During infection of humans and animals, pathogenic mycobacteria manipulate the host cell causing severe diseases such as tuberculosis and leprosy. To understand the basis of mycobacterial pathogenicity, it is crucial to identify the molecular virulence mechanisms. In this study, we address the contribution of ESX-1 and ESX-5-two homologous type VII secretion systems of mycobacteria that secrete distinct sets of immune modulators-during the macrophage infection cycle. Using wild-type, ESX-1- and ESX-5-deficient mycobacterial strains, we demonstrate that these secretion systems differentially affect subcellular localization and macrophage cell responses. We show that in contrast to ESX-1, the effector proteins secreted by ESX-5 are not required for the translocation of Mycobacterium tuberculosis or Mycobacterium marinum to the cytosol of host cells. However, the M. marinum ESX-5 mutant does not induce inflammasome activation and IL-1 beta activation. The ESX-5 system also induces a caspase-independent cell death after translocation has taken place. Importantly, by means of inhibitory agents and small interfering RNA experiments, we reveal that cathepsin B is involved in both the induction of cell death and inflammasome activation upon infection with wild-type mycobacteria. These results reveal distinct roles for two different type VII secretion systems during infection and shed light on how virulent mycobacteria manipulate the host cell in various ways to replicate and spread. The Journal of Immunology, 2011, 187: 4744-4753.@en

Vitreous cryo-section-induced compression influences the interpretation and the reliability of electron microscopy images and tomographic reconstructions. Previous studies of this deformation have been focused at the cellular level where considerable compression occurs, yet the degree of possible intracellular macromolecular deformation has remained unclear. Here, electron cryo-tomographic reconstructions of vitreous cryo-sections show that SOS ribosomes, both intracellular and in an isolated state, appear able to resist section-induced compression. Our observations indicate that vitreous section-induced compression is non-uniform between whole cells that have been sectioned and their intracellular macromolecular complexes. We conclude that electron cryo-tomography of vitreous cryo-sections, in spite of section-induced compression, is a suitable technique for charting the structural organization of cellular nanomachines, such as ribosomes, in a cellular environment.@en

A major objective of modern structural biology is to appreciate the cellular organization by elucidating the spatial arrangement of macromolecular complexes within a cell. Cryogenic sample preparation, combined with cryo-ultramicrotomy, enables large cells and pieces of biological tissues to be thinned for electron cryo-tomography, which provides a three-dimensional view of the biological sample. There are, however, limitations associated with the technique that must be realized, addressed and overcome for the procedure to become mainstream. Here, we provide perspectives on the continued advancements in cryogenic sample preparation for vitreous cryo-sectioning, image collection and post-image processing that have expanded the attainable information limit within the three-dimensional reconstructions of cells and pieces of biological tissues.@en

Cryo-ultramicrotomy can be used to obtain ultrathin cryo-sections from cryo-fixed or aldehyde-fixed cryo-protected vitreous biologic samples. For immuno-gold EM, cryo-sections are retrieved from the cryo-chamber on a droplet of a pick-up solution (paste-like and almost frozen) to which the sections attach. The sections are then placed on an EM specimen grid at room temperature. This procedure compromises the ultrastructure, resulting in folds, holes, and loss of the original material. In this paper we show the critical influence of humidity, stretching, and relief of compression during thawing of the sections. We show a new lift-up hinge device for semi-automated retrieval of cryo-sections that results in significantly improved section quality. This approach was also applied successfully to vitreous sections from high pressure frozen samples. An important advance is that these vitreous cryo-sections can now successfully be post-fixed and immunolabelled after thawing: this allows cryo-EM comparison with adjacent ribbons of sections still in the frozen hydrated state. These findings call for technical innovations aiming at automated cryo-ultramicrotomy in a fully controlled environment for improved localization of proteins within their 'close to native' cellular context and correlative electron cryo-tomography of consecutive ribbons of sections of one frozen hydrated sample@en

During prion disease, cellular prion protein (PrPC) is refolded into a pathogenic isoform (PrPSc) that accumulates in the central nervous system and causes neurodegeneration and death. We used immunofluorescence, quantitative cryo-immunogold EM, and tomography to detect nascent, full-length PrPSc in the hippocampus of prion-infected mice from early preclinical disease stages onward. Comparison of uninfected and infected brains showed that sites containing full-length PrPSc could be recognized in the neuropil by bright spots and streaks of immunofluorescence on semi-thin (200-nm) sections, and by clusters of cryo-immunogold EM labeling. PrPSc was found mainly on neuronal plasma membranes, most strikingly on membrane invaginations and sites of cell-to-cell contact, and was evident by 65 days postinoculation, or 54% of the incubation period to terminal disease. Both axons and dendrites in the neuropil were affected. We hypothesize that closely apposed plasma membranes provide a favorable environment for prion conversion and intercellular prion transfer. Only a small proportion of clustered PrP immunogold labeling was found at synapses, indicating that synapses are not targeted specifically in prion disease.@en

Dark field TEM imaging using a stop of the central beam (DF-000) is reported, It is shown that a strong enhancement in the contrast can be obtained for graphene as example of weak phase object and endocytic multivescilar body as example of an unstained biological sample. No charging or significant contamination of the central beam stop is observed. For graphene, a resolution beyond 1 angstrom(-1) was easily obtained. DF-000 imaging can be considered as a good and easy to use alternative of a phase pla@en

After oral exposure, prions are thought to enter Peyer's patches via M cells and accumulate first upon follicular dendritic cells (FDCs) before spreading to the nervous system. How prions are actually initially acquired from the gut lumen is not known. Using high-resolution immunofluorescence and cryo-immunogold electron microscopy, we report the trafficking of the prion protein (PrP) toward Peyer's patches of wild-type and PrP-deficient mice. PrP was transiently detectable at 1 day post feeding (dpf) within large multivesicular LAMP1-positive endosomes of enterocytes in the follicle-associated epithelium (FAE) and at much lower levels within M cells. Subsequently, PrP was detected on vesicles in the late endosomal compartments of macrophages in the subepithelial dome. At 7-21 dpf, increased PrP labelling was observed on the plasma membranes of FDCs in germinal centres of Peyer's patches from wild-type mice only, identifying FDCs as the first sites of PrP conversion and replication. Detection of PrP on extracellular vesicles displaying FAE enterocyte-derived A33 protein implied transport towards FDCs in association with FAE-derived vesicles. By 21 dpf, PrP was observed on the plasma membranes of neurons within neighbouring myenteric plexi. Together, these data identify a novel potential M cell-independent mechanism for prion transport, mediated by FAE enterocytes, which acts to initiate conversion and replication upon FDCs and subsequent infection of enteric nerves.@en