INsPECT, an Open-Source and Versatile Software for Automated Quantification of (Leishmania) Intracellular Parasites

Intracellular protozoan parasites are causative agents of infectious diseases that constitute major health problems for developing countries. Leishmania sp., Trypanosoma cruzi or Toxoplasma gondii are all obligate intracellular protozoan parasites that reside and multiply within the host cells of mammals, including humans. Following up intracellular parasite proliferation is therefore an essential and a quotidian task for many laboratories working on primary screening of new natural and synthetic drugs, analyzing drug susceptibility or comparing virulence properties of natural and genetically modified strains. Nevertheless, laborious manual microscopic counting of intracellular parasites is still the most commonly used approach. Here, we present INsPECT (Intracellular ParasitE CounTer), an open-source and platform independent software dedicated to automate infection level measurement based on fluorescent DNA staining. It offers the possibility to choose between different types of analyses (fluorescent DNA acquisitions only or in combination with phase contrast image set to further separate intra-from extracellular parasites), and software running modes (automatic or custom). A proof-of-concept study with intracellular Leishmania infantum parasites stained with DAPI (49,6-diamidino-2-phenylindole) confirms a good correspondence between digital results and the "gold standard" microscopic counting method with Giemsa. Interestingly, this software is versatile enough to accurately detect intracellular T. gondii parasites on images acquired with High Content Screening (HCS) systems. In conclusion, INsPECT software is proposed as a new fast and simple alternative to the classical intracellular Leishmania quantification methods and can be adapted for mid to large-scale drug screening against different intracellular parasites

Prion Replication in the Hematopoietic Compