B. mallei is resistant to the oxidative burst of macrophages despite its marked susceptibility to ROS We investigated the possible mechanism for the apparent dispensability of the NADPH oxidase in the intracellular killing of B. mallei. B. mallei was found to be susceptible to authentic H2O2 (fig. 6A). As little as 100 mM H2O2 killed over 90% of B. mallei after 2 h. A kinetic study revealed that the viability of B. mallei was markedly reduced 90 min upon exposure to 200 mM H2O2, declining further thereafter (fig. 6B). These findings demonstrate that B. mallei are extraordinarily susceptible to ROS-mediated killing. The ability of Burkholderia to escape into the cytosol [26,27] could rationalize the apparent dispensability of NADPH oxidase in the anti-B. mallei arsenal of macrophages. To test the intracellular localization of B. mallei, the ability of the bacterium to form actin- tails was evaluated by fluorescence microscopy. As shown in figure 6C–E, B. mallei do not appear to polymerize actin tails at the early times examined here, although polymerization of actin was evident in about 10% of the B. mallei-containing phagosomes (figure 6E). Electron microscopy studies revealed that the totality of B. mallei were found within phagosomes in both unstimulated and IFNc-primed macrophages (fig. 6F and G). Collectively, the fluorescence and electron microscopy studies indicate that B. mallei remain within the confines of phagosomal membranes during the NO killing of B. mallei early stages of infection studied herein, irrespective of whether the phagocytes are stimulated with IFNc (fig. 6F & G). We tested whether the seeming dispensability of the NADPH oxidase in the anti-B. mallei activity of macrophages was due to a poor respiratory burst. Estimates of O2N2 production by the superoxide dismutase- inhibitable reduction of cytochrome c revealed that B. mallei was a poor stimulant of the respiratory burst as compared to an aflagellated fliC fljB Salmonella control (fig. 6H). Discussion O2-dependent host defenses are the best-characterized compo- nents of the antimicrobial arsenal of professional phagocytes. O2 is used by NADPH oxidase and iNOS hemoproteins for the generation of O2N2 and NO, which are the precursors of a plethora of ROS and RNS. Pathogenic microorganisms show a spectrum of susceptibilities to the antimicrobial actions of RNS. For example, RNS are essential for resistance to Mycobacterium tuberculosis and Leishmania major [12,28], but appear to be dispensable in host defense against Legionella pneumophila and Pseudomonas aeruginosa [25]. Similar to Mycobacterium and Leishmania, our investigations have shown that the intracellular pathogen B. mallei is highly susceptible to RNS-mediated host defenses. We find Figure 6. Susceptibility of Burkholderia to ROS generated by macrophages. The susceptibility of B. mallei to increasing concentrations of H2O2 is shown in A. Kinetic analysis of the viability of B. mallei in response to 200 mM H2O2 is shown in B. The cellular localization of phalloidin-labeled actin filaments (C) and GFP-expressing B. mallei (D) was examined by fluorescence microscopy. The host cell nucleus labeled with DAPI (blue), B. mallei (green) and actin (red) are visualized in the merge panel (E). The micrograph is representative of a total of 285 bacteria examined from 33 cells collected in 2 independent experiments. The location of B. mallei in the cytoplasm of control (F) and IFNc-primed (G) macrophages from C57BL/6 mice was visualized by transmission electron microscopy 3.5 h after infection. Bars in F and G denote 2 and 0.5 mm, respectively. The micrographs are representative of 97 Burkholderia-containing phagosomes from 5 independent experiments. Production of superoxide by B. mallei-infected macrophages isolated from C57BL/6 mice was assessed spectrophotometrically as the superoxide dismutase-inhibitable reduction of cytochrome c (H). Data in panels A, B and G represent the mean 6 sem from 6 independent observations. doi:10.1371/journal.pone.0001976.g006 PLoS ONE | www.plosone.org 7 April 2008 | Volume 3 | Issue 4 | e1976