A multi-parametric analysis of Trypanosoma cruzi infection: common pathophysiologic patterns beyond extreme heterogeneity of host responses

The extreme genetic diversity of the protozoan Trypanosoma cruzi has been proposed to be associated with the clinical outcomes of the disease it provokes: Chagas disease (CD). To address this question, we analysed the similarities and differences in the CD pathophysiogenesis caused by different parasite strains. Using syngeneic mice infected acutely or chronically with 6 distant parasite strains, we integrated simultaneously 66 parameters: parasite tropism (7 parameters), organ and immune responses (local and systemic; 57 parameters), and clinical presentations of CD (2 parameters). While the parasite genetic background consistently impacts most of these parameters, they remain highly variable, as observed in patients, impeding reliable one-dimensional association with phases, strains, and damage. However, multi-dimensional statistics overcame this extreme intra-group variability for each individual parameter and revealed some pathophysiological patterns that accurately allow defining (i) the infection phase, (ii) the infecting parasite strains, and (iii) organ damage type and intensity. Our results demonstrated a greater variability of clinical outcomes and host responses to T. cruzi infection than previously thought, while our multi-parametric analysis defined common pathophysiological patterns linked to clinical outcome of CD, conserved among the genetically diverse infecting strains

calcium-binding protein 1 of entamoeba histolytica transiently associates with phagocytic cups in a calcium-independent manner

EhCaBP1, a calcium-binding protein of the parasite Entamoeba histolytica, is known to participate in cellular processes involving actin filaments. This may be due to its direct interaction with actin. In order to understand the kinetics of EhCaBP1 in such processes, its movement was studied in living cells expressing GFP-EhCaBP1. The results showed that EhCaBP1 accumulated at phagocytic cups and pseudopods transiently. The time taken for appearance and disappearance of EhCaBP1 was found to be around 12 s. Site-directed mutagenesis was used to generate an EhCaBP1 mutant with reduced Ca2+- and G-actin binding ability without any defect in its ability to bind F-actin. The overexpression of this mutant EhCaBP1 in the E. histolytica trophozoites resulted in the impairment of erythrophagocytosis, uptake of bacterial cells, killing of target cells but not fluid-phase pinocytosis. However, the mutant protein was still found to transiently localize with f-actin at the phagocytic cups and pseudopods. The mutant protein displayed reduced ability to activate endogenous kinase(s) suggesting that phagosome formation may require Ca2+-EhCaBP1 transducing downstream signalling but initiation of phagocytosis may be independent of its intrinsic ability to bind Ca2+. The results suggest a dynamic association of EhCaBP1 with F-actin-mediated processes

Use of Bacterially Expressed dsRNA to Downregulate Entamoeba histolytica Gene Expression

BACKGROUND:Modern RNA interference (RNAi) methodologies using small interfering RNA (siRNA) oligonucleotide duplexes or episomally synthesized hairpin RNA are valuable tools for the analysis of gene function in the protozoan parasite Entamoeba histolytica. However, these approaches still require time-consuming procedures including transfection and drug selection, or costly synthetic molecules. PRINCIPAL FINDINGS:Here we report an efficient and handy alternative for E. histolytica gene down-regulation mediated by bacterial double-stranded RNA (dsRNA) targeting parasite genes. The Escherichia coli strain HT115 which is unable to degrade dsRNA, was genetically engineered to produce high quantities of long dsRNA segments targeting the genes that encode E. histolytica beta-tubulin and virulence factor KERP1. Trophozoites cultured in vitro were directly fed with dsRNA-expressing bacteria or soaked with purified dsRNA. Both dsRNA delivery methods resulted in significant reduction of protein expression. In vitro host cell-parasite assays showed that efficient downregulation of kerp1 gene expression mediated by bacterial dsRNA resulted in significant reduction of parasite adhesion and lytic capabilities, thus supporting a major role for KERP1 in the pathogenic process. Furthermore, treatment of trophozoites cultured in microtiter plates, with a repertoire of eighty-five distinct bacterial dsRNA segments targeting E. histolytica genes with unknown function, led to the identification of three genes potentially involved in the growth of the parasite. CONCLUSIONS:Our results showed that the use of bacterial dsRNA is a powerful method for the study of gene function in E. histolytica. This dsRNA delivery method is also technically suitable for the study of a large number of genes, thus opening interesting perspectives for the identification of novel drug and vaccine targets

