Mapping and Identification of the Major Cell Wall-Associated Components of Mycobacterium leprae

Abstract

Mycobacterium leprae, an obligate intracellular pathogen, can be derived only from host tissue and thus affords the opportunity to study in vivo-expressed products responsible for the particular pathogenesis of leprosy. Despite considerable progress in the characterization of the proteins and secondary gene products ofM. leprae, there is little information on the nature of the proteins associated with the cell envelope. M. leprae has been fractionated into its major subcellular components, cell wall, cytoplasmic membrane, and soluble cytosol. A number of biochemical markers, including diaminopimelic acid content, monosaccharide composition, mycolic acid, and glycolipid distribution, were applied to their characterization, and two-dimensional gel electrophoresis was used to map the component proteins. A total of 391 major proteins spots were resolved, and 8 proteins were identified based on their reactivity to a panel of monoclonal antibodies and/or relative pI size. Microsequencing of six protein spots present in the cell wall fraction allowed identification of new proteins, including the protein elongation factor EF-Tu and a homolog for the Mycobacterium tuberculosis MtrA response regulator. These results, together with previous studies, contribute to the progressive knowledge of the composition of the in vivo-expressed proteins of M. leprae.

Use of a whole blood assay to evaluatein vitroT cell responses to new leprosy skin test antigens in leprosy patients and healthy subjects

“…There did not appear to be any group-specific effect to suggest that recognition of one fraction was associated with a particular group of subjects. MLCwA appears to be a more potent antigen for detecting exposure to M. leprae than MLSA-LAM Protein composition of MLSA-LAM and MLCwA SDS-PAGE gel electrophoresis and Western blotting using antibodies of known specificity [27] were used to identify the major proteins of MLSA-LAM and MLCwA ( [28][29][30]; C. Pessolani, unpublished data). Each fraction was found to contain a wide array of proteins.…”

“…These in vitro results suggest that further fractionation will be required to give preparations for use in a highly specific test of exposure to M. leprae. Comparison of T cell responses to the fractions with individual M. leprae proteins showed that the 65-kD and 35-kD proteins may be immunodominant antigens in these fractions, which is supported by the abundance of these two proteins in such preparations [30]. These and other proteins present in these fractions have previously been demonstrated to provoke type 1 responses in paucibacillary leprosy patients and healthy contacts: 10 kD [34], 18 kD [35], 28 kD [36], 30-31 kD (the Antigen 85 complex; [37]), 35 kD [38], 45-kD protein [39,40] and 65 kD [41].…”

“…This is supported by the observation that responses to whole M. leprae (in which the protein components are contained within the glycolipid cell wall, and require greater processing prior to presentation) were much lower than to M. leprae sonicate (or either of the fractions). However, [29][30][31]. †Coefficients of correlation are given as R values, with significance of correlation (P); significant associations are shown in bold.…”

High Resolution Electroelution of Polyacrylamide Gels for the Purification of Single Proteins from Mycobacterium tuberculosis Culture Filtrate

“…For TB18.6, database searches did not reveal the presence of structural elements which could indicate a biological function of this protein. A M. leprae homologue of TB18.6 has recently been identified as a minor spot in 2-DE analysis of the M.leprae cell wall preparation [39], and the position of this protein corresponds well with the position of TB18.6 in our M. tuberculosis 2-DE reference map.…”

Advances in Proteomics of Mycobacterium leprae

“…Better knowledge of the main antigens involved in induction of immune responses in patients with leprosy [2][3][4][5][6][7][8][9][10][11] can pave way for developing a vaccine against leprosy and for selecting immunodiagnostic reagents. Over the years, several studies [12][13][14][15][16][17] have been carried out to define the proteome of M. leprae. Highlights from various approaches used in these studies are summarized in Table 1; in addition, we review important findings obtained through proteomics of M. leprae.…”