Direct Visualization by Cryo-EM of the Mycobacterial Capsular Layer: A Labile Structure Containing ESX-1-Secreted Proteins

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, α-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

Metabolic Network for the Biosynthesis of Intra- and Extracellular alpha-Glucans Required for Virulence of Mycobacterium tuberculosis

Mycobacterium tuberculosis synthesizes intra- and extracellular alpha-glucans that were believed to originate from separate pathways. The extracellular glucose polymer is the main constituent of the mycobacterial capsule that is thought to be involved in immune evasion and virulence. However, the role of the alpha-glucan capsule in pathogenesis has remained enigmatic due to an incomplete understanding of alpha-glucan biosynthetic pathways preventing the generation of capsule-deficient mutants. Three separate and potentially redundant pathways had been implicated in alpha-glucan biosynthesis in mycobacteria: the GlgC-GlgA, the Rv3032 and the TreS-Pep2-GlgE pathways. We now show that alpha-glucan in mycobacteria is exclusively assembled intracellularly utilizing the building block alpha-maltose-1-phosphate as the substrate for the maltosyltransferase GlgE, with subsequent branching of the polymer by the branching enzyme GlgB. Some alpha-glucan is exported to form the alpha-glucan capsule. There is an unexpected convergence of the TreS-Pep2 and GlgC-GlgA pathways that both generate alpha-maltose-1-phosphate. While the TreS-Pep2 route from trehalose was already known, we have now established that GlgA forms this phosphosugar from ADP-glucose and glucose 1-phosphate 1000-fold more efficiently than its hitherto described glycogen synthase activity. The two routes are connected by the common precursor ADPglucose, allowing compensatory flux from one route to the other. Having elucidated this unexpected configuration of the metabolic pathways underlying alpha-glucan biosynthesis in mycobacteria, an M. tuberculosis double mutant devoid of alpha-glucan could be constructed, showing a direct link between the GlgE pathway, alpha-glucan biosynthesis and virulence in a mouse infection model

Identification of the surface-exposed lipids on the cell envelopes of Mycobacterium tuberculosis and other mycobacterial species.

The surface-exposed lipids of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium kansasii, Mycobacterium gastri, Mycobacterium smegmatis, and Mycobacterium aurum were isolated by gentle mechanical treatment of cells with glass beads. Analysis of the exposed lipids demonstrated a selective location of classes of ubiquitous lipids on the surfaces of mycobacteria. While phosphatidylethanolamine and phosphatidylinositol mannosides were exposed in all the species examined, dimycoloyl trehalose ("cord factor") was identified in the surface components of M. aurum only. Furthermore, monomycoloyl trehaloses and triacylglycerides were identified in the surface-exposed lipids of M. avium and M. smegmatis but not in those of the other mycobacterial species examined. The species- and type-species specific lipids were present on the mycobacterial cell surface: phenolic glycolipids, dimycocerosates of phthiocerols, and lipooligosaccharides were identified in the surface-exposed materials of M. tuberculosis (Canetti), M. kansasii, and M. gastri, whereas glycopeptidolipids were identified in the outermost lipid constituents of M. avium and M. smegmatis. This difference in the surface exposure of lipids of various mycobacterial species may reflect differences in their cell envelope organizations. Brief treatments of M. tuberculosis with Tween 80 prior to the use of glass beads led to erosion of regions of the capsule to expose gradually both cord factor and other lipids on the cell surface of the tubercle bacillus, demonstrating that the latter lipids are buried more deeply in the cell envelope and leading to the proposal of a scheme for the location of the capsular lipids of the tubercle bacillus