Direct visualization by cryo-EM of the mycobacterial capsular layer: a labile structure containing ESX-1-secreted
Musa Sani1, Edith N G Houben, Jeroen Geurtsen
1Division of Cell Biology-B6, Netherlands Cancer Institute-Antoni van Leeuwenhoek Hospital (NKI-AVL), Amsterdam, the Netherlands.
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, 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.
Insights
Pathogenic mycobacteria possess a thick, detergent-labile capsule containing specific carbohydrates and ESX-1 secreted proteins. This capsule aids in host cell binding and reduces inflammatory responses during infection.
Area of Science:
- Microbiology
- Cell Biology
- Structural Biology
Background:
- Mycobacteria, Gram-positive bacteria, have a complex cell envelope including mycolic acids.
- The presence and composition of a mycobacterial capsule remain poorly understood.
- Previous studies debated the existence and characteristics of the mycobacterial capsule.
Purpose of the Study:
- To visualize and characterize the mycobacterial capsule.
- To identify the molecular components of the capsule.
- To determine the functional role of the capsule in host-pathogen interactions.
Main Methods:
- Plunge freezing cryo-electron microscopy for high-resolution imaging.
- Biochemical analysis of detergent-extracted capsule components.
- Immunogenic and proteomic analyses to identify proteins.
- Cell infection experiments to assess functional roles.
Main Results:
- Pathogenic mycobacteria produce a thick, labile capsule under unperturbed growth conditions.
- The capsule is composed of arabinomannan, alpha-glucan, and oligomannosyl-capped glycolipids.
- Mycobacterium marinum capsule is rich in proteins secreted via the ESX-1 pathway.
- The capsule facilitates bacterial binding to host cells and suppresses pro-inflammatory cytokine release.
Conclusions:
- Direct visualization confirms the existence of a labile mycobacterial capsule.
- The capsule contains specific glycans, lipids, and ESX-1 secreted proteins.
- The capsule plays a crucial role in host cell attachment and immune modulation.
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