title: Differential T-cell recognition of native and recombinant Mycobacterium tuberculosis GroES.
abstract: Mycobacterium tuberculosis GroES was purified from culture filtrate, and its identity was confirmed by immunoblot analysis and N-terminal sequencing. Comparing the immunological recognition of native and recombinant GroES, we found that whereas native GroES elicited a strong proliferative response and release of gamma interferon-gamma by peripheral blood mononuclear cells from healthy tuberculin reactors, the recombinant protein failed to do so. The same difference in immunological recognition was observed in a mouse model of TB infection. Both the native and recombinant preparations were recognized by mice immunized with the recombinant protein. Biochemical characterization including sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two-dimensional electrophoresis, and mass spectrometry analysis of both proteins demonstrated no differences between the native and recombinant forms of GroES except for the eight additional N-terminal amino acids derived from the fusion partner in recombinant GroES. The recombinant fusion protein, still tagged with the maltose binding protein, was recognized by T cells isolated from TB-infected mice if mixed with culture filtrate before affinity purification on an amylose column. The maltose binding protein treated in the same manner as a control preparation was not recognized. Based on the data presented, we suggest that the association of biologically active molecules from culture filtrate with the chaperone GroES may be responsible for the observed T-cell recognition of the native preparation.
authors.affiliation: Department of TB Immunology, Statens Serum Institut, Copenhagen, Odense, Denmark.
authors.forename: I
authors.initials: I
authors.lastname: Rosenkrands
authors.forename: K
authors.initials: K
authors.lastname: Weldingh
authors.forename: P
authors.initials: P
authors.lastname: Ravn
authors.forename: L
authors.initials: L
authors.lastname: Brandt
authors.forename: P
authors.initials: P
authors.lastname: Højrup
authors.forename: P B
authors.initials: PB
authors.lastname: Rasmussen
authors.forename: A R
authors.initials: AR
authors.lastname: Coates
authors.forename: M
authors.initials: M
authors.lastname: Singh
authors.forename: P
authors.initials: P
authors.lastname: Mascagni
authors.forename: P
authors.initials: P
authors.lastname: Andersen
mesh_terms: D000818:Animals; D018835:Chaperonin 10 / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D018835:Chaperonin 10; D011993:Recombinant Fusion Proteins