Physiopathologie de l'amibiase intestinale

PARIS-BIUP (751062107) / SudocSudocFranceF

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

Construction and characterization of a protective antigen-deficient Bacillus anthracis strain

International audienceThe pag gene coding for protective antigen (PA), one of the three toxin components of Bacillus anthracis, has been cloned into the mobilizable shuttle vector pAT187 and transferred by conjugation from Escherichia coli to B. anthracis. Using this strategy, an insertionally mutated pag gene constructed and characterized in E. coli, was introduced into B. anthracis Sterne strain. This transconjugant was used to select a recombinant clone (RP8) carrying the inactivated pag gene on the toxin-encoding piasmid, pXO1. Strain RP8 was deficient for PA while still producing the two other toxin components, i.e. lethal factor (LF) and edema factor (EF). In contrast to spores from the wild-type Sterne strain, spores prepared from RP8 were totally non-lethal in mice. These results clearly establish the central role played by PA in B. anthracis pathogenicity

Distribution of a Potential p21-Activated Serine/Threonine Kinase (PAK) in Entamoeba histolytica

International audienc

Germination of Bacillus anthracis spores within alveolar macrophages

International audienceThe fatal character of the infection caused by inhalation of Bacillus anthracis spores results from a complex pathogenic cycle involving the synthesis of toxins by the bacterium. We have shown using immunofluorescent staining, confocal scanning laser microscopy and image cytometry analysis that the alveolar macrophage was the primary site of B. anthracis germination in a murine inhalation infection model. Bacillus anthracis germinated inside murine macrophage-like RAW264.7 cells and murine alveolar macrophages. Germination occurred in vesicles derived from the phagosomal compartment. We have also demonstrated that the toxin genes and their trans-activator, AtxA, were expressed within the macrophages after germination

A Min-Max Based Hyperparameter Estimation For Domain-Adapted Segmentation Of Amoeboid Cells

International audienc

3-D Active Meshes: Fast Discrete Deformable Models for Cell Tracking in 3-D Time-Lapse Microscopy

International audienceVariational deformable models have proven over the past decades a high efficiency for segmentation and tracking in 2-D sequences. Yet, their application to 3-D time-lapse images has been hampered by discretization issues, heavy computational loads and lack of proper user visualization and interaction, limiting their use for routine analysis of large data-sets. We propose here to address these limitations by reformulating the problem entirely in the discrete domain using 3-D active meshes, which express a surface as a discrete triangular mesh, and minimize the energy functional accordingly. By performing computations in the discrete domain, computational costs are drastically reduced, whilst the mesh formalism allows to benefit from real-time 3-D rendering and other GPU-based optimizations. Performance evaluations on both simulated and real biological data sets show that this novel framework outperforms current state-of-the-art methods, constituting a light and fast alternative to traditional variational models for segmentation and tracking applications

Domain Adapted Multi-task Learning For Segmenting Amoeboid Cells in Microscopy

International audienceThe method proposed in this paper is a robust combination of multi-task learning and unsupervised domain adaptation for segmenting amoeboid cells in microscopy. A highlight of this work is the manner in which the model’s hyperparameters are estimated. The detriments of ad-hoc parameter estimation are well known, but this issue remains largely unaddressed in the context of CNN-based segmentation. Using a novel min-max formulation of the segmentation cost function our proposed method analytically estimates the model’s hyperparameters, while simultaneously learning the CNN weights during training. This end-to-end framework provides a consolidated mechanism to harness the potential of multi-task learning to isolate and segment clustered cells from low contrast brightfield images, and it simultaneously leverages deep domain adaptation to segment fluorescent cells without explicit pixel-level re-annotation of the data. Experimental validations on multi-cellular images strongly suggest the effectiveness of the proposed technique, and our quantitative results show at least 15% and 10% improvement in cell segmentation on brightfield and fluorescence images respectively compared to contemporary supervised segmentation methods

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

Characterization of ATP and calmodulin-binding properties of a truncated form of Bacillus anthracis adenylate cyclase

International audienceThe Bacillus anthracis cya gene encodes a calmodulin-dependent adenylate cyclase. A deletion cya gene product obtained by removing 261 codons at the 5' end was expressed in a protease-deficient loti' E. coli strain and purified to homogeneity. This truncated enzyme (CYA 62) exhibits catalytic and calmodulin-binding properties similar to the properties of wild-type adenylate cyclase from B. anthracis culture supernatants, i.e., a kcat of 1100 s"1 at 30 °C and pH 8, an apparent Km for ATP of 0.25 mM, and a Kd for bovine brain calmodulin of 23 nM. The calmodulin-binding domain of the CYA 62 truncated enzyme was labeled with a cleavable radioactive photoaffinity cross-linker coupled to calmodulin. The labeled CYA 62 protein was then cleaved with cyanogen bromide and A-chlorosuccinimide. We show that the calmo- dulin-binding domain of B. anthracis adenylate cyclase is located within the last 150 amino acid residues of the protein. A further deletion at the 3' end of the CYA 62 coding sequence yielded an adenylate cyclase species (CYA 57) lacking 127 C-terminal amino residues. CYA 57, still sensitive to activation by high concentrations of calmodulin, exhibits less than 0.1% of the specific activity of CYA 62. Binding of 3'dATP (a competitive inhibitor) to CYA 62 was determined by equilibrium dialysis. In the absence of calmodulin, binding of the ATP analogue to this truncated protein was severely impaired, which explains, at least in part, the absolute requirement for calmodulin for the catalytic activity of B. anthracis adenylate cyclase