Macrophage activation determines the outcome of infection by Mycobacterium tuberculosis (Mtb). Interferon-γ (IFN-γ) activates macrophages by driving Janus tyrosine kinase (JAK)/signal transducer and activator of transcription–dependent induction of transcription and PKR-dependent suppression of translation. Microarray-based experiments reported here enlarge this picture. Exposure to IFN-γ and/or Mtb led to altered expression of 25% of the monitored genome in macrophages. The number of genes suppressed by IFN-γ exceeded the number of genes induced, and much of the suppression was transcriptional. Five times as many genes related to immunity and inflammation were induced than suppressed. Mtb mimicked or synergized with IFN-γ more than antagonized its actions. Phagocytosis of nonviable Mtb or polystyrene beads affected many genes, but the transcriptional signature of macrophages infected with viable Mtb was distinct. Studies involving macrophages deficient in inducible nitric oxide synthase and/or phagocyte oxidase revealed that these two antimicrobial enzymes help orchestrate the profound transcriptional remodeling that underlies macrophage activation.

SUMMARY Mycobacterium tuberculosis (Mtb) adapts to persist in a nutritionally limited macrophage compartment. Lipoamide dehydrogenase (Lpd), the third enzyme (E3) in Mtb’s pyruvate dehydrogenase complex (PDH), also serves as E1 of peroxynitrite reductase/peroxidase (PNR/P), which helps Mtb resist host reactive nitrogen intermediates. In contrast to Mtb lacking dihydrolipoamide acyltransferase (DlaT), the E2 of PDH and PNR/P, Lpd-deficient Mtb is severely attenuated in wild type and immunodeficient mice. This suggests that Lpd has a function that DlaT does not share. When DlaT is absent, Mtb upregulates an Lpd-dependent branched chain keto-acid dehydrogenase (BCKADH) encoded by pdhA, pdhB, pdhC and lpdC. Without Lpd, Mtb cannot metabolize branched chain amino acids and potentially toxic branched chain intermediates accumulate. Mtb deficient in both DlaT and PdhC phenocopies Lpd-deficient Mtb. Thus, Mtb critically requires BCKADH along with PDH and PNR/P for pathogenesis. These findings position Lpd as a potential target for anti-infectives against Mtb.

Macrophages are activated from a resting state by a combination of cytokines and microbial products. Microbes are often sensed through Toll-like receptors signaling through MyD88. We used large-scale microarrays in multiple replicate experiments followed by stringent statistical analysis to compare gene expression in wild-type (WT) and MyD88−/− macrophages. We confirmed key results by quantitative reverse transcription polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay. Surprisingly, many genes, such as inducible nitric oxide synthase, IRG-1, IP-10, MIG, RANTES, and interleukin 6 were induced by interferon (IFN)-γ from 5- to 100-fold less extensively in MyD88−/− macrophages than in WT macrophages. Thus, widespread, full-scale activation of macrophages by IFN-γ requires MyD88. Analysis of the mechanism revealed that MyD88 mediates a process of self-priming by which resting macrophages produce a low level of tumor necrosis factor. This and other factors lead to basal activation of nuclear factor κB, which synergizes with IFN-γ for gene induction. In contrast, infection by live, virulent Mycobacterium tuberculosis (Mtb) activated macrophages largely through MyD88-independent pathways, and macrophages did not need MyD88 to kill Mtb in vitro. Thus, MyD88 plays a dynamic role in resting macrophages that supports IFN-γ–dependent activation, whereas macrophages can respond to a complex microbial stimulus, the tubercle bacillus, chiefly by other routes.