Trypanosoma cruzi strains cause different myocarditis patterns in infected mice

Aims. Chagas disease pathology is dependent on the infecting T. cruzi strain. However, the relationship between the extent and type of myocarditis caused by different T. cruzi strains in the acute and chronic phases of infection has not been studied in detail. To address this, we infected mice with three genetically distant T. cruzi strains as well as infected in vitro different cell types. Methods and Results. Parasitemia was detected in mice infected with the Y and VFRA strains, but not with the Sc43 strain; however, only the Y strain was lethal. When infected with VFRA, mice showed higher inflammation and parasitism in the heart than with Sc43 strain. Y and VFRA caused homogeneous pancarditis with inflammatory infiltrates along the epicardium, whereas Sc43 caused inflammation preferentially in the auricles in association with intracellular parasite localization. We observed intramyocardic perivasculitis in mice infected with the VFRA and Y strains, but not with Sc43, during the acute phase, which suggests that endothelial cells may be involved in heart colonization by these more virulent strains. In in vitro infection assays, the Y strain had the highest parasite-cell ratio in epithelial, macrophage and endothelial cell lines, but Y and VFRA strains were higher than Sc43 in cardiomyocytes. Conclusions. This study supports parasite variability as a cause for the diverse cardiac outcomes observed in Chagas disease, and suggests that endothelial cells could be involved in heart infection during the acute phase.This work was partially supported by grants from “Fondo de Investigaciones Sanitarias” (PS09/00538 and PI12/00289); “Ministerio de Ciencia e Innovación” (SAF2010-18733); The European Union (ChagasEpiNet); “Comunidad de Madrid” S2010/BMD-2332; RED RECAVA RD06/0014/1013;RED RICET RD12/0018/004 and an institutional grant from “Fundación Ramon Areces

Reassessing the Role of Entamoeba gingivalis in Periodontitis

The protozoan Entamoeba gingivalis resides in the oral cavity and is frequently observed in the periodontal pockets of humans and pets. This species of Entamoeba is closely related to the human pathogen Entamoeba histolytica, the agent of amoebiasis. Although E. gingivalis is highly enriched in people with periodontitis (a disease in which inflammation and bone loss correlate with changes in the microbial flora), the potential role of this protozoan in oral infectious diseases is not known. Periodontitis affects half the adult population in the world, eventually leads to edentulism, and has been linked to other pathologies, like diabetes and cardiovascular diseases. As aging is a risk factor for the disorder, it is considered an inevitable physiological process, even though it can be prevented and cured. However, the impact of periodontitis on the patient's health and quality of life, as well as its economic burden, are underestimated. Commonly accepted models explain the progression from health to gingivitis and then periodontitis by a gradual change in the identity and proportion of bacterial microorganisms in the gingival crevices. Though not pathognomonic, inflammation is always present in periodontitis. The recruitment of leukocytes to inflamed gums and their passage to the periodontal pocket lumen are speculated to fuel both tissue destruction and the development of the flora. The individual contribution to the disease of each bacterial species is difficult to establish and the eventual role of protozoa in the fate of this disease has been ignored. Following recent scientific findings, we discuss the relevance of these data and propose that the status of E. gingivalis be reconsidered as a potential pathogen contributing to periodontitis

Endoplasmic Reticulum Stress-Sensing Mechanism Is Activated in Entamoeba histolytica upon Treatment with Nitric Oxide

