Identification of proteins from Mycobacterium tuberculosis missing in attenuated Mycobacterium bovis BCG strains.

A proteome approach, combining high-resolution two-dimensional electrophoresis (2-DE) with mass spectrometry, was used to compare the cellular protein composition of two virulent strains of Mycobacterium tuberculosis with two attenuated strains of Mycobacterium bovis Bacillus Calmette-Guerin (BCG), in order to identify unique proteins of these strains. Emphasis was given to the identification of M. tuberculosis specific proteins, because we consider these proteins to represent putative virulence factors and interesting candidates for vaccination and diagnosis of tuberculosis. The genome of M. tuberculosis strain H37Rv comprises nearly 4000 predicted open reading frames. In contrast, the separation of proteins from whole mycobacterial cells by 2-DE resulted in silver-stained patterns comprising about 1800 distinct protein spots. Amongst these, 96 spots were exclusively detected either in the virulent (56 spots) or in the attenuated (40 spots) mycobacterial strains. Fifty-three of these spots were analyzed by mass spectrometry, of which 41 were identified, including 32 M. tuberculosis specific spots. Twelve M. tuberculosis specific spots were identified as proteins, encoded by genes previously reported to be deleted in M. bovis BCG. The remaining 20 spots unique for M. tuberculosis were identified as proteins encoded by genes that are not known to be missing in M. bovis BCG.

Isolation of RNA from mycobacteria grown under in vitro and in vivo conditions.

Isolation of RNA from mycobacteria is very difficult to perform, and the yields are generally very low. We describe an approach to isolate RNA from mycobacterial species which combines the disruption of mycobacterial cells by a silica/ceramic matrix in a reciprocal shaker with the ease and efficiency of subsequent RNA purification on spin columns with silica gel-based membranes. This method is rapid, easy to perform and yields high amounts of pure, intact total RNA. Due to its safety, this method is applicable even to group 3 biological hazard organisms like Mycobacterium tuberculosis. By combining a method for the isolation of phagosomal bacteria from infected primary macrophages with the novel RNA isolation technique, we are able to monitor gene expression during infection even in bacteria which are rather resistant to genetic manipulation, like Mycobacterium bovis.

Intersection of group I CD1 molecules and mycobacteria in different intracellular compartments of dendritic cells.

Human CD1a, CD1b, and CD1c molecules can present mycobacterial glycolipids to T cells. Because phagosomes containing viable mycobacteria represent early endosomal compartments, we studied where mycobacterial glycolipids intersect with CD1 molecules in infected APC. CD1b and CD1c, but not CD1a, localized to late endosomes/lysosomes. CD1a and CD1c were predominantly expressed on the cell surface and in mycobacterial phagosomes of the early endosomal stage. In contrast, CD1b was present in a subset of mycobacterial phagosomes representing mature phagolysosomes. Released mycobacterial glycolipids including lipoarabinomannan and phosphatidylinositol mannosides were transported from the phagosome into late endosomes/lysosomes and to uninfected bystander cells. The macrophage mannose receptor, which has been implicated in glycolipid uptake by APC for CD1b-mediated presentation, was absent from mycobacterial phagosomes and may therefore not be involved in trafficking of glycolipids between phagosomes and late endosomes/lysosomes. In conclusion, all three CD1 molecules have access to mycobacteria and glycolipids thereof, but at different intracellular sites. This allows sampling by CD1a, CD1b, and CD1c of mycobacterial glycolipids from different intracellular sites of the infected cell, which has important implications for processing and presentation of such Ags during mycobacterial infections.

Comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains: towards functional genomics of microbial pathogens.

In 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 identified by mass spectrometry and introduced into a dynamic 2-DE database (http://www.mpiib-berlin.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 identified. 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 identified 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.