B-Cell Epitopes and Quantification of the ESAT-6 Protein of Mycobacterium tuberculosis

ESAT-6 is an important T-cell antigen recognized by protective T cells in animal models of infection with Mycobacterium tuberculosis. In an enzyme-linked immunosorbent assay (ELISA) with overlapping peptides spanning the sequence of ESAT-6, monoclonal antibody HYB76-8 reacted with two peptides in the N-terminal region of the molecule. Assays with synthetic truncated peptides allowed a precise mapping of the epitope to the residues EQQWNFAGIEAAA at positions 3 to 15. Hydrophilicity plots revealed one hydrophilic area at the N terminus and two additional areas further along the polypeptide chain. Antipeptide antibodies were generated by immunization with synthetic 8-mer peptides corresponding to these two regions coupled to keyhole limpet hemocyanin. Prolonged immunization with a 23-mer peptide (positions 40 to 62) resulted in the formation of antibodies reacting with the peptide as well as native ESAT-6. A double-antibody ELISA was then developed with monoclonal antibody HYB76-8 as a capture antibody, antigen for testing in the second layer, and antipeptide antibody in the third layer. The assay was suitable for quantification of ESAT-6 in M. tuberculosis antigen preparations, showing no reactivity with M. bovis BCG Tokyo culture fluid, used as a negative control, or with MPT64 or antigen 85B, previously shown to cross-react with HYB76-8. This capture ELISA permitted the identification of ESAT-6 expression from vaccinia virus constructs containing the esat-6 gene; this expression could not be identified by standard immunoblotting.     

MPB70 and MPB83 as Indicators of Protein Localization in Mycobacterial Cells

Culture fluids after growth of Mycobacterium bovis BCG on Sauton medium contain actively secreted proteins and proteins released by bacterial lysis. BCG culture fluids and sonicates of Mycobacterium tuberculosis and Mycobacterium paratuberculosis were tested after separation by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The localization of marker proteins was determined by enzyme-linked immunosorbent assay and Western blotting with selected monoclonal antibodies of known specificities. Soluble secreted proteins (MPB64 and proteins of the antigen 85 complex) and three heat shock proteins (DnaK, GroEL, and GroES) were recovered in a single peak after gel filtration, indicating their occurrence as a free monomer in the culture fluid and cytosol, respectively. Other constituents eluted in two distinct peaks during gel filtration. The first peak corresponded to the void volume, indicating complex formation between several proteins or attachment to lipids in the surface layer or the cytoplasmic membrane; the second peak corresponded to the expected monomer size indicated by SDS-PAGE under conditions that separate proteins from each other during sample preparation. The two-peak group contained constituents with known lipid contents, the 19- and 38-kDa lipoproteins and lipoarabinomannan. The 26-kDa form of MPB83 behaved similarly. After extraction with Triton X-114, these constituents entered into the detergent phase, confirming the lipoprotein nature of 26-kDa MPB83. The MPB83 molecule was shown to be available on the surface of BCG Tokyo bacilli for reaction with monoclonal antibody MBS43 by flow cytometry.

A multitude of different proteins are synthesized by the mycobacterial cell. It is often valuable to consider these proteins in different broad categories based on common characteristics, such as physicochemical properties, localization in the mycobacterial cell, and active secretion during culture. In turn, these distinct properties are closely related to their functional properties and the tendency to interact with the immune system of the host after infection.MPB70 was initially isolated by Nagai et al. (37). This secreted protein is of particular interest since it is highly species specific (13). It is consistently present in virulent Mycobacterium bovis, whereas it is synthesized and secreted in markedly different amounts by various substrains of avirulent M. bovis BCG (13, 32, 35).We recently studied the closely related MPB83 protein and isolated three peptides derived from it after CNBr cleavage, showing that MPB70 and MPB83 are homologous but clearly distinct proteins and are therefore encoded by different genes (15). The heterogeneity between different substrains of BCG in regard to the synthesis of MPB70 and MPB83 proteins is clearly greater than previously realized (53). Another type of heterogeneity has also previously been identified; 26- and 23-kDa fractions of a protein that was presumed to be MPB70 differed markedly in carbohydrate content (9). The available data indicate that MPB83 occurs in 26- and 23-kDa forms, both glycosylated, whereas MPB70 (at 22 kDa) is nonglycosylated.The term MPB was introduced (37) for the designation of a protein purified from M. bovis BCG, with the number after MPB denoting the relative mobility by electrophoresis on a 7.7% polyacrylamide gel run at pH 9.5. MPT is used for similar designations of proteins purified from M. tuberculosis (38). The designations mpb70 and mpt70 denote the corresponding genes.Cloning of mpb70 (43) revealed the sequence of a polypeptide chain preceded by a signal peptide that is typical of secreted proteins. In contrast, both mpb83 from M. bovis BCG (33, 45) and mpt83 from M. tuberculosis (20) revealed a typical lipoprotein consensus element in the signal sequence. The relative concentrations of the 26- and 23-kDa components of these proteins vary markedly between sonicates and culture fluids of BCG bacilli. In sonicates, the 26-kDa component, consisting of MPB83, dominates. In culture fluids, the reverse is observed, with a markedly higher concentration of the 22- to 23-kDa components in BCG Sweden and BCG Russia (18).The purpose of the present work was to investigate these properties at the level of native proteins in relation to the localization of these and other marker proteins in the mycobacterial cell. Using monoclonal antibody (MAb) MBS43, which reacts with MPB83 but not with MPB70 (53), permitted more precise distinctions between these proteins than previously obtained.     

Children's conceptions of their own play

This paper described a research project, Play and Learning, to investigate the mental processes that underlie children's activities in “free play” and “structured learning” situations in preschool. Interviews of children about “what they did” were used to investigate children's own views of play and learning.