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Infection and Immunity, November 1999, p. 5552-5558, Vol. 67, No. 11
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Differential T-Cell Recognition of Native and Recombinant Mycobacterium tuberculosis GroES

Ida Rosenkrands,1 Karin Weldingh,1 Pernille Ravn,1 Lise Brandt,1 Peter Højrup,2 Peter Birk Rasmussen,1 Anthony R. Coates,3 Mahavir Singh,4 Paolo Mascagni,5 and Peter Andersen1,*

Department of TB Immunology, Statens Serum Institut, Copenhagen,1 and Department of Molecular Biology, Odense University, Odense,2 Denmark; Department of Medical Microbiology, St. George's Hospital Medical School, London, United Kingdom3; Department of Chemistry, GBF Center for Biotechnology, Braunschweig, Germany4; and Italfarmaco Research Centre, Cinisello Balsamo, Milan, Italy5

Received 11 March 1999/Returned for modification 7 May 1999/Accepted 9 August 1999

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.

* Corresponding author. Mailing address: Department of TB Immunology, Statens Serum Institut, 5 Artillerivej, DK-2300 Copenhagen S, Denmark. Phone: 45 32 68 34 62. Fax: 45 32 68 30 35. E-mail: tbimm{at}ssi.dk.

Infection and Immunity, November 1999, p. 5552-5558, Vol. 67, No. 11
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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