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.
Highlights
Mycobacteria are the causative agent of tuberculosis and other chronic diseases such as leprosy and causes about 1.7 million deaths annually
The mycobacterial cell envelope has a complex structure composed of a typical phospholipid bilayer plasma membrane (PM), an outer membrane and an outermost layer known as the capsule in the case of pathogenic species [2]
We demonstrated the presence of the capsular layer in the cell envelope of both pathogenic and nonpathogenic mycobacterial species and determined its thickness to be around 30 nm (Table S1), taking into account the instability of the capsule in vitro; both groups have been previously shown to differ in the amount of extracellular material found in static and non-detergent-treated medium, which is interpreted as representing part of the capsule shed from growing cells [10,11]
Summary
Mycobacteria are the causative agent of tuberculosis and other chronic diseases such as leprosy and causes about 1.7 million deaths annually. The mycobacterial cell envelope has a complex structure composed of a typical phospholipid bilayer plasma membrane (PM), an outer membrane and an outermost layer known as the capsule in the case of pathogenic species [2]. The capsule is visible as an electron transparent zone (ETZ) surrounding the mycobacterial cell envelope [2,7] in conventional EM preparations [8,9]. This layer was not, observed by cryo-EM [5,6], questioning somehow its existence. We have characterized the mycobacterial capsule layer by immunological and
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