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The capacity of infected cells to undergo apoptosis upon insult with a pathogen is an ancient innate immune defense mechanism. Consequently, the ability of persisting, intracellular pathogens such as the human pathogen Mycobacterium tuberculosis (Mtb) to inhibit infection-induced apoptosis of macrophages is important for virulence. The nuoG gene of Mtb, which encodes the NuoG subunit of the type I NADH dehydrogenase, NDH-1, is important in Mtb-mediated inhibition of host macrophage apoptosis, but the molecular mechanism of this host pathogen interaction remains elusive. Here we show that the apoptogenic phenotype of MtbDnuoG was significantly reduced in human macrophages treated with caspase-3 and -8 inhibitors, TNF-a-neutralizing antibodies, and also after infection of murine TNF2/2 macrophages. Interestingly, incubation of macrophages with inhibitors of reactive oxygen species (ROS) reduced not only the apoptosis induced by the nuoG mutant, but also its capacity to increase macrophage TNF-a secretion. The MtbDnuoG phagosomes showed increased ROS levels compared to Mtb phagosomes in primary murine and human alveolar macrophages. The increase in MtbDnuoG induced ROS and apoptosis was abolished in NOX-2 deficient (gp912/2) macrophages. These results suggest that Mtb, via a NuoG-dependent mechanism, can neutralize NOX2-derived ROS in order to inhibit TNF-a-mediated host cell apoptosis. Consistently, an Mtb mutant deficient in secreted catalase induced increases in phagosomal ROS and host cell apoptosis, both of which were dependent upon macrophage NOX-2 activity. In conclusion, these results serendipitously reveal a novel connection between NOX2 activity, phagosomal ROS, and TNF-a signaling during infection-induced apoptosis in macrophages. Furthermore, our study reveals a novel function of NOX2 activity in innate immunity beyond the initial respiratory burst, which is the sensing of persistent intracellular pathogens and subsequent induction of host cell apoptosis as a second line of defense. Citation: Miller JL, Velmurugan K, Cowan MJ, Briken V (2010) The Type I NADH Dehydrogenase of Mycobacterium tuberculosis Counters Phagosomal NOX2 Activity to Inhibit TNF-a-Mediated Host Cell Apoptosis. PLoS Pathog 6(4): e1000864. doi:10.1371/journal.ppat.1000864 Editor: Vojo Deretic, University of New Mexico, United States of America Received August 27, 2009; Accepted March 18, 2010; Published April 22, 2010 Copyright: 2010 Miller 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: The research described was supported by NIH/NIAID grants R56AI072584 and RO1AI072584 (V.B.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: vbriken@umd.edu ¤ Current address: AERAS TB Foundation, Rockville, Maryland, United States of America

Introduction The phagocytic NADPH-oxidase (NOX2-complex or phox) resides on phagosomes and has been shown to be involved in microcidal activity in phagocytes. NOX2 is the original member of the NOX family of reactive oxygen species (ROS)-generating NADPH oxidases, which now includes NOX1-NOX5, DUOX1 and DUOX2 [1,2]. The multicomponent NOX2 complex consists of two transmembrane proteins, gp91phox and gp22 phox, and three cytosolic components, p40 phox , p47 phox and p67 phox [1,2]. Additionally, the cytosolic GTPase Rac has to be recruited in order to form a fully active NOX2 complex [1]. The gp91phox subunit, which is constitutively associated with gp22 phox, is a transmembrane redox chain that generates phagosomal superoxide by transferring electrons from cytosolic NADPH to phagosomal oxygen [1]. NOX2-generated superoxide can then be converted into a multitude of microcidal oxidants, including hydrogen peroxide and hypochlorous acid, which are important components of the bactericidal activity of the macrophage phagosome [3]. However, NOX2 activity seems to serve a different function in phagosomes of dendritic cells, where it is important for efficient crosspresentation of antigens [4]. The significance of the NOX2-complex for innate immune response is illustrated by the development of chronic granulomatous disease (CGD) in human subjects that have genetic defects in components of the complex.

CGD is characterized by greatly increased susceptibility to fungal and bacterial infections [5]. Correspondingly, mice deficient in the NOX2 subunits are much more susceptible to infections with bacterial pathogens such as Salmonella typhimurium for example [3,5]. Not surprisingly, some pathogens have developed strategies to counter the NOX2 response by either inhibiting NOX2 assembly on the phagosome, as is the case for S. typhimurium [3] and Helicobacter pylori [6], or reducing steady-state levels of NOX2 components as illustrated by Anaplasma phagocytophila [7] or Ehrlichia chaffeensis [8] (for review [9]). PLoS Pathogens | www.plospathogens.org 1 April 2010 | Volume 6 | Issue 4 | e1000864 Programmed cell death (PCD), or apoptosis, plays an important role in the innate immune response (IR) against pathogens, a defense strategy that is evolutionarily conserved and extends even into the plant world[10]. Inhibition of host cell apoptosis has been extensively studied and there are numerous examples of viral proteins directly interfering with host cell apoptosis signaling[11]. Furthermore, an increasing number of protozoal pathogens have been shown to manipulate PCD signaling of infected host cells[12]. Finally, prokaryotic pathogens such as Chlamydia, Legionella, Rickettsia, and Mycobacterium among others have the capacity to inhibit host cell apoptosis signaling [13,14]. Mycobacterium tuberculosis (Mtb) is an extremely successful human pathogen that manipulates host cells via multiple pathways in order to achieve survival[15,16,17]. The inhibition of host cell apoptosis by Mtb has been implicated as a potential virulence mechanism[18]. Indeed, an inverse correlation between the virulence of a mycobacterial species and their capacity to induce apoptosis of primary human alveolar macrophages was demonstrated[19]. Cells infected with virulent Mtb have also been shown to be more resistant to various apoptosis stimuli when compared to uninfected or avirulent strains of Mtb[18]. For example, Mtbinfected macrophages secrete soluble TNF-a-receptor in order to inhibit TNF-a-mediated host cell apoptosis induction [20].

