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Mycobacterium tuberculosis -infected macrophages and dendritic cells are limited in their ability to present antigen to CD4+ T cells suggesting that other mechanism of antigen presentation are driving the robust T cell response observed during an M. tuberculosis infection. These mechanisms could include antigens present in apoptotic bodies, necrotic debris, exosomes or even release of non-vesicular antigen from infected cells. However, there is limited data to support any of these mechanisms as important in driving T cell activation in vivo . In the present study we use Rab27a-deficient mice which show diminished trafficking of mycobacterial components to exosomes as well as M. tuberculosis strains that express recombinant proteins which traffic or fail to traffic to exosomes. We observed that exosomes released during a mouse M. tuberculosis infection contribute significantly to its T cell response. These finding imply that exosomes function to promote T cell immunity during a bacterial infection and are an important source of extracellular antigen.

Activation of both CD4(+) and CD8(+) T cells is required for an effective immune response to an M. tuberculosis infection. However, infected macrophages are poor antigen presenting cells and may be spatially separated from recruited T cells, thus limiting antigen presentation within a granuloma. Our previous studies showed that infected macrophages release from cells small membrane-bound vesicles called exosomes which contain mycobacterial lipid components and showed that these exosomes could stimulate a pro-inflammatory response in naïve macrophages. In the present study we demonstrate that exosomes stimulate both CD4(+) and CD8(+) splenic T cells isolated from mycobacteria-sensitized mice. Although the exosomes contain MHC I and II as well as costimulatory molecules, maximum stimulation of T cells required prior incubation of exosomes with antigen presenting cells. Exosomes isolated from M. bovis and M. tuberculosis infected macrophages also stimulated activation and maturation of mouse bone marrow-derived dendritic cells. Interestingly, intranasal administration of mice with exosomes isolated from M. bovis BCG infected macrophages induce the generation of memory CD4(+) and CD8(+) T cells. The isolated T cells also produced IFN-gamma upon restimulation with BCG antigens. The release of exosomes from infected macrophages may overcome some of the defects in antigen presentation associated with mycobacterial infections and we suggest that exosomes may be a promising M. tuberculosis vaccine candidate.

Retinoic acid inducible gene I (Rig-I) is a cytosolic pattern recognition receptor canonically described for its important role in sensing viral RNAs. Increasingly, bacterially-derived RNA from intracellular bacteria such as Mycobacterium tuberculosis , have been shown to activate the same host Rig-I/Mitochondrial antiviral sensing protein (MAVS) signaling pathway to drive a type-I interferon response that contributes to bacterial pathogenesis in vivo . In M . tuberculosis , this response is mediated by the protein secretion system SecA2, but little is known about whether this process is conserved in other pathogenic mycobacteria or the mechanism by which these nucleic acids gain access to the host cytoplasm. Because the M . tuberculosis and M . marinum SecA2 protein secretion systems share a high degree of genetic and functional conservation, we hypothesized that Rig-I/MAVS activation and subsequent induction of IFN-β secretion by host macrophages will also be conserved between these two mycobacterial species. To test this, we generated a Δ secA2 M . marinum strain along with complementation strains expressing either the M . marinum or M . tuberculosis secA2 genes. Our results suggest that the Δ secA2 strain has a growth defect in vitro but not in host macrophages. These intracellular growth curves also suggested that the calculation applied to estimate the number of bacteria added to macrophage monolayers in infection assays underestimates bacterial inputs for the Δ secA2 strain. Therefore, to better examine secreted IFN-β levels when bacterial infection levels are equal across strains we plated bacterial CFUs at 2hpi alongside our ELISA based infections. This enabled us to normalize secreted levels of IFN-β to a standard number of bacteria. Applying this approach to both WT and MAVS -/- bone marrow derived macrophages we observed equal or higher levels of secreted IFN-β from macrophages infected with the Δ secA2 M . marinum strain as compared to WT. Together our findings suggest that activation of host Rig-I/MAVS cytosolic sensors and subsequent induction of IFN-β response in a SecA2-dependent manner is not conserved in M . marinum under the conditions tested.

Mycobacterial infection leads to activation of the RIG-I/MAVS/TBK1 RNA sensing pathway in macrophages but the consequences of this activation remains poorly defined. In this study, we determined that activation of this RNA sensing pathway stimulates ICAM-1 expression in M.avium-infected macrophage through the inhibition of the E3 ubiquitin ligase CRL4COP1/DET1. CRL4 when active targets the transcription factor ETV5 for degradation by the ubiquitin-proteasome system. In the absence of the ETV5 transcription factor, ICAM-1 expression is significantly decreased. The M.avium-induced ICAM-1 production is required for the formation of immune synapse between infected macrophages and antigen-specific CD4+ T lymphocytes, and is essential for CD4+ T lymphocyte-mediated mycobacterial killing in vitro and in mice. This study demonstrates a previously undefined mechanism by which a host cytosolic RNA sensing pathway contributes to the interplay between mycobacteria infected macrophages and antigen-specific T lymphocytes.

