Caractérisation moléculaire et cellulaire de composants amibiens et humains influençant la migration d'Entamoeba histolytica lors du franchissement de la barrière intestinale

Entamoeba histolytica est l agent étiologique de l amibiase. Au sein de l espèce E. histolytica, une souche pathogène (HMI), responsable de la destruction de la barrière intestinale et une souche non pathogène (Rahman) ont été identifiées. Mon projet de thèse vise d une part, à déterminer les facteurs qui régissent le phénotype non pathogène et le phénotype virulent et d autre part, d étudier le remodelage de la matrice extracellulaire lors de la migration tissulaire d E. histolytica. Un modèle d explants de colon humain permettant l étude de l amibiase intestinale ainsi que des techniques avancées en génomique et en imagerie dynamique ont été utilisés. En contact avec le colon humain, le paysage transcriptomique d E. histolytica HMI est caractérisé par la surexpression de gènes codant des enzymes de la glycolyse ainsi que des glycosyl hydrolases. Celui d E. histolytica Rahman, contient des gènes liés au métabolisme des lipides. Ceci suggère que lors de l invasion du mucus, seule E. histolytica HMI est capable de cliver les résidus saccharidiques des mucines, rendant accessible le corps protéique des mucines aux protéases du parasite. Nous avons mis en évidence que E. histolytica est doué d une migration amoeboide doté d une activité collagénolytique. L étude de l invasion d un explant de colon humain par E. histolytica a révélé que d une part, les structures de collagène fibrillaire présentes dans le colon imposent une route d invasion aux trophozoïtes et que d autre part la pénétration de la lamina propria requiert une destruction du réseau de collagène. Nous avons montré que CP-A5 est requise pour la dégradation du réseau de collagène in situ.Entamoeba histolytica is the causative agent of amoebiasis. Among E. histolytica species, two different strains have been identified. The pathogenic strain HMI is responsible for the destruction of the intestinal barrier whereas the Rahman strain remains non pathogenic. My PhD project aims at determining the factors regulating the virulent and the commensal phenotypes. Moreover, this study is intended to determine the remodeling of the extracellular matrix upon migration of E. histolytica within the tissues. A model based on human colonic explants enabling the analysis of early steps in intestinal amoebiasis has been used along with advanced techniques in genomics and dynamic imaging. Upon contact with the human colon, the transcriptomic landscape of E. histolytica HMI is characterized by the overexpression of genes encoding glycolysis enzymes as well as glycosyl hydrolases. Conversely, E. histolytica Rahman transcriptomic landscape displays genes linked to the lipid metabolism. This suggests that upon mucus invasion, only E. histolytica HMI is able to cleave carbohydrate residues on the mucins. This cleavage would uncover the proteic backbone of the mucins, enabling the cystein proteases of the parasite to further deplete the mucus layer. We have shown that E. histolytica is displaying an amoeboid migration combined to a collagenolytic activity. The study of the invasion of human colonic explants by E. histolytica has revealed that fibrillar collagen structures of the colon force an invasion route on the parasite. Moreover, penetration of the lamina propria requires the destruction of the collagen network, carried on by CP-A5 in situ.VERSAILLES-BU Sciences et IUT (786462101) / SudocSudocFranceF

Crosstalk between Entamoeba histolytica and the human intestinal tract during amoebiasis

International audienceThe protozoan parasite Entamoeba histolytica is the microbial agent of amoebiasis-an infection that is endemic worldwide and is associated with high morbidity and mortality rates. As the disease develops, virulent E. histolytica deplete the mucus layer, interact with the intestinal epithelium, and then degrade the colonic mucosa and disrupt the extracellular matrix (ECM). Our research demonstrated that virulent parasites with an invasive phenotype display rapid, highly specific changes in their transcriptome (notably for essential factors involved in carbohydrate metabolism and the processing of glycosylated residues). Moreover, combined activation of parasite and host lytic enzymes leads to the destruction of the intestinal parenchyma. Together, these enzymes degrade the mucus layer and the ECM, and trigger the inflammatory response essential to the development of amoebiasis