The Endoplasmic Reticulum stores calcium and is a site of protein synthesis and modification. Changes in ER homeostasis lead to stress responses with an activation of the unfolded protein response (UPR). The Entamoeba histolytica endomembrane system is simple compared to those of higher eukaryotes, as a canonical ER is not observed. During amoebiasis, an infection of the human intestine and liver by E. histolytica, nitric oxide (NO) triggers an apoptotic-like event preceded by an impairment of energy production and a loss of important parasite pathogenic features. We address the question of how this ancient eukaryote responds to stress induced by immune components (i.e. NO) and whether stress leads to ER changes and subsequently to an UPR. Gene expression analysis suggested that NO triggers stress responses marked by (i) dramatic up-regulation of hsp genes although a bona fide UPR is absent; (ii) induction of DNA repair and redox gene expression and iii) up-regulation of glycolysis-related gene expression. Enzymology approaches demonstrate that NO directly inhibits glycolysis and enhance cysteine synthase activity. Using live imaging and confocal microscopy we found that NO dramatically provokes extensive ER fragmentation. ER fission in E. histolytica appears as a protective response against stress, as it has been recently proposed for neuron self-defense during neurologic disorders. Chronic ER stress is also involved in metabolic diseases including diabetes, where NO production reduces ER calcium levels and activates cell death. Our data highlighted unique cellular responses of interest to understand the mechanisms of parasite death during amoebiasis

Analyse moléculaire et cellulaire du rôle de KERP1 dans le processus pathogène d'Entamoeba histolytica et développement d'un test de diagnostic de l'amibiase

La colonisation de l intestin humain par le parasite Entamoeba histolytica, l agent étiologique de l amibiase, demeure le plus fréquemment asymptomatique. Suite à des stimuli inconnus, le parasite devient invasif et détruit la barrière muco-intestinale ; ceci conduit à la dysenterie amibienne et aux formes cliniques graves : l amibome intestinal et l abcès hépatique amibien, qui sont les causes d une centaine de milliers de morts par an.Par la comparaison des transcriptomes d amibes virulentes et avirulentes de même fond génétique, nous avons identifié une réponse au stress et à l oxydation chez les parasites virulents, ainsi qu une famille de protéines riches en lysine. L inhibition de la production de KERP1, un de ces facteurs, bloque la formation des foyers inflammatoires dans un modèle animal. Par ailleurs, l expression de kerp1 est modulée au cours de l infection hépatique, ainsi que par le monoxyde d azote, un effecteur de la réponse inflammatoire, et par un choc thermique. La mise en évidence de l implication de KERP1 dans la digestion des globules rouges phagocytés par l amibe et dans l adhérence aux cellules périsinusoïdales hépatiques nous a conduits à proposer un modèle expliquant le rôle de cette protéine au cours de l infection : KERP1, par son association à la membrane plasmique des cellules de l hôte, permettrait de les lyser et servirait de ligand pour des facteurs amibiens. Autrement, KERP1 permettrait le trafic vers la cellule cible de facteurs accomplissant ces activités.KERP1 ne partage d homologie avec aucune protéine connue et les anticorps générés pour notre étude permettent d identifier exclusivement le parasite E. histolytica ; ainsi, nous les avons utilisés pour développer un test de diagnostic de l amibiase.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Host-Microbe Interactions and Defense Mechanisms in the Development of Amoebic Liver Abscesses

Summary: Amoebiasis by Entamoeba histolytica is a major public health problem in developing countries and leads to several thousand deaths per year. The parasite invades the intestine (provoking diarrhea and dysentery) and the liver, where it forms abscesses (amoebic liver abscesses [ALAs]). The liver is the organ responsible for filtering blood coming from the intestinal tract, a task that implies a particular structure and immune features. Amoebae use the portal route and break through the sinusoidal endothelial barrier to reach the hepatic parenchyma. When faced with systemic and cell-mediated defenses, trophozoites adapt to their new environment and modulate host responses, leading to parasite survival and the formation of inflammatory foci. Cytopathogenic effects and the onset of inflammation may be caused by diffusible products originating from parasites and/or immune cells either by their secretion or by their release after cell death. Liver infection thus results from the interplay between E. histolytica and hepatic cells. Despite its importance in terms of public health burden, the lack of integrated data on ALA genesis means that we have only an incomplete description of the initiation and development of hepatic amoebiasis. Here, we review the main steps of ALA development as well as the responses triggered in both the host and the parasite. Transcriptome studies highlighted parasite factors involved in adherence to human cells, cytopathogenic effects, and adaptative and stress responses. An understanding of their role in ALA development will help to unravel the host-pathogen interactions and their evolution throughout the infection

parodontite et autres maladies

peer reviewe