM; TNFα, 50 ng/ml; and IFN, 250 μg/ml) with Cu(II)-based NO-detecting probe (CuFL) at 2 and 18 h after infection. The relative intensity of the fluorescent signal was calculated by IPLab Scientific image processing software.

Fig. 4.

B. anthracis NO protects bacteria from the macrophage-inflicted oxidative damage. bNOS-dependent bacterial DNA protection during macrophage infection. Chromosomal damage was monitored by qPCR. A representative agarose gel shows a 3.6-kb PCR fragment amplified from the Sterne or Δnos chromosome. Genomic DNA was isolated from J774A.1 macrophages at 2 h after infection. Control DNA was isolated from bacteria grown exponentially in BHI media without (lanes 3 and 4) or with (lanes 5 and 6) 10 mM H₂O₂ treatment. M, 1 kb DNA marker. % indicates the fraction of the full size PCR products. Values are the mean ± SD from three experiments.

Fig. 5.

The proposed mechanism of the NO-mediated defense system in B. anthracis. Upon germination, bNOS, which has been accumulated in the spore during the sporulation phase (32, 33), generates NO that instantly protects the pathogen from H₂O₂ toxicity by a dual mechanism. NO interrupts the production of damaging hydroxyl radicals from the Fenton reaction and directly activates catalase (Kat) (8), which has already been shown to be a part of the exosporium (34).