Macrophages are central to host defense against microbes, but intracellular pathogens have evolved to evade their antimicrobial functions. Mycobacterium tuberculosis (MTB) has successfully exploited macrophages as its primary niche in vivo, but the bacterial genome-wide requirements that promote its intracellular survival remain undefined. Here we comprehensively identify the MTB genes required for survival by screening for transposon mutants that fail to grow within primary macrophages. We identify mutants showing decreased growth in macrophage environments that model stages of the host immune response. By systematically analyzing several biologically relevant data sets, we have been able to identify putative pathways that could not be predicted by genome organization alone. In one example, phosphate transport, requiring physically unlinked genes, was found to be critical for MTB growth in macrophages and important for establishing persistent infection in lungs. Remarkably, the majority of MTB genes found by this analysis to be required for survival are constitutively expressed rather than regulated by macrophages, revealing the host-adapted lifestyle of an evolutionarily selected intracellular pathogen. mutagenesis U p to one-third of humans world-wide harbor Mycobacterium tuberculosis (MTB) in a latent asymptomatic state and are thus at risk for tuberculosis when immune-compromised (1). Macrophages are critical both for permitting the survival of MTB and in linking innate and adaptive immunity in the host (2). They promote T cell activation and recruitment, crucial for containing MTB within granulomas in the lung. Virulent MTB can replicate within the hostile environment of macrophages, sequestered in poorly acidified phagosomes that fail to fuse with lysosomes (3-5). Although phagocytosis by IFN-␥-activated murine macrophages results in some bacterial killing, in part via nitric oxide-dependent mechanisms (6), MTB can evade macrophage bactericidal function by inhibiting IFN-␥-mediated signaling (7). MTB is also reported to interfere with antigen presentation, multiple signaling pathways, and transcriptional responses within the macrophage (2). However, the mycobacterial genes involved are largely unknown.We sought to systematically identify the MTB genes required for growth in macrophages. We used transposon site hybridization (TraSH) (8), a microarray-based technique that comprehensively identifies genes from large pools of transposon mutants that are essential for growth under different conditions (described in Fig. 1a). We devised screens to identify MTB mutants that fail to survive prolonged infection of primary murine macrophages and are thus attenuated for growth. We have identified 126 genes as necessary for survival in macrophages under conditions that model the immune response. Comparative analyses with biologically relevant data sets allow association of genes into functional groups, provide insights into gene regulation in mycobacteria, and highlight key gene products for further detailed study. W...

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Proteins of the nuclear factor of activated T cells (NFAT) family of transcription factors are critical for lymphocyte activation in the immune system. In particular, NFATs are important regulators of inducible IL-4 gene expression. Interferon regulatory factor 4 (IRF4) is an immune system–restricted interferon regulatory factor that is required for lymphocyte activation, but its molecular functions in the T lineage remain to be elucidated. We demonstrate that IRF4 potently synergizes with NFATc2 to specifically enhance NFATc2-driven transcriptional activation of the IL-4 promoter. This function is dependent on the physical interaction of IRF4 with NFATc2. IRF4 synergizes with NFATc2 and the IL-4–inducing transcription factor, c-maf, to augment IL-4 promoter activity as well as to elicit significant levels of endogenous IL-4 production. Furthermore, naïve T helper cells from mice lacking IRF4 are compromised severely for the production of IL-4 and other Th2 cytokines. The identification of IRF4 as a partner for NFATc2 in IL-4 gene regulation provides an important molecular function for IRF4 in T helper cell differentiation.

Nuclear factor of activated T cells (NFAT) is a critical regulator of early gene transcription in response to TCR-mediated signals. Here, we show that mice lacking both NFATp and NFAT4 develop a profound lymphoproliferative disorder likely due to a lowered threshold for TCR signaling coupled with increased resistance to apoptosis secondary to defective FasL expression. NFAT mutant mice also have allergic blepharitis, interstitial pneumonitis, and a 10(3) to 10(4) fold increase in serum IgG1 and IgE levels, secondary to a dramatic and selective increase in Th2 cytokines. This phenotype may be ascribed to unopposed occupancy of the IL-4 promoter by NFATc. Our data demonstrate that lymphoid homeostasis and Th2 activation require a critical balance among NFAT family members.

Tuberculosis (TB) is a global pandemic, partially due to the failure of vaccination approaches. Novel anti-TB vaccines are therefore urgently required. Here we show that aerosol immunization of macaques with the Mtb mutant in SigH (MtbΔsigH) results in significant recruitment of inducible bronchus-associated lymphoid tissue as well as CD4+ and CD8+ T cells expressing activation and proliferation markers to the lungs. Further, the findings indicate that pulmonary vaccination with MtbΔsigH elicited strong central memory CD4+ and CD8+ T cell responses in the lung. Vaccination with MtbΔsigH results in significant protection against a lethal TB challenge, as evidenced by a ~three log reduction in bacterial burdens, significantly diminished clinical manifestations and granulomatous pathology and characterized by the presence of profound iBALT. This highly protective response is virtually absent in unvaccinated and BCG-vaccinated animals after challenge. These results suggest that future TB vaccine candidates can be developed based on MtbΔsigH.