A systematic review of Leptospira in water and soil environments

International audienceBackground: Leptospirosis, caused by pathogenic Leptospira, is a zoonosis of global distribution. This infectious disease is mainly transmitted by indirect exposure to urine of asymptomatic animals via the environment. As human cases generally occur after heavy rain, an emerging hypothesis suggests that rainfall re-suspend leptospires together with soil particles. Bacteria are then carried to surface water, where humans get exposed. It is currently assumed that pathogenic leptospires can survive in the environment but do not multiply. However, little is known on their capacity to survive in a soil and freshwater environment.Methods: We conducted a systematic review on Leptospira and leptospirosis in the environment in order to collect current knowledge on the lifestyle of Leptospira in soil and water. In total, 86 scientific articles retrieved from online databases or institutional libraries were included in this study. Principals findings/significance: This work identified evidence of survival of Leptospira in the environment but major gaps remain about the survival of virulent species associated with human and animal diseases. Studies providing quantitative data on Leptospira in soil and water are a very recent trend, but must be interpreted with caution because of the uncertainty in the species identification. Several studies mentioned the presence of Leptospira in soils more frequently than in waters, supporting the hypothesis of the soil habitat and dispersion of Leptospira with re-suspended soil particles during heavy rain. In a near future, the growing use of high throughput sequencing will offer new opportunities to improve our understanding of the habitat of Leptospira in the environment. This better insight into the risk of leptospirosis will allow implementing efficient control measures and prevention for the human and animal populations exposed

In Entamoeba histolytica, a BspA family protein is required for chemotaxis toward tumour necrosis factor

Background: [i]Entamoeba histolytica[/i] cell migration is essential for the development of human amoebiasis (an infectious disease characterized by tissue invasion and destruction). The tissue inflammation associated with tumour necrosis factor (TNF) secretion by host cells is a well-documented feature of amoebiasis. Tumour necrosis factor is a chemoattractant for E. histolytica, and the parasite may have a TNF receptor at its cell surface. Methods: confocal microscopy, RNA Sequencing, bioinformatics, RNA antisense techniques and histological analysis of human colon explants were used to characterize the interplay between TNF and E. histolytica. Results: an antibody against human TNF receptor 1 (TNFR1) stained the E. histolytica trophozoite surface and (on immunoblots) binds to a 150-kDa protein. Proteome screening with the TNFR1 sequence revealed a BspA family protein in E. histolytica that carries a TNFR signature domain and six leucine-rich repeats (named here as “cell surface protein”, CSP, in view of its cellular location). Cell surface protein shares structural homologies with Toll-Like receptors, colocalizes with TNF and is internalized in TNF-containing vesicles. Reduction of cellular CSP levels abolished chemotaxis toward TNF and blocked parasite invasion of human colon. Conclusions: there is a clear link between TNF chemotaxis, CSP and pathogenesis

Assessment of the interplay between scaffold geometry, induced shear stresses and cell proliferation within a packed bed perfusion bioreactor

International audienceBy favoring cell proliferation and differentiation, perfusion bioreactors proved efficient at optimizing cell culture. The aim of this study was to quantify cell proliferation within a perfusion bioreactor and correlate it to the wall shear stress (WSS) distribution by combining 3-D imaging and computational fluid dynamics simulations.NIH-3T3 fibroblasts were cultured onto a scaffold model made of impermeable polyacetal spheres or Polydimethylsiloxane cubes. After 1, 2, and 3 weeks of culture, constructs were analyzed by micro-computed tomography (mu CT) and quantification of cell proliferation was assessed. After 3 weeks, the volume of cells was found four times higher in the stacking of spheres than in the stacking of cube.3D-mu CT reconstruction of bioreactors was used as input for the numerical simulations. Using a lattice-Boltzmann method, we simulated the fluid flow within the bioreactors. We retrieved the WSS distribution (PDF) on the scaffolds surface at the beginning of cultivation and correlated this distribution to the local presence of cells after 3 weeks of cultivation. We found that the WSS distributions strongly differ between spheres and cubes even if the porosity and the specific wetted area of the stackings were very similar. The PDF is narrower and the mean WSS is lower for cubes (11 mPa) than for spheres (20 mPa). For the stacking of spheres, the relative occupancy of the surface sites by cells is maximal when WSS is greater than 20 mPa. For cubes, the relative occupancy is maximal when the WSS is lower than 10 mPa. The discrepancies between spheres and cubes are attributed to the more numerous sites in stacking of spheres that may induce 3-D (multi-layered) proliferation

The parasite Entamoeba histolytica exploits the activities of human matrix metalloproteinases to invade colonic tissue

International audienceIntestinal invasion by the protozoan parasite Entamoeba histolytica is characterized by remodelling of the extracellular matrix (ECM). The parasite cysteine proteinase A5 (CP-A5) is thought to cooperate with human matrix metalloproteinases (MMPs) involved in ECM degradation. Here, we investigate the role CP-A5 plays in the regulation of MMPs upon mucosal invasion. We use human colon explants to determine whether CP-A5 activates human MMPs. Inhibition of the MMPs' proteolytic activities abolishes remodelling of the fibrillar collagen structure and prevents trophozoite invasion of the mucosa. In the presence of trophozoites, MMPs-1 and-3 are overexpressed and are associated with fibrillar collagen remodelling. In vitro, CP-A5 performs the catalytic cleavage needed to activate pro-MMP-3, which in turn activates pro-MMP-1. Ex vivo, incubation with recombinant CP-A5 was enough to rescue CP-A5-defective trophozoites. Our results suggest that MMP-3 and/or CP-A5 inhibitors may be of value in further studies aiming to treat intestinal amoebiasis

