The generation of cell-mediated immunity against many infectious pathogens involves the production of interleukin-12 (IL-12), a key signal of the innate immune system. Yet, for many pathogens, the molecules that induce IL-12 production by macrophages and the mechanisms by which they do so remain undefined. Here it is shown that microbial lipoproteins are potent stimulators of IL-12 production by human macrophages, and that induction is mediated by Toll-like receptors (TLRs). Several lipoproteins stimulated TLR-dependent transcription of inducible nitric oxide synthase and the production of nitric oxide, a powerful microbicidal pathway. Activation of TLRs by microbial lipoproteins may initiate innate defense mechanisms against infectious pathogens.

The dominant exported proteins and protective antigens of Mycobacterium tuberculosis are a triad of related gene products called the antigen 85 (Ag85) complex. Each has also been implicated in disease pathogenesis through its fibronectin-binding capacities. A carboxylesterase domain was found within the amino acid sequences of Ag85A, B, and C, and each protein acted as a mycolyltransferase involved in the final stages of mycobacterial cell wall assembly, as shown by direct enzyme assay and site-directed mutagenesis. Furthermore, the use of an antagonist (6-azido-6-deoxy-alpha, alpha'-trehalose) of this activity demonstrates that these proteins are essential and potential targets for new antimycobacterial drugs.

The mammalian innate immune system retains from Drosophila a family of homologous Toll-like receptors (TLRs) that mediate responses to microbial ligands. Here, we show that TLR2 activation leads to killing of intracellular Mycobacterium tuberculosis in both mouse and human macrophages, through distinct mechanisms. In mouse macrophages, bacterial lipoprotein activation of TLR2 leads to a nitric oxide-dependent killing of intracellular tubercle bacilli, but in human monocytes and alveolar macrophages, this pathway was nitric oxide-independent. Thus, mammalian TLRs respond (as Drosophila Toll receptors do) to microbial ligands and also have the ability to activate antimicrobial effector pathways at the site of infection.

Live Mycobacterium tuberculosis persists in macrophage phagosomes by interfering with phagolysosome biogenesis. Here, using four-dimensional microscopy and in vitro assays, we report the principal difference between phagosomes containing live and dead mycobacteria. Phosphatidylinositol 3-phosphate (PI3P), a membrane trafficking regulatory lipid essential for phagosomal acquisition of lysosomal constituents, is retained on phagosomes harboring dead mycobacteria but is continuously eliminated from phagosomes with live bacilli. We show that the exclusion of PI3P from live mycobacterial phagosomes can be only transiently reversed by Ca 2؉ fluxes, and that live M. tuberculosis secretes a lipid phosphatase, SapM, that hydrolyzes PI3P, inhibits phagosome-late endosome fusion in vitro, and contributes to inhibition of phagosomal maturation.macrophage ͉ phagosome ͉ tuberculosis ͉ lysosome ͉ phosphatidylinositol 3-phosphate T he infectious cycle of Mycobacterium tuberculosis rests upon the ability of this potent pathogen to parasitize host mononuclear phagocytic cells (1). In infected macrophages, M. tuberculosis resides within a phagosome that avoids the default maturation pathway leading to phagolysosome formation (2). The salient characteristics of the mycobacterial phagosome include (i) paucity of vacuolar H ϩ ATPase (3), (ii) attendant inefficient luminal acidification (3); and (iii) inadequate levels of mature lysosomal hydrolases (3, 4). These and additional (4-6) properties of the M. tuberculosis phagosome promote intracellular survival and growth of the tubercle bacilli and help avoid their immunological detection (1).The arrest of M. tuberculosis phagosome maturation has been studied at the membrane-trafficking level (2), with a focus on the small GTP-binding proteins, including Rab GTPases (7-9). Rabs direct intracellular trafficking by regulating activity and recruitment to organellar membranes of Rab-interacting partners and downstream effectors (10). The initial analyses of Rabs on mycobacterial phagosomes have indicated that the M. tuberculosis phagolysosome biogenesis arrest occurs between the stages controlled by the early endosomal GTPase Rab5 and its late endosomal counterpart Rab7 (7). A number of follow-up studies have indicated critical contributions of Rab5 effectors in mycobacterial phagosome maturation arrest, with a prominent role for the phosphatidylinositol 3-kinase (PI3K) hVPS34, its product phosphatidylinositol 3-phosphate (PI3P), and an array of PI3P-binding proteins (11)(12)(13)(14). PI3P affects localization and function of proteins containing the PI3P-binding domains (FYVE, PH, and PX) (15). These proteins in turn execute various steps in membrane trafficking, endosomal protein sorting, and multisubunit enzyme assembly at the membrane, including phagosomal maturation (11, 16), early endosomal homotypic fusion (17), delivery of internalized plasma membrane receptors to late endosomes (18), formation of internal vesicles within late endosomal multivesicular bodies involved in termination of...

The antimycobacterial compound ethambutol [Emb; dextro-2,2'-(ethylenediimino)-di-1-

Tuberculosis remains a severe worldwide health threat. A thorough understanding of Mycobacterium tuberculosis pathogenesis will facilitate the development of new treatments for tuberculosis. Numerous bacterial pathogens possess specialized protein secretion systems that are dedicated to the export of virulence factors. Mycobacterium tuberculosis is part of a developing group of pathogenic bacteria that share the uncommon property of possessing two secA genes (secA1 and secA2). In mycobacteria, SecA1 is the essential 'housekeeping' SecA protein whereas SecA2 is an accessory secretion factor. Here we demonstrate that SecA2 contributes to the pathogenesis of M. tuberculosis. A deletion of the secA2 gene in M. tuberculosis attenuates the virulence of the organism in mice. By comparing the profile of proteins secreted by wild-type M. tuberculosis and the DeltasecA2 mutant, we identified superoxide dismutase A (SodA) as a protein dependent on SecA2 for secretion. SodA lacks a classical signal sequence for protein export. Our data suggests that SecA2-dependent export is a new type of secretion pathway that is part of a virulence mechanism of M. tuberculosis to elude the oxidative attack of macrophages.

The significance of fever in response to a bacterial infection has been investigated using the lizard Dipsosaurus dorsalis as an animal model. These lizards develop a fever of about 2 degrees C after injection with the bacterium Aeromonas hydrophila. To determine whether this elevation in body temperature increases the resistance of the host to this infection, as measured by survival, lizards were infected with the live bacteria and placed in a neutral (38 degrees C), low (34 degrees or 36 degrees C), or high (40 degrees or 42 degrees C) ambient temperature. An elevation in temperature following experimental bacterial infection results in a significant increase in host survival.

Previous studies in mice and humans have suggested an important role for CD8+ T cells in host defense to Mtb. Recently, we have described human, Mtb-specific CD8+ cells that are neither HLA-A, B, or C nor group 1 CD1 restricted, and have found that these cells comprise the dominant CD8+ T cell response in latently infected individuals. In this report, three independent methods are used to demonstrate the ability of these cells to recognize Mtb-derived antigen in the context of the monomorphic HLA-E molecule. This is the first demonstration of the ability of HLA-E to present pathogen-derived antigen. Further definition of the HLA-E specific response may aid development of an effective vaccine against tuberculosis.