that the inactivation of [Fe-S] clusters is a major component of the anti-B. mallei activity of RNS. Poor killing of B. mallei by IFNc-treated macrophages lacking iNOS, irrespective of the NADPH oxidase status of the cell, suggests that the RNS-mediated killing of B. mallei does not depend on host-derived ONOO2 synthesis. NO itself, nitrogen oxides derived from the autooxidation of NO in the presence of O2, or ONOO2 generated in the bacterial cytoplasm from the reaction of NO with endogenous O2N2 represent a few of the RNS that may be responsible for the iNOS-dependent killing of B. mallei. B. mallei are extraordinarily susceptible to RNS as shown by the profound killing seen after treatment with 10 mM spermine NONOate. NO donors also exert antimicrobial activity against phylogenetically diverse microbes such as Candida albicans, Salmonella enterica and Escherichia coli [29–31]. However, NO donors, even when used at high micromolar or low millimolar concentrations, are cytostatic for these microbial pathogens. Therefore, our studies identify B. mallei as one of the few pathogens for which host-derived RNS are microbicidal. Aconitase is inactivated upon incubation of B. mallei with NO congeners, possibly reflecting the RNS-dependent oxidation of the solvent exposed Fea in the [Fe-S] cluster [32,33]. It is therefore not surprising that genes encoding for iron-sulfur cluster assembly are highly transcribed in NO-treated B. mallei. RNS-mediated oxidation of [Fe-S] clusters of dehydratases [33–36] has for a long time been seen as a concomitant event of cytotoxic, NO- producing macrophages [32]. However, a causative relationship between the oxidation of [Fe-S] clusters and killing by RNS has not been previously demonstrated. The rapid loss in viability and aconitase activity of an iscR mutant exposed to sublethal NO concentrations demonstrate a direct relationship between [Fe-S] cluster damage and the bactericidal activity of RNS against B. mallei. Oxidation of [Fe-S] prosthetic groups of dehydratases critical for intermediate metabolism may explain the bactericidal activity of RNS against the obligate aerobe B. mallei. According to our model, the overt resistance of facultative anaerobes to NO reflects the ability of these microbes to generate energy by switching from oxidative phosphorylation heavily dependent on RNS-modifiable dehydratases to fermentative pathways resilient to nitrosative stress. Future experiments will be needed to test this hypothesis. Our data indicate that the NADPH oxidase is dispensable for intracellular killing of B. mallei. These findings might seem striking since B. mallei are readily killed upon exposure to H2O2. Microbes have devised an assortment of strategies to avoid NADPH oxidase- dependent cytotoxicity. For example, detoxification of oxyradicals by superoxide dismutases, catalases and hydroperoxydases or scavengers such as low molecular weight thiols are efficient ways to diminish oxidative stress [6]. The antimicrobial activity of the NADPH oxidase is best manifested against intracellular pathogens confined within endocytic membranes [37,38]. At the early times examined here, B. mallei do not escape into the cytosol as has been shown for various Burkholderia spp. at later time points after infection [27,39]. The poor respiratory burst elicited by B. mallei represents a mechanism for minimizing exposure to the NADPH oxidase during the time that this intracellular pathogen remains in the phagosome. Analogous to other microorganisms [40], the putative polysaccharide extracellular capsule [41] may help B. mallei avoid signaling cascades that stimulate a productive References NO killing of B. mallei respiratory burst. The resistance of B. mallei to the NADPH oxidase-mediated antimicrobial activity of macrophages does not, however, eliminate the possibility that this hemoprotein may play a role in the purulent phase of the host response as has been shown for B. cepacia and B. pseudomallei [12,14]. O2-independent mechanisms are a significant component of the innate killing of B. mallei as indicated by the fact that macrophages from doubly immunodeficient mice lacking both NADPH oxidase and iNOS hemoproteins efficiently kill this bacterium. Innate activation of the autophagic pathway as described for the respiratory pathogen Legionella pneumophila [42–44] is one possible mechanism for the O2-independent killing of B. mallei seen in macrophages lacking both gp91phox and iNOS. Recent studies have shown that small GTPases induced in response to IFNc stimulate killing of M. tuberculosis by promoting autophagosome formation [45,46]. Most of the enhanced antimicrobial activity stimulated by IFNc against M. tuberculosis and Salmonella enterica is nonetheless associated with the production of RNS [21]. In contrast to these two intracellular pathogens, the enhanced killing of B. mallei by IFNc-primed macrophages is exclusively dependent on the expression of functional iNOS. This role for RNS in the bactericidal activity of IFNc-primed macrophages against B. mallei contrasts with the apparent dispensability of iNOS to the early innate host defenses of unstimulated macrophages. The short time course of the infection studied herein likely prevented innate production of nitrogen oxides in B. mallei-infected macrophages. Given the extraordinary susceptibility of B. mallei to RNS (studies herein) and the stimulation of TLR4 signaling by LPS purified from B. mallei [47], it is not surprising that NO produced at later times of the innate response contributes to the killing of this intracellular pathogen [15]. In summary, our studies indicate that despite its susceptibility to ROS and RNS, B. mallei are selectively killed by RNS generated by IFNc-activated macrophages. These investigations place B. mallei among the limited number of microorganisms for which NO congeners are microbicidal. Our biochemical and genetic studies indicate that [Fe-S] clusters are critical targets of RNS-mediated killing of B. mallei. The extraordinary hypersusceptibility of B. mallei to RNS raises the intriguing possibility that NO donors could be explored as antibiotics to treat glanders, as shown in the experimental treatment of cutaneous mycosis and leishmaniasis [48,49]. However, analogous to herpes simplex virus and toxoplasma (reviewed in 49), the possibility exists that despite exerting potent antimicrobial activity, NO could still exacerbate pathology. Future experiments will be needed to determine the value of NO donors in the treatment of glanders. Acknowledgments We thank Dr. Brad T. Cookson for providing the DfliC DfljB Salmonella strain BC696, Dr. Maroof Husain for help with the aconitase assays and Dot Dill for assistance with the electron microscopy. Author Contributions Conceived and designed the experiments: AV JJ. Performed the experiments: JJ JL AS. Analyzed the data: AV JJ MV. Contributed reagents/materials/analysis tools: MH. Wrote the paper: AV JJ MV JL MH. 1. Lopez J, Copps J, Wilhelmsen C, Moore R, Kubay J, et al. (2003) Characterization of experimental equine glanders. Microbes Infect 5: 1125–1131. 2. Lehavi O, Aizenstien O, Katz LH, Hourvitz A (2002) Glanders-a potential disease for biological warfare in humans and animals. Harefuah 141 Spec No: 88–91, pp 119. PLoS ONE | www.plosone.org 8 April 2008 | Volume 3 | Issue 4 | e1976