Direct visualization by cryo-EM of the mycobacterial capsular layer: a labile structure containing ESX-1-secreted proteins

• Published in: PLoS pathogens Vol. 6; no. 3; p. e1000794
• Main Authors: Sani, Musa, Houben, Edith N G, Geurtsen, Jeroen, Pierson, Jason, de Punder, Karin, van Zon, Maaike, Wever, Brigitte, Piersma, Sander R, Jiménez, Connie R, Daffé, Mamadou, Appelmelk, Ben J, Bitter, Wilbert, van der Wel, Nicole, Peters, Peter J
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 01-03-2010; Public Library of Science (PLoS)
• Subjects: Bacteria; Bacterial Capsules - metabolism; Bacterial Capsules - ultrastructure; Bacterial Outer Membrane Proteins - metabolism; Bacterial Outer Membrane Proteins - secretion; Bacterial Outer Membrane Proteins - ultrastructure; Bacterial proteins; Bacteriology; Cell Biology; Cryoelectron Microscopy; Cytokines; Cytokines - metabolism; Electron microscopy; Experiments; Health aspects; Humans; Infectious Diseases/Bacterial Infections; Lipids; Macrophages - immunology; Macrophages - metabolism; Macrophages - microbiology; Microbiology; Microbiology/Cellular Microbiology and Pathogenesis; Microscopy; Microscopy, Immunoelectron; Mycobacteria; Mycobacterium; Mycobacterium - metabolism; Mycobacterium - ultrastructure; Mycobacterium bovis - metabolism; Mycobacterium bovis - ultrastructure; Mycobacterium marinum - metabolism; Mycobacterium marinum - ultrastructure; Mycobacterium smegmatis - metabolism; Mycobacterium smegmatis - ultrastructure; Mycobacterium tuberculosis - metabolism; Mycobacterium tuberculosis - ultrastructure; Physiological aspects; Proteins; Structure; Vaccines; Netherlands
• ISSN: 1553-7374; 1553-7366; 1553-7374
• DOI: 10.1371/journal.ppat.1000794

The cell envelope of mycobacteria, a group of Gram positive bacteria, is composed of a plasma membrane and a Gram-negative-like outer membrane containing mycolic acids. In addition, the surface of the mycobacteria is coated with an ill-characterized layer of extractable, non-covalently linked glycans, lipids and proteins, collectively known as the capsule, whose occurrence is a matter of debate. By using plunge freezing cryo-electron microscopy technique, we were able to show that pathogenic mycobacteria produce a thick capsule, only present when the cells were grown under unperturbed conditions and easily removed by mild detergents. This detergent-labile capsule layer contains arabinomannan, alpha-glucan and oligomannosyl-capped glycolipids. Further immunogenic and proteomic analyses revealed that Mycobacterium marinum capsule contains high amounts of proteins that are secreted via the ESX-1 pathway. Finally, cell infection experiments demonstrated the importance of the capsule for binding to cells and dampening of pro-inflammatory cytokine response. Together, these results show a direct visualization of the mycobacterial capsular layer as a labile structure that contains ESX-1-secreted proteins.