Mtbinfection reduces the cell surface expression of Fas receptors, resulting in the resistance of the host cells to Fas-ligand induce cell death[21]. Infection with Mtb also induces the upregulation of the anti-apoptosis gene mcl-1, which confers resistance of cells to apoptosis induction via the host cell mitochondria[22]. Finally, it has recently been shown that Mtb can manipulate the surface of infected macrophages in order to favor a necrotic, rather than apoptotic, cell death outcome[23]. In macrophages infected with virulent MtbH37Rv, but not avirulent MtbH37Ra, the aminoterminal domain of annexin-1 is removed by proteolysis, preventing completion of the apoptotic envelop [24]. Similarly, cells infected with Mtb are less likely to induce host cell membrane repair, which is important for the induction of apoptosis and supports the induction of necrotic cells death and the subsequent dissemination of bacteria[24,25]. While there is substantial evidence supporting the ability of Mtb to inhibit host cell apoptosis, a causal link between apoptosis inhibition and virulence of Mtb had not been established due to the lack of defined pro-apoptosis mutants. We have recently performed a ‘‘gain-of-function’’ genetic screen and identified three independent regions in the genome of Mtb that contain antiapoptosis genes[26]. The deletion of one of the identified genes, the nuoG gene of Mtb, which encodes one of the 14 subunits of the type I NADH dehydrogenase (NDH-1), increased infectioninduced apoptosis of macrophages and significantly reduced bacterial virulence in mice. These findings support a direct causal relationship between virulence of pathogenic mycobacteria and their ability to inhibit macrophage apoptosis[26]. Our findings are consistent with the identification of another anti-apoptosis gene (superoxide dismutase A) that plays an important role in the virulence of Mtb[27]. Finally, a third gene (Protein kinase E) with anti-apoptosis capacity has recently been described, but the impact of the deletion of this gene on bacterial virulence has not been established[28]. Altogether, the identification of multiple antiapoptosis genes suggests that Mtb utilizes several strategies to inhibit the apoptotic response of the host cell; however the molecular mechanisms of these interactions have not been investigated. The present study describes the investigation of the molecular mechanisms by which NuoG of Mtb inhibits host cell apoptosis. The use of TNF-a-neutralizing antibodies and specific caspase inhibitors on human macrophage cell lines, as well as the infection of bone-marrow derived macrophages (BMDM) of wild-type and TNF2/2 mice demonstrated that NuoG is involved in inhibiting an extrinsic, TNF-a-dependent, apoptosis pathway. Furthermore, the pro-apoptotic phenotype of the nuoG mutant was abolished in the presence of both ROS scavengers and in the absence of a functional NOX2 system as demonstrated in BMDM and primary human alveolar macrophages. Altogether, our results reveal a novel function of the NOX2 system in helping the host macrophage in sensing persistent intracellular mycobacteria via increased phagosomal ROS levels and the subsequent induction of host cell apoptosis. This may constitute a second line of defense of the macrophage and it is intriguing to speculate that this NOX2 mediated apoptosis induction is equally important in the defense against other intracellular pathogens. Results The MtbDnuoG mutant induces apoptosis via an extrinsic, caspase-dependent pathway We previously demonstrated that a DnuoG mutant of Mtb induced more apoptosis in host cells than wild type bacteria [26]. In order to analyze the mechanism of the NuoG/NDH-1 mediated host cell apoptosis inhibition, we first determined the involvement of caspases in the pro-apoptotic phenotype of MtbDnuoG using specific caspase inhibitors. PMA-differentiated THP-1 cells were pre-treated with Caspase-3 inhibitor (C3I) or a chemical analog with no inhibitor activity (C3I-A) at 20 mM for 3 h before infection, during infection, and after infection. Cells were either left uninfected, or were infected with Mtb or MtbDnuoG. After five days cells were harvested and stained for genomic DNA degradation using the fluorescent Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) assay. The percentage of TUNEL positive cells was determined by flow cytometry analysis. This analysis revealed that the uninfected population contained low percentage of apoptotic cells (2.360.3%), Mtb infection slightly increased this amount to 1160.6%. As expected from previously published results [26], cells infected with the nuoG mutant showed a very significant increase in apoptosis (67.567.0%). Interestingly, treatment of THP-1 cells Author Summary Mycobacterium tuberculosis, the causative agent of tuberculosis, is highly adapted to survive in macrophages of its human host. Host cell suicide is an ancient host cell defense mechanism employed by organisms to wall off invading pathogens. M. tuberculosis manipulates infected cells to inhibit host cell death but the molecular mechanism of this interaction has not been elucidated. Here we describe that M. tuberculosis uses an enzyme complex (NDH-1) usually needed for energy generation in order to neutralize the NOX-2 enzyme-mediated production of toxic oxygen radicals (ROS) by the host cell. We demonstrate that an M. tuberculosis mutant deficient in NDH-1 accumulates ROS inside the macrophage which induces the secretion of an inflammatory cytokine (TNF-a) and subsequent host cell death. The increase of ROS is dependent upon functional NOX-2, since host cells missing a NOX-2 component do not undergo cell death upon infection with the mutant. We propose that a novel function of the host cell NOX-2 complex is to allow sensing of intracellular pathogens by the host cell in order to commit suicide and thus limit bacterial survival.

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