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

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          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.

          Author Summary

          The genus Mycobacterium contains a number of important pathogens, such as Mycobacterium tuberculosis. The highly characteristic cell envelope of these bacteria plays a crucial role in the infection process. The most apparent difference with other bacteria is the recently described outer membrane composed of unique (glyco)lipids. However, on top of this membrane mycobacteria also have an ill-defined capsular layer. In this paper, we studied this capsular layer using different electron microscopy techniques and mass spectrometry. Using close to native state preparation method, we show that both pathogenic and non-pathogenic mycobacteria have a labile capsular layer that covers the outer membrane. This capsular layer, in addition to containing arabinogalactan, glycan and mannose-containing glyco-lipids, also surprisingly contains a large amount of ESX-1-secreted proteins in Mycobacterium marinum. Furthermore, we also show that the capsule plays a role in the binding of macrophages and the induction of cytokines. Collectively, these results show for the first time that the capsule can be visualized on both pathogenic and non-pathogenic mycobacteria. In addition, growing mycobacteria under standard laboratory conditions in the presence of detergent with agitation promotes capsular shedding and influences the biological characteristics of the bacteria.

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          Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti.

          Although large human populations have been safely immunized against tuberculosis with two live vaccines, Mycobacterium bovis BCG or Mycobacterium microti, the vole bacillus, the molecular basis for the avirulence of these vaccine strains remains unknown. Comparative genomics has identified a series of chromosomal deletions common to both virulent and avirulent species but only a single locus, RD1, that has been deleted from M. bovis BCG and M. microti. Restoration of RD1, by gene knock-in, resulted in a marked change in colonial morphology towards that of virulent tubercle bacilli. Three RD1-encoded proteins were localized in the cell wall, and two of them, the immunodominant T-cell antigens ESAT-6 and CFP-10, were also found in culture supernatants. The BCG::RD1 and M. microti::RD1 knock-ins grew more vigorously than controls in immunodeficient mice, inducing extensive splenomegaly and granuloma formation. Increased persistence and partial reversal of attenuation were observed when immunocompetent mice were infected with the BCG::RD1 knock-in, whereas BCG controls were cleared. Knocking-in five other RD loci did not affect the virulence of BCG. This study describes a genetic lesion that contributes to safety and opens new avenues for vaccine development.
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            Disclosure of the mycobacterial outer membrane: cryo-electron tomography and vitreous sections reveal the lipid bilayer structure.

            The cell walls of mycobacteria form an exceptional permeability barrier, and they are essential for virulence. They contain extractable lipids and long-chain mycolic acids that are covalently linked to peptidoglycan via an arabinogalactan network. The lipids were thought to form an asymmetrical bilayer of considerable thickness, but this could never be proven directly by microscopy or other means. Cryo-electron tomography of unperturbed or detergent-treated cells of Mycobacterium smegmatis embedded in vitreous ice now reveals the native organization of the cell envelope and its delineation into several distinct layers. The 3D data and the investigation of ultrathin frozen-hydrated cryosections of M. smegmatis, Myobacterium bovis bacillus Calmette-Guérin, and Corynebacterium glutamicum identified the outermost layer as a morphologically symmetrical lipid bilayer. The structure of the mycobacterial outer membrane necessitates considerable revision of the current view of its architecture. Conceivable models are proposed and discussed. These results are crucial for the investigation and understanding of transport processes across the mycobacterial cell wall, and they are of particular medical relevance in the case of pathogenic mycobacteria.
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              Acute infection and macrophage subversion by Mycobacterium tuberculosis require a specialized secretion system.

              Although many bacterial pathogens use specialized secretion systems for virulence, no such systems have been described for Mycobacterium tuberculosis, a major pathogen of humans that proliferates in host macrophages. In a screen to identify genes required for virulence of M. tuberculosis, we have discovered three components and two substrates of the first Sec-independent secretion pathway described in M. tuberculosis, which we designate the Snm pathway. Here we demonstrate that the proteins Snm1, -2, and -4 are required for the secretion of ESAT-6 and CFP-10, small proteins previously identified as major T cell antigens. Snm2, a member of the AAA ATPase family, interacts with substrates and with Snm1, another AAA ATPase. We show that M. tuberculosis mutants lacking either the Snm system or these substrates exhibit defects in bacterial growth during the acute phase of a mouse infection and are attenuated for virulence. Strikingly, snm mutants fail to replicate in cultured macrophages and to inhibit macrophage inflammatory responses, two well established activities of wild-type M. tuberculosis bacilli. Thus, the Snm secretion pathway works to subvert normal macrophage responses and is a major determinant of M. tuberculosis virulence.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                March 2010
                March 2010
                5 March 2010
                : 6
                : 3
                : e1000794
                Affiliations
                [1 ]Division of Cell Biology-B6, Netherlands Cancer Institute–Antoni van Leeuwenhoek Hospital (NKI-AVL), Amsterdam, the Netherlands
                [2 ]Department of Medical Microbiology and Infection Control, VU University Medical Centre, Amsterdam, the Netherlands
                [3 ]Department of Medical Oncology, VU University Medical Centre, Amsterdam, the Netherlands
                [4 ]CNRS, Institut de Pharmacologie et de Biologie Structurale, Département Mécanismes Moléculaires des Infections Mycobactériennes, Toulouse, France
                [5 ]Kavli Institute of Nanoscience, Delft University of Technology, Delft, the Netherlands
                University of Washington, United States of America
                Author notes
                [¤]

                Current address: Université de Toulouse (Toulouse III), Toulouse, France

                Conceived and designed the experiments: MS ENGH JG MD BJA WB NvdW PJP. Performed the experiments: MS ENGH JG JP KdP MvZ BW SRP CRJ. Analyzed the data: MS ENGH JG KdP NvdW. Wrote the paper: MS ENGH JG SRP MD BJA WB NvdW PJP.

                Article
                09-PLPA-RA-1839R2
                10.1371/journal.ppat.1000794
                2832766
                20221442
                92e3cd44-998d-42aa-aa1a-800af6db096e
                Sani et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 15 October 2009
                : 26 January 2010
                Page count
                Pages: 10
                Categories
                Research Article
                Cell Biology
                Infectious Diseases/Bacterial Infections
                Microbiology/Cellular Microbiology and Pathogenesis

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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