Protective immunity against Mycobacterium tuberculosis involves major histocompatibility complex class I (MHC-I)- and CD1-restricted CD8 T cells, but the mechanisms underlying antigen delivery to antigen-presenting molecules remain enigmatic. Macrophages, the primary host cells for mycobacteria, are CD1-negative. Here we show that M. tuberculosis phagosomes are secluded from the cytosolic MHC-I processing pathway and that mycobacteria-infected cells lose their antigen-presenting capacity. We also show that mycobacteria induce apoptosis in macrophages, causing the release of apoptotic vesicles that carry mycobacterial antigens to uninfected antigen-presenting cells (APCs). Inhibition of apoptosis reduced transfer of antigens to bystander cells and activation of CD8 T cells. Uninfected dendritic cells, which engulfed extracellular vesicles, were indispensable for subsequent cross-presentation of antigens, through MHC-I and CD1b, to T cells from mycobacteria-sensitized donors. This new 'detour' pathway for presentation of antigens from a phagosome-contained pathogen shows the functional significance of infection-induced apoptosis in the activation of CD8 T cells specific for both protein and glycolipid antigens in tuberculosis.
SummaryIn 1993, the WHO declared tuberculosis a global emergency on the basis that there are 8 million new cases per year. The complete genome of the strain H37Rv of the causative microorganism, Mycobacterium tuberculosis, comprising 3924 genes has been sequenced. We compared the proteomes of two non-virulent vaccine strains of M. bovis BCG (Chicago and Copenhagen) with two virulent strains of M. tuberculosis (H37Rv and Erdman) to identify protein candidates of value for the development of vaccines, diagnostics and therapeutics. The mycobacterial strains were analysed by two-dimensional electrophoresis (2-DE) combining non-equilibrium pH gradient electrophoresis (NEPHGE) with SDS±PAGE. Distinct and characteristic proteins were identi®ed by mass spectrometry and introduced into a dynamic 2-DE database (http:/ /www.mpiibberlin.mpg.de/2D-PAGE). Silver-stained 2-DE patterns of mycobacterial cell proteins or culture supernatants contained 1800 or 800 spots, respectively, from which 263 were identi®ed. Of these, 54 belong to the culture supernatant. Sixteen and 25 proteins differing in intensity or position between M. tuberculosis H37Rv and Erdman, and H37Rv and M. bovis BCG Chicago, respectively, were identi®ed and categorized into protein classes. It is to be hoped that the availability of the mycobacterial proteome will facilitate the design of novel measures for prevention and therapy of one of the great health threats, tuberculosis.
As a hallmark of tuberculosis (TB), Mycobacterium tuberculosis (MTB) induces granulomatous lung lesions and systemic inflammatory responses during active disease. Molecular regulation of inflammation is associated with inflammasome assembly. We determined the extent to which MTB triggers inflammasome activation and how this impacts on the severity of TB in a mouse model. MTB stimulated release of mature IL-1b in macrophages while attenuated M. bovis BCG failed to do so. Tubercle bacilli specifically activated the NLRP3 inflammasome and this propensity was strictly controlled by the virulenceassociated RD1 locus of MTB. However, Nlrp3-deficient mice controlled pulmonary TB, a feature correlated with NLRP3-independent production of IL-1b in infected lungs. Our studies demonstrate that MTB activates the NLRP3 inflammasome in macrophages in an ESX-1-dependent manner. However, during TB, MTB promotes NLRP3-and caspase-1-independent IL-1b release in myeloid cells recruited to lung parenchyma and thus overcomes NLRP3 deficiency in vivo in experimental models.
Classical proteomics combined two-dimensional gel electrophoresis (2-DE) for the separation and quantification of proteins in a complex mixture with mass spectrometric identification of selected proteins. More recently, the combination of liquid chromatography (LC), stable isotope tagging, and tandem mass spectrometry (MS/MS) has emerged as an alternative quantitative proteomics technology. We have analyzed the proteome of Mycobacterium tuberculosis, a major human pathogen comprising about 4,000 genes, by (i) 2-DE and mass spectrometry (MS) and by (ii) the isotope-coded affinity tag (ICAT) reagent method and MS/MS. The data obtained by either technology were compared with respect to their selectivity for certain protein types and classes and with respect to the accuracy of quantification. Initial datasets of 60,000 peptide MS/MS spectra and 1,800 spots for the ICAT-LC/MS and 2-DE/MS methods, respectively, were reduced to 280 and 108 conclusively identified and quantified proteins, respectively. ICAT-LC/MS showed a clear bias for high M r proteins and was complemented by the 2-DE/MS method, which showed a preference for low M r proteins and also identified cysteine-free proteins that were transparent to the ICAT-LC/MS method. Relative quantification between two strains of the M. tuberculosis complex also revealed that the two technologies provide complementary quantitative information; whereas the ICAT-LC/MS method quantifies the sum of the protein species of one gene product, the 2-DE/MS method quantifies at the level of resolved protein species, including post-translationally modified and processed polypeptides. Our data indicate that different proteomic technologies applied to the same sample provide complementary types of information that contribute to a more complete understanding of the biological system studied. with mass spectrometry (MS) and have revealed about 1,800 distinct spots separated by 2-DE (1). About 350 of these were identified, and the comparison of the protein patterns of virulent and attenuated strains identified several proteins that are being studied further as potential vaccine candidates (1, 2). More than two million deaths/year and eight million new infections are caused by M. tuberculosis, the bacterium responsible for tuberculosis (3). Genomics, transcriptomics, and proteomics, which form the rationale basis to develop new therapeutic and preventive strategies, have been applied to combat this disease. The complete genome of M. tuberculosis comprises about 4,000 genes, which were classified in six protein classes and 30 subclasses (4).The smallest unit of the proteome, the protein species, is defined by its chemical structure (5). Therefore, each modification of a protein leads to a new protein species. These are successfully resolved by 2-DE if they differ by at least one charge or by at least several hundred daltons in mass. Quantification by 2-DE requires a high degree of pattern reproducibility, which is difficult to achieve in a multistep and parallel procedure.To alleviate limi...
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