title: 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?
abstract: 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 beta-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 beta-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.
authors.affiliation: Italfarmaco Research Centre, Cinisello Balsamo 20092, Milan, Italy.
authors.forename: Gianluca
authors.initials: G
authors.lastname: Fossati
authors.forename: Gaetano
authors.initials: G
authors.lastname: Izzo
authors.forename: Emanuele
authors.initials: E
authors.lastname: Rizzi
authors.forename: Emanuela
authors.initials: E
authors.lastname: Gancia
authors.forename: Daniela
authors.initials: D
authors.lastname: Modena
authors.forename: Maria Luisa
authors.initials: ML
authors.lastname: Moras
authors.forename: Neri
authors.initials: N
authors.lastname: Niccolai
authors.forename: Elena
authors.initials: E
authors.lastname: Giannozzi
authors.forename: Ottavia
authors.initials: O
authors.lastname: Spiga
authors.forename: Letizia
authors.initials: L
authors.lastname: Bono
authors.forename: Piero
authors.initials: P
authors.lastname: Marone
authors.forename: Eugenio
authors.initials: E
authors.lastname: Leone
authors.forename: Francesca
authors.initials: F
authors.lastname: Mangili
authors.forename: Stephen
authors.initials: S
authors.lastname: Harding
authors.forename: Neil
authors.initials: N
authors.lastname: Errington
authors.forename: Christopher
authors.initials: C
authors.lastname: Walters
authors.forename: Brian
authors.initials: B
authors.lastname: Henderson
authors.forename: Michael M
authors.initials: MM
authors.lastname: Roberts
authors.forename: Anthony R M
authors.initials: AR
authors.lastname: Coates
authors.forename: Bruno
authors.initials: B
authors.lastname: Casetta
authors.forename: Paolo
authors.initials: P
authors.lastname: Mascagni
mesh_terms: D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D018835:Chaperonin 10 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D006801:Humans; D008264:Macrophages / Q000378:metabolism / Q000382:microbiology*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D008854:Microscopy, Electron; D009169:Mycobacterium tuberculosis / Q000378:metabolism* / Q000472:pathogenicity; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D010588:Phagosomes / Q000378:metabolism* / Q000382:microbiology; D017433:Protein Structure, Secondary; D011817:Rabbits; D012997:Solvents; D013329:Structure-Activity Relationship
chemical_list: D018835:Chaperonin 10; D010455:Peptides; D012997:Solvents