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

Abstract

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.

Unexpected Link between Lipooligosaccharide Biosynthesis and Surface Protein Release in Mycobacterium marinum

“…Colonies with reduced amounts of secreted PE_PGRS proteins were selected and re-checked for PE_PGRS secretion defects in a second double filter assay. In a similar assay, a selection of mutants was also tested with antiserum directed against the surface protein EspE (4,25).…”

“…The natural antibiotic resistance of M. tuberculosis is partially due to its exceptionally hydrophobic and complex cell envelope structure. Although mycobacteria are classified as high GC Gram-positives, recent cryo-transmission electron microscopy unequivocally showed that the mycobacterial cell envelope consists of two membranes: a cytoplasmic membrane and a unique outer membrane (2)(3)(4). The inner leaflet of this atypical outer membrane consists of long chain fatty acids called mycolic acids, which are covalently linked to a periplasmic arabinogalactan polymer.…”

Latently and uninfected healthcare workers exposed to TB make protective antibodies against Mycobacterium tuberculosis

“…If the target of protective antibodies was expressed on the mycobacterial surface, we reasoned that standard culture of mycobacteria in our experiments, which includes axenic growth in media containing the mild detergent Tween-80, might strip away some of the mycobacterial capsule components (28). We, therefore, grew Mtb either on 7H10 agar plated in the absence of Tween-80 or in axenic culture in supplemented 7H9 medium, which includes Tween-80, and then used the bacteria in the WBA.…”

General secretion signal for the mycobacterial type VII secretion pathway

“…In pathogenic mycobacteria, several different ESX systems exist and we were interested in identifying the secretion signal for these systems as well. Recently, we identified a putative ESX-1-dependent PPE substrate by proteomic analysis, i.e., M. marinum PPE68_1 (hereafter referred to as MmPPE68_1) (15). MmPPE68_1 is adjacent to PE35 (hereafter referred to as MmPE35) in a two-gene operon (Mmar_0185-Mmar_0186).…”

“…The archetypical T7S system, ESX-1, is responsible for the secretion of several proteins, including the ESX-1-encoded T-cell antigens ESAT-6 (early secreted antigenic target of 6 kDa) and CFP-10 (culture filtrate protein of 10 kDa) (7,(11)(12)(13)(14)(15). ESX-1 and its substrates play an important, albeit not yet fully understood role in M. tuberculosis virulence.…”