Ammunition for the TB Wars: Tuberculosis is a major human disease of global importance resulting from infection with the air-borne pathogen Mycobacterium tuberculosis , which is becoming increasingly resistant to all available drugs. An antituberculosis benzothiazinone compound kills mycobacterium in infected cells and in mice. Makarov et al. (p. 801 ) have identified a sulfur atom and nitro residues important for benzothiazinone's activity and used genetic methods and biochemical analysis to identify its target in blocking arabinogalactan biosynthesis during cell-wall synthesis. The compound affects the same pathway as ethambutol, and thus a benzothiazinone drug has the potential to become an important part of treatment of drug-resistant disease and, possibly, replace the less effective ethambutol in the primary treatment of tuberculosis.

Tuberculous sputum provides a sample of bacilli that must be eliminated by chemotherapy and that may go on to transmit infection. A preliminary observation that Mycobacterium tuberculosis cells contain triacylglycerol lipid bodies in sputum, but not when growing in vitro, led us to investigate the extent of this phenomenon and its physiological basis. Microscopy-positive sputum samples from the UK and The Gambia were investigated for their content of lipid body–positive mycobacteria by combined Nile red and auramine staining. All samples contained a lipid body–positive population varying from 3% to 86% of the acid-fast bacilli present. The recent finding that triacylglycerol synthase is expressed by mycobacteria when they enter in vitro nonreplicating persistence led us to investigate whether this state was also associated with lipid body formation. We found that, when placed in laboratory conditions inducing nonreplicating persistence, two M. tuberculosis strains had lipid body levels comparable to those found in sputum. We investigated these physiological findings further by comparing the M. tuberculosis transcriptome of growing and nonreplicating persistence cultures with that obtained directly from sputum samples. Although sputum has traditionally been thought to contain actively growing tubercle bacilli, our transcript analyses refute the hypothesis that these cells predominate. Rather, they reinforce the results of the lipid body analyses by revealing transcriptional signatures that can be clearly attributed to slowly replicating or nonreplicating mycobacteria. Finally, the lipid body count was highly correlated (R² = 0.64, p < 0.03) with time to positivity in diagnostic liquid cultures, thereby establishing a direct link between this cytological feature and the size of a potential nonreplicating population. As nonreplicating tubercle bacilli are tolerant to the cidal action of antibiotics and resistant to multiple stresses, identification of this persister-like population of tubercle bacilli in sputum presents exciting and tractable new opportunities to investigate both responses to chemotherapy and the transmission of tuberculosis.

Mycobacterium tuberculosis thrives within macrophages by residing in phagosomes and preventing them from maturing and fusing with lysosomes. A parallel transcriptional survey of intracellular mycobacteria and their host macrophages revealed signatures of heavy metal poisoning. In particular, mycobacterial genes encoding heavy metal efflux P-type ATPases CtpC, CtpG, and CtpV, and host cell metallothioneins and zinc exporter ZnT1, were induced during infection. Consistent with this pattern of gene modulation, we observed a burst of free zinc inside macrophages, and intraphagosomal zinc accumulation within a few hours postinfection. Zinc exposure led to rapid CtpC induction, and ctpC deficiency caused zinc retention within the mycobacterial cytoplasm, leading to impaired intracellular growth of the bacilli. Thus, the use of P(1)-type ATPases represents a M. tuberculosis strategy to neutralize the toxic effects of zinc in macrophages. We propose that heavy metal toxicity and its counteraction might represent yet another chapter in the host-microbe arms race.

Genetic defects in the IFN-γ response pathway cause unique susceptibility to intracellular pathogens, particularly mycobacteria, but are rare and do not explain mycobacterial disease in the majority of affected patients. We postulated that acquired defects in macrophage activation by IFN-γ may cause a similar immunological phenotype and thus explain the occurrence of disseminated intracellular infections in some patients without identifiable immune deficiency. Macrophage activation in response to IFN-γ and IFN-γ production were studied in whole blood and PBMCs of 3 patients with severe, unexplained nontuberculous mycobacterial infection. In all 3 patients, IFN-γ was undetectable following mitogen stimulation of whole blood, but significant quantities were detectable in the supernatants of PBMCs when stimulated in the absence of the patients’ own plasma. The patients’ plasma inhibited the ability of IFN-γ to increase production of TNF-α by both autologous and normal donor PBMCs, and recovery of exogenous IFN-γ from the patients’ plasma was greatly reduced. Using affinity chromatography, surface-enhanced laser desorption/ionization mass spectrometry, and sequencing, we isolated an IFN-γ–neutralizing factor from the patients’ plasma and showed it to be an autoantibody against IFN-γ. The purified anti–IFN-γ antibody was shown to be functional first in blocking the upregulation of TNF-α production in response to endotoxin; second in blocking induction of IFN-γ–inducible genes (according to results of high-density cDNA microarrays); and third in inhibiting upregulation of HLA class II expression on PBMCs. Acquired defects in the IFN-γ pathway may explain unusual susceptibility to intracellular pathogens in other patients without underlying, genetically determined immunological defects.

Transcriptional profiling using microarrays provides a unique opportunity to decipher host pathogen cross-talk on the global level. Here, for the first time, we have been able to investigate gene expression changes in both Mycobacterium tuberculosis, a major human pathogen, and its human host cells, macrophages and dendritic cells. In addition to common responses, we could identify eukaryotic and microbial transcriptional signatures that are specific to the cell type involved in the infection process. In particular M. tuberculosis shows a marked stress response when inside dendritic cells, which is in accordance with the low permissivity of these specialized phagocytes to the tubercle bacillus and to other pathogens. In contrast, the mycobacterial transcriptome inside macrophages reflects that of replicating bacteria. On the host cell side, differential responses to infection in macrophages and dendritic cells were identified in genes involved in oxidative stress, intracellular vesicle trafficking and phagosome acidification. This study provides the proof of principle that probing the host and the microbe transcriptomes simultaneously is a valuable means to accessing unique information on host pathogen interactions. Our results also underline the extraordinary plasticity of host cell and pathogen responses to infection, and provide a solid framework to further understand the complex mechanisms involved in immunity to M. tuberculosis and in mycobacterial adaptation to different intracellular environments.

Interferons are key modulators of the immune system, and are central to the control of many diseases. The response of immune cells to stimuli in complex populations is the product of direct and indirect effects, and of homotypic and heterotypic cell interactions. Dissecting the global transcriptional profiles of immune cell populations may provide insights into this regulatory interplay. The host transcriptional response may also be useful in discriminating between disease states, and in understanding pathophysiology. The transcriptional programs of cell populations in health therefore provide a paradigm for deconvoluting disease-associated gene expression profiles. We used human cDNA microarrays to (1) compare the gene expression programs in human peripheral blood mononuclear cells (PBMCs) elicited by 6 major mediators of the immune response: interferons α, β, ω and γ, IL12 and TNFα; and (2) characterize the transcriptional responses of purified immune cell populations (CD4⁺ and CD8⁺ T cells, B cells, NK cells and monocytes) to IFNγ stimulation. We defined a highly stereotyped response to type I interferons, while responses to IFNγ and IL12 were largely restricted to a subset of type I interferon-inducible genes. TNFα stimulation resulted in a distinct pattern of gene expression. Cell type-specific transcriptional programs were identified, highlighting the pronounced response of monocytes to IFNγ, and emergent properties associated with IFN-mediated activation of mixed cell populations. This information provides a detailed view of cellular activation by immune mediators, and contributes an interpretive framework for the definition of host immune responses in a variety of disease settings.