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Journal of Bacteriology, July 2003, p. 4256-4267, Vol. 185, No. 14
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.14.4256-4267.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Mycobacterium tuberculosis Chaperonin 10 Is Secreted in the Macrophage Phagosome: Is Secretion Due to Dissociation and Adoption of a Partially Helical Structure at the Membrane?

Gianluca Fossati,1 Gaetano Izzo,1 Emanuele Rizzi,1 Emanuela Gancia,1 Daniela Modena,1 Maria Luisa Moras,1 Neri Niccolai,2 Elena Giannozzi,2 Ottavia Spiga,2 Letizia Bono,3 Piero Marone,3 Eugenio Leone,4 Francesca Mangili,4 Stephen Harding,5 Neil Errington,5 Christopher Walters,5 Brian Henderson,6 Michael M. Roberts,7 Anthony R. M. Coates,7 Bruno Casetta,8 and Paolo Mascagni1*

Italfarmaco Research Centre, Cinisello Balsamo 20092,1 Anatomia Patologica, Facoltà di Medicina e Chirurgia, Università Milano Bicocca,4 Applied Biosystems, 20052 Monza, Milan,8 Biomolecular Structure Research Centre and Department of Molecular Biology, University of Siena, Siena,2 IRCCS S. Matteo Hospital, Pavia, Italy,3 Department of Applied Biochemistry and Food Science, University of Nottingham, Nottingham,5 Cellular Microbiology Research Group, Eastman Dental Institute, University College London,6 Department of Medical Microbiology, St George's Hospital Medical School, London, United Kingdom7

Received 4 February 2003/ Accepted 28 April 2003

To confirm that Mycobacterium tuberculosis chaperonin 10 (Cpn10) is secreted outside the live bacillus, infected macrophages were examined by electron microscopy. This revealed that the mycobacterial protein accumulates both in the wall of the bacterium and in the matrix of the phagosomes in which ingested mycobacteria survive within infected macrophages. To understand the structural implications underlying this secretion, a structural study of M. tuberculosis Cpn10 was performed under conditions that are generally believed to mimic the membrane environment. It was found that in buffer-organic solvent mixtures, the mycobacterial protein forms two main species, namely, a partially helical monomer that prevails in dilute solutions at room temperature and a dimer that folds into a ß-sheet-dominated structure and prevails in either concentrated protein solutions at room temperature or in dilute solutions at low temperature. A partially helical monomer was also found and was completely associated with negatively charged detergents in a micelle-bound state. Remarkably, zwitterionic lipids had no effect on the protein structure. By using N- and C-truncated forms of the protein, the C- and N-terminal sequences were identified as possessing an amphiphilic helical character and as selectively associating with acidic detergent micelles. When the study was extended to other chaperonins, it was found that human Cpn10 is also monomeric and partially helical in dilute organic solvent-buffer mixtures. In contrast, Escherichia coli Cpn10 is mostly dimeric and predominately ß-sheet in both dilute and concentrated solutions. Interestingly, human Cpn10 also crosses biological membranes, whereas the E. coli homologue is strictly cytosolic. These results suggest that dissociation to partially helical monomers and interaction with acidic lipids may be two important steps in the mechanism of secretion of M. tuberculosis Cpn10 to the external environment.

* Corresponding author. Mailing address: Italfarmaco Research Centre, via dei Lavoratori 54, Cinisello Balsamo 20092, Milan, Italy. Phone: 390264433000. Fax: 390266011579. E-mail: p.mascagni{at}italfarmaco.com.

Journal of Bacteriology, July 2003, p. 4256-4267, Vol. 185, No. 14
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.14.4256-4267.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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