Isolation and partial characterization of major protein antigens in the culture fluid of Mycobacterium tuberculosis

Abstract

Five actively secreted proteins (MPT32, MPT45, MPT51, MPT53, and MPT63) and the MPT46 protein were purified to homogeneity from Mycobacterium tuberculosis culture fluid and compared with proteins previously purified by ourselves and other investigators. Antisera were obtained by immunization of rabbits with all of the newly isolated proteins identified to be immunogenic. Two-dimensional electrophoresis of culture fluids obtained each week for 2 to 10 weeks of culturing of M. tuberculosis revealed characteristic changes, permitting identification of two distinct groups of proteins being actively secreted from the mycobacterial cells or appearing later in the culture fluids as a result of the release of soluble proteins from the cytosol after lysis of bacteria. The N-terminal amino acid sequences of five MPTs were shown to be identical to those of proteins previously isolated by other investigators and given different designations, and five new sequences are given. These sequences and the use of the antisera may serve to identify these proteins with mycobacterial constituents isolated by other investigators. The previously identified but not isolated MPT45 protein was shown to correspond to the C component of the antigen 85 complex. The 27-kDa MPT51 protein was demonstrated to cross-react with the three components of the antigen 85 complex, and the N-terminal amino acid sequences of MPT51 and MPT59 showed 60% homology. This finding and the extensive cross-reactivity between the components of the antigen 85 complex may indicate that there is a family of closely related secreted proteins in mycobacteria.

Expression of Mycobacterium tuberculosis MPT64 in recombinant Myco. smegmatis: purification, immunogenicity and application to skin tests for tuberculosis

“…tuberculosis (0 . 3 mg/l; S. Nagai, personal communication [19] ). Purification of nMPT64 from culture filtrate, in which it constitutes only 0 .…”

“…Purification of nMPT64 from culture filtrate, in which it constitutes only 0 . 1% of the total protein, involves the use of at least four serial chromatographic procedures [19]. By contrast, purification from Myco.…”

Identification of the new T-cell-stimulating antigens fromMycobacterium tuberculosisculture filtrate

“…The cytokine production in response to all of the antigens tested was signi¢cantly greater in healthy tuberculin reactors than in negative subjects (P 6 0.05). The mean IFN-Q production in rMTSP11-stimulated PPD(+) PBMCs (305.7 þ 18.4 pg ml 31 ) was similar to that of Ag85B-and Ag85C-stimulated PPD(+) PBMCs (296.0 þ 32.4 and 348.3 þ 34.6 pg ml 31 , respectively) and was higher than that of rMTSP17-stimulated PBMCs (226.6 þ 10.4 pg ml 31 ) (Fig. 4B).…”

Mycobacterium tuberculosispellicles express unique proteins recognized by the host humoral response

“…The technique has been applied to MTB in conjunction with mass spectrometry (MS) to demonstrate differential protein responses to a variety of stressors such as heat, reactive oxygen/nitrogen species, or hypoxia. Much of the 2DGE proteomics studies on MTB have focused on culture filtrates to identify secreted virulence factors or diagnostic targets (Nagai et al, 1991;Naito et al, 1998;Malen et al, 2008). One study by Florczyk et al (2001) examined the difference between the proteome of shaken and standing cultures of BCG Pasteur demonstrating a group of five standing-culture-specific proteins.…”