SummaryMycobacterium tuberculosis (Mtb) persists for prolonged periods in macrophages, where it must adapt to metabolic limitations and oxidative/nitrosative stress. However, little is known about Mtb's intermediary metabolism or antioxidant defences. We recently identified a peroxynitrite reductase-peroxidase complex in Mtb that included products of the genes sucB and lpd , which are annotated to encode the dihydrolipoamide succinyltransferase (E2) and lipoamide dehydrogenase (E3) components of a a a a -ketoglutarate dehydrogenase (KDH). However, we could detect no KDH activity in Mtb lysates, nor could we reconstitute KDH by combining the recombinant proteins SucA (annotated as the E1 component of KDH), SucB and Lpd. We therefore renamed the sucB product dihydrolipoamide acyltransferase (DlaT). Mtb lysates contained pyruvate dehydrogenase (PDH) activity, which was lost when the dlaT gene (formerly, sucB ) was disrupted. Purification of PDH from Mtb yielded AceE, annotated as an E1 component of PDH, along with DlaT and Lpd. Moreover, anti-DlaT antibody coimmunoprecipitated AceE. Finally, recombinant AceE, DlaT and Lpd, although encoded by genes that are widely separated on the chromosome, reconstituted PDH in vitro with K m values typical of bacterial PDH complexes. In sum, Mtb appears to lack KDH. Instead, DlaT and Lpd join with AceE to constitute PDH.

Mycobacterium tuberculosis has evolved to persist in host macrophages, where it faces a nutrient-poor environment and is exposed to oxidative and nitrosative stress. To defend itself against oxidative/nitrosative stress, M. tuberculosis expresses an NADH-dependent peroxidase and peroxynitrite reductase that is encoded by ahpC, ahpD, lpd, and dlaT. In addition to its central role in the peroxynitrite reductase complex, dlaT (Rv2215) also encodes the E2 component of pyruvate dehydrogenase. Here we demonstrate that inactivation of dlaT in the chromosome of H37Rv resulted in a mutant (H37Rv⌬dlaT) that displayed phenotypes associated with DlaT's role in metabolism and in defense against nitrosative stress. The H37Rv⌬dlaT strain showed retarded growth in vitro and was highly susceptible to killing by acidified sodium nitrite. Mouse macrophages readily killed intracellular H37Rv⌬dlaT organisms, and in mice dlaT was required for full virulence.Mycobacterium tuberculosis is a major global health threat. Nearly 2 billion people have been infected with M. tuberculosis, and ϳ10% of these individuals are predicted to develop active disease at some time in their lives (7). During infection, M. tuberculosis is phagocytosed by alveolar macrophages in the lungs. The bacteria either grow to cause primary tuberculosis or enter a state of latency in which they can persist, sometimes for decades, within the host. The mechanisms that allow M. tuberculosis to survive and persist in the face of a strong host immune response remain largely unidentified.There is considerable evidence that the production of reactive nitrogen intermediates (RNI) by macrophages is important in the generation of effective immunity against mycobacteria (3, 14, 18). In vitro, RNI are mycobacteriocidal (6, 13), and gamma interferon (IFN-␥)-activated mouse macrophages can kill M. tuberculosis in an inducible nitric oxide synthase (iNOS)-dependent manner (8). Mice deficient in iNOS display markedly enhanced susceptibility to M. tuberculosis (14). In patients with tuberculosis, the expression of iNOS and nitrotyrosine has been demonstrated in the lungs, indicating NO production (4, 22).The ability of M. tuberculosis to resist the toxicity of RNI has been suggested by several studies. The expression of M. tuberculosis noxR1 in Escherichia coli and Mycobacterium smegmatis conferred resistance to RNI during in vitro culture and during ex vivo growth in macrophages (9); however, an M. tuberculosis noxR1 deletion strain did not show decreased virulence in mice (23). M. tuberculosis noxR3 protected Salmonella enterica serovar Typhimurium from the NO donor S-nitrosoglutathione, acidified nitrite, and hydrogen peroxide (19), but its role during in vivo infection has not been addressed. M. tuberculosis encodes three annotated NO dioxygenase homologues (Hmp, GlbN, and GlbO). Hmp is induced in response to RNI stress (12), but NO dioxygenase activity has not been reported for Hmp. In contrast, the truncated hemoglobins GlbN and GlbO possess NO dioxygenase activity in vitro an...