Inactivation of [Fe-S] Metalloproteins Mediates Nitric Oxide-Dependent Killing of Burkholderia mallei Jessica Jones-Carson1,2, James Laughlin1, Mohammed A. Hamad 1 , Amanda L. Stewart1, Martin I. Voskuil1, Andre´ s Va´ zquez-Torres1* 1 Department of Microbiology, University of Colorado Health Sciences Center, Aurora, Colorado, United States of America, 2 Department of Medicine, University of Colorado Health Sciences Center, Aurora, Colorado, United States of America Abstract Background: Much remains to be known about the mechanisms by which O2-dependent host defenses mediate broad antimicrobial activity. Methodology/Principal Findings: We show herein that reactive nitrogen species (RNS) generated by inducible nitric oxide (NO) synthase (iNOS) account for the anti-Burkholderia mallei activity of IFNc-primed macrophages. Inducible NOS-mediated intracellular killing may represent direct bactericidal activity, because B. mallei showed an exquisite sensitivity to NO generated chemically. Exposure of B. mallei to sublethal concentrations of NO upregulated transcription of [Fe-S] cluster repair genes, while damaging the enzymatic activity of the [Fe-S] protein aconitase. To test whether [Fe-S] clusters are critical targets for RNS-dependent killing of B. mallei, a mutation was constructed in the NO-induced, [Fe-S] cluster repair regulator iscR. Not only was the iscR mutant hypersusceptible to iNOS-mediated killing, but its aconitase pool was readily oxidized by NO donors as compared to wild-type controls. Although killed by authentic H2O2, which also oxidizes [Fe-S] clusters, B. mallei appear to be resilient to NADPH oxidase-mediated cytotoxicity. The poor respiratory burst elicited by this bacterium likely explains why the NADPH oxidase is nonessential to the killing of B. mallei while it is still confined within phagosomes. Conclusions/Significance: Collectively, these findings have revealed a disparate role for NADPH oxidase and iNOS in the innate macrophage response against the strict aerobe B. mallei. To the best of our knowledge, this is the first instance in which disruption of [Fe-S] clusters is demonstrated as cause of the bactericidal activity of NO congeners. Citation: Jones-Carson J, Laughlin J, Hamad MA, Stewart AL, Voskuil MI, et al. (2008) Inactivation of [Fe-S] Metalloproteins Mediates Nitric Oxide-Dependent Killing of Burkholderia mallei. PLoS ONE 3(4): e1976. doi:10.1371/journal.pone.0001976 Editor: Alejandro Aballay, Duke University Medical Center, United States of America Received February 11, 2008; Accepted March 7, 2008; Published April 9, 2008 Copyright: 2008 Jones-Carson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Support of this work was provided by the Rocky Mountain Regional Center of Excellence. Competing Interests: The authors have declared that no competing interests exist. * E-mail: andres.vazquez-torres@uchsc.edu Introduction The gram negative, nonmotile B. mallei bacillus is the causative agent of glanders, a disease that can be transmitted to humans upon cutaneous, mucosal or aerosol exposure to mucopurulent discharge from the eyes, nose and lips of infected solipeds [1]. The clinical presentation of glanders is characterized by an acute or chronic suppurative syndrome involving the upper and lower respiratory tract. If untreated, the local signs of glanders often evolve into full-blown sepsis, multiorgan system failure and shock. The poor understanding of the pathogenesis of glanders, the severity and clinical diversity of the infection and a lack of vaccines make B. mallei a desirable bioterrorist agent as has been demonstrated empirically in humans and animals in both World War I and II [2]. Pathogens adapted to an intracellular lifestyle avoid competition with other microbes that colonize host surfaces, while taking advantage of the nutritional resources provided by the cell. The cost of intracellular parasitism is the potential exposure to a variety of host defenses. For example, intracellular pathogens may encounter the O2-dependent antimicrobial mechanisms that are coupled to NADPH oxidase and iNOS enzymatic activities. The NADPH phagocyte oxidase is assembled on endocytic or plasma cell membranes, whereupon it catalyzes the NADPH-dependent univalent reduction of O2 to superoxide (O2N2). This radical serves as precursor to a variety of reactive oxygen species (ROS) endowed with widespread antimicrobial activity [3]. The electro- genic nature of the NADPH oxidase may also contribute to the host cell arsenal by promoting the release of cationic proteases from the proteoglycan matrix into the phagosomal lumen [4]. Professional phagocytes independently use O2 and NADPH in the oxidation of L-arginine with the consequent generation of L- citrulline and NO [5]. Similar to ROS, products of the reaction of NO with O2, O2N2 and metals are endowed with antimicrobial activity against phylogenetically diverse microorganisms [6]. Recent investigations have demonstrated that members of the genus Burkholderia survive within mononuclear phagocytes [7], prompting an interest in the role of ROS and RNS in defense against these bacteria [8–12]. The importance that the NADPH oxidase plays in resistance to some Burkholderia spp. is made evident by the fact that B. cepacia is the second most lethal infection in chronic granulomatous disease patients carrying mutations in PLoS ONE | www.plosone.org 1 April 2008 | Volume 3 | Issue 4 | e1976