Tuberculosis remains a global threat due in part to the long treatment regimen and the increased prevalence of drug resistant M. tuberculosis strains. Therefore, new drug regimens are urgently required to combat this deadly disease. We previously synthesized and evaluated a series of new anti-tuberculosis compounds which belong to the family of imidazo[1,2-a]pyridines. This family of compounds showed low nM MIC (minimal inhibitory concentration) values against M. tuberculosis in vitro. In this study, a derivative of imidazo[1,2-a]pyridines, (N-(4-(4-chlorophenoxy)benzyl)-2,7-dimethylimidazo[1,2-a]pyridine-3-carboxamide) (ND-09759), was selected as a promising lead compound to determine its protective efficacy using a mouse infection model. Pharmacokinetic analysis of ND-09759 determined that at a dosage of 30 mg/kg mouse body weight (PO) gave a maximum serum drug concentration (Cmax) of 2.9 µg/ml and a half-life of 20.1 h. M. tuberculosis burden in the lungs and spleens was significantly decreased in mice treated once daily 6 days per week for 4-weeks with ND-09759 compared to untreated mice and this antibiotic activity was equivalent to isoniazid (INH) and rifampicin (RMP), two first-line anti-TB drugs. We observed slightly higher efficacy when using a combination of ND-09759 with either INH or RMP. Finally, the histopathological analysis revealed that infected mice treated with ND-09759 had significantly reduced inflammation relative to untreated mice. In conclusion, our findings indicate ND-09759 might be a potent candidate for the treatment of active TB in combination with current standard anti-TB drugs.

Pulmonary infections with Mycobacterium avium occur in susceptible individuals following exposure to the bacterium in the environment, where it often persists in biofilms. Many methods have been used to generate biofilms of M. avium, and it is unknown whether different approaches generate similar structures and cell phenotypes. To make a parallel comparison of in vitro biofilm ultrastructure, extracellular matrix (ECM) composition, and the drug susceptibility of biofilm resident bacteria, we used two published methods to generate M. avium biofilms: four-week incubation in M63 medium or 24 h exposure to dithiothreitol (DTT). Scanning electron microscopy revealed differences in the biofilm ultrastructure between the two methods, including variation in the appearance of ECM materials and morphology of resident cells, while light microscopy and staining with calcofluor white indicated that both biofilms contained polysaccharides characteristic of cellulose. Measuring the susceptibility of biofilms to degradation by enzymes suggested differences in structurally important ECM molecules, with DTT biofilms having important protein and, to a lesser extent, cellulose components, and M63 biofilms having moderate protein, cellulose, and DNA components. Both biofilms conferred resistance to the bactericidal effects of amikacin and clarithromycin, with resident cells being killed at greater than 10-fold lower rates than planktonic cells at almost all concentrations. These comparisons indicate differences in biofilm responses by M. avium under differing conditions, but also suggest common features of biofilm formation, including cellulose production and antimicrobial resistance.

Exosomes and other extracellular microvesicles (ExMVs) have important functions in intercellular communication and regulation. During the course of infection, these vesicles can convey pathogen molecules that serve as antigens or agonists of innate immune receptors to induce host defense and immunity, or that serve as regulators of host defense and mediators of immune evasion. These molecules may include proteins, nucleic acids, lipids, and carbohydrates. Pathogen molecules may be disseminated by incorporation into vesicles that are created and shed by host cells, or they may be incorporated into vesicles shed from microbial cells. Involvement of ExMVs in the induction of immunity and host defense is widespread among many pathogens, whereas their involvement in immune evasion mechanisms is prominent among pathogens that establish chronic infection and is found in some that cause acute infection. Because of their immunogenicity and enrichment of pathogen molecules, exosomes may also have potential in vaccine preparations and as diagnostic markers. Additionally, the ability of exosomes to deliver molecules to recipient cells raises the possibility of their use for drug/therapy delivery. Thus, ExMVs play a major role in the pathogenesis of infection and provide exciting potential for the development of novel diagnostic and therapeutic approaches.

Activation of both CD4.sup.+ and CD8.sup.+ T cells is required for an effective immune response to an M. tuberculosis infection. However, infected macrophages are poor antigen presenting cells and may be spatially separated from recruited T cells, thus limiting antigen presentation within a granuloma. Our previous studies showed that infected macrophages release from cells small membrane-bound vesicles called exosomes which contain mycobacterial lipid components and showed that these exosomes could stimulate a pro-inflammatory response in naïve macrophages. In the present study we demonstrate that exosomes stimulate both CD4.sup.+ and CD8.sup.+ splenic T cells isolated from mycobacteria-sensitized mice. Although the exosomes contain MHC I and II as well as costimulatory molecules, maximum stimulation of T cells required prior incubation of exosomes with antigen presenting cells. Exosomes isolated from M. bovis and M. tuberculosis infected macrophages also stimulated activation and maturation of mouse bone marrow-derived dendritic cells. Interestingly, intranasal administration of mice with exosomes isolated from M. bovis BCG infected macrophages induce the generation of memory CD4.sup.+ and CD8.sup.+ T cells. The isolated T cells also produced IFN-[gamma] upon restimulation with BCG antigens. The release of exosomes from infected macrophages may overcome some of the defects in antigen presentation associated with mycobacterial infections and we suggest that exosomes may be a promising M. tuberculosis vaccine candidate.

Tuberculosis is the leading cause of death due to an infectious organism, killing an estimated 3 million people annually. Mycobacterium tuberculosis, the causative agent of tuberculosis, and other pathogenic mycobacteria require entry into host macrophages to initiate infection. An invasion mechanism was defined that was shared among pathogenic mycobacteria including M. tuberculosis, M. leprae, and M. avium but not by nonpathogenic mycobacteria or nonmycobacterial intramacrophage pathogens. This pathway required the association of the complement cleavage product C2a with mycobacteria resulting in the formation of a C3 convertase. The mycobacteria-associated C2a cleaved C3, resulting in C3b opsonization of the mycobacteria and recognition by macrophages.