In vitro conversion of normal prion protein into pathologic isoforms

This article describes the various available in vitro systems used to study transmissible spongiform encephalopathy (TSE) replication and the processes involved in prion protein (PrP) conversion. Advantages and disadvantages of these in vitro systems and the underlying molecular mechanisms of PrP conversion are discussed. Several applications of these systems are outlined more extensively, including their species specificity, polymorphism specificity, strain specificity, and the potential that these systems have in screening and discovering TSE therapeutics

Major differential gene regulation in Coxiella burnetii between in vivo and in vitro cultivation models

BackgroundCoxiella burnetii is the causative agent of the zoonotic disease Q fever. As it is an intracellular pathogen, infection by C. burnetii requires adaptation to its eukaryotic host and intracellular environment. The recently developed cell-free medium also allows the bacteria to propagate without host cells, maintaining its infection potential. The adaptation to different hosts or extracellular environments has been assumed to involve genome-wide modulation of C. burnetii gene expression. However, little is currently known about these adaptation events which are critical for understanding the intracellular survival of C. burnetii.ResultsWe studied C. burnetii genome–wide transcriptional patterns in vivo (mice spleen) and in cell and cell-free in vitro culture models to examine its metabolic pathways and virulence associated gene expression patterns that are required to colonize and persist in different environments. Within each model, the gene expression profiles of the Dutch C. burnetii outbreak strain (602) and NM reference strains were largely similar. In contrast, modulation of gene-expression was strongly influenced by the cultivation method, indicating adaptation of the bacterium to available components. Genome–wide expression profiles of C. burnetii from in vitro cell culture were more similar to those seen for in vivo conditions, while gene expression profiles of cell-free culture were more distant to in vivo. Under in vivo conditions, significant alterations of genes involved in metabolism and virulence were identified. We observed that C. burnetii under in vivo conditions predominantly uses glucose as a carbon source (mostly for biosynthetic processes) and fatty acids for energy generation. C. burnetii experienced nutrient limitation and anaerobiosis as major stressors, while phosphate limitation was identified as an important signal for intracellular growth inside eukaryotic host cells. Finally, the in vivo environment significantly induced expression of several virulence genes, including those implicated in LPS synthesis, colonization, host component modulation and DNA repair mechanisms.ConclusionOur study shows that C. burnetii, with its relative small genome, requires only a subset of core gene functions to survive under in vitro conditions, but requires the induction of full repertoire of genes for successful pathogenesis and thriving in harsh environments in vivo

ESX-1-mediated translocation to the cytosol controls virulence of mycobacteria.

Item does not contain fulltextMycobacterium species, including Mycobacterium tuberculosis and Mycobacterium leprae, are among the most potent human bacterial pathogens. The discovery of cytosolic mycobacteria challenged the paradigm that these pathogens exclusively localize within the phagosome of host cells. As yet the biological relevance of mycobacterial translocation to the cytosol remained unclear. In this current study we used electron microscopy techniques to establish a clear link between translocation and mycobacterial virulence. Pathogenic, patient-derived mycobacteria species were found to translocate to the cytosol, while non-pathogenic species did not. We were further able to link cytosolic translocation with pathogenicity by introducing the ESX-1 (type VII) secretion system into the non-virulent, exclusively phagolysosomal Mycobacterium bovis BCG. Furthermore, we show that translocation is dependent on the C-terminus of the early-secreted antigen ESAT-6. The C-terminal truncation of ESAT-6 was shown to result in attenuation in mice, again linking translocation to virulence. Together, these data demonstrate the molecular mechanism facilitating translocation of mycobacteria. The ability to translocate from the phagolysosome to the cytosol is with this study proven to be biologically significant as it determines mycobacterial virulence.1 augustus 201