Deciphering the unexplored Leptospira diversity from soils uncovers genomic evolution to virulence

International audienceDespite recent advances in our understanding of Leptospira genomics, little is known on how virulence has emerged in this heterogeneous bacterial genus as well as on the lifestyle of pathogenic Leptospira outside animal hosts. Here, we isolated 12 novel Leptospira species from tropical soils, significantly increasing the number of known species to 35 and evidencing a highly unexplored biodiversity in the genus. Extended comparative phylogenomics and pan-genome analyses at the genus level by incorporating 26 new genomes, revealed that, the traditional leptospiral " pathogens " cluster, as defined by their phylogenetic position, can be split in two groups with distinct virulence potential and accessory gene patterns. These genomic distinctions are strongly linked to the abilit

Identification of the Virulence Landscape Essential for Entamoeba histolytica Invasion of the Human Colon

International audienceEntamoeba histolytica is the pathogenic amoeba responsible for amoebiasis, an infectious disease targeting human tissues. Amoebiasis arises when virulent trophozoites start to destroy the muco-epithelial barrier by first crossing the mucus, then killing host cells, triggering inflammation and subsequently causing dysentery. The main goal of this study was to analyse pathophysiology and gene expression changes related to virulent (i.e. HM1:IMSS) and non-virulent (i.e. Rahman) strains when they are in contact with the human colon. Transcriptome comparisons between the two strains, both in culture conditions and upon contact with human colon explants, provide a global view of gene expression changes that might contribute to the observed phenotypic differences. The most remarkable feature of the virulent phenotype resides in the up-regulation of genes implicated in carbohydrate metabolism and processing of glycosylated residues. Consequently, inhibition of gene expression by RNA interference of a glycoside hydrolase (b-amylase absent from humans) abolishes mucus depletion and tissue invasion by HM1:IMSS. In summary, our data suggest a potential role of carbohydrate metabolism in colon invasion by virulent E. histolytica

BioFlow: a non-invasive, image-based method to measure speed, pressure and forces inside living cells

International audienceCell motility is governed by a complex molecular machinery that converts physico-chemical cues into whole-cell movement. Understanding the underlying biophysical mechanisms requires the ability to measure physical quantities inside the cell in a simple, reproducible and preferably non-invasive manner. To this end, we developed BioFlow, a computational mechano-imaging method and associated software able to extract intracellular measurements including pressure, forces and velocity everywhere inside freely moving cells in two and three dimensions with high spatial resolution in a non-invasive manner. This is achieved by extracting the motion of intracellular material observed using fluorescence microscopy, while simultaneously inferring the parameters of a given theoretical model of the cell interior. We illustrate the power of BioFlow in the context of amoeboid cell migration, by modelling the intracellular actin bulk flow of the parasite Entamoeba histolytica using fluid dynamics, and report unique experimental measures that complement and extend both theoretical estimations and invasive experimental measures. Thanks to its flexibility, BioFlow is easily adaptable to other theoretical models of the cell, and alleviates the need for complex or invasive experimental conditions, thus constituting a powerful tool-kit for mechano-biology studies. BioFlow is open-source and freely available via the Icy software. The ability of cells to define and alter their shape, maintain cell-cell contact, initiate and regulate movement is central to numerous fundamental biological processes including development, microbial infection, immune response, and cancer metastasis 1. The mechanisms underlying cell shape and motility involve complex molecular machinery that senses and translates both internal and external signals (mechanical and chemical) into physical quantities. At the mechanical level, deciphering how cells deform and migrate requires a better understanding of the biophysical quantities driving intracellular dynamics, including intracellular pressure, stiffness, viscosity and forces 2. Unfortunately, many of these quantities cannot be measured directly with current methodologies, and are typically estimated using various indirect or invasive experimental approaches 3. Many such methods operate at the extracellular level, and typically involve interacting with the cell surface. This can be done either actively, e.g. using micro-pipette aspiration 4 , Atomic Force Microscopy 5 and micro-particle insertion 6 , or passively , e.g. using Traction Force Microscopy, where the cells freely interact with engineered substrates formed either of micro-pillars of known properties 7 or filled with fluorescent beads 8, 9. At the intracellular level however , biophysical measurements remain scarce and limited by experimental constraints. Foreign particles can be inserted inside the cell and tracked through video-microscopy in order to characterise intracellular dynamics (Particle Tracking Velocimetry 10, 11). This technique generally requires controlled manipulation of the particles, which is usually achieved via magnetic 12 or optical 13 tweezers. Unfortunately, these methods are highly localise