MPB70 AND MPB83 AS INDICATORS OF PROTEIN LOCALIZATION IN MYCOBACTERIAL CELLS

Culture fluids after growth of Mycobacterium bovis BCG on Sauton mediu m contain actively secreted proteins and proteins released by bacteria l lysis, BCG culture fluids and sonicates of Mycobacterium tuberculosi s and Mycobacterium paratuberculosis were tested after separation by g el filtration and sodium dodecyl sulfate-polyacrylamide gel electropho resis (SDS-PAGE), The localization of marker proteins was determined b y enzyme-linked immunosorbent assay and Western blotting with selected monoclonal antibodies of known specificities. Soluble secreted protei ns (MPB64 and proteins of the antigen 85 complex) and three heat shock proteins (DnaK, GroEL, and GroES) were recovered in a single peak aft er gel filtration, indicating their occurrence as a free monomer in th e culture fluid and cytosol, respectively. Other constituents eluted i n two distinct peaks during gel filtration, The first peak corresponde d to the void volume, indicating complex formation between several pro teins or attachment to lipids in the surface layer or the cytoplasmic membrane; the second peak corresponded to the expected monomer size in dicated by SDS-PAGE under conditions that separate proteins from each other during sample preparation. The two-peak group contained constitu ents with known lipid contents, the 19- and 38-kDa lipoproteins and li poarabinomannan. The 26-kDa form of MPB83 behaved similarly, After ext raction with Triton X-114, these constituents entered into the deterge nt phase, confirming the lipoprotein nature of 26-kDa MPB83, The MPB83 molecule was shown to be available on the surface of BCG Tokyo bacill i for reaction with monoclonal antibody MBS43 by flow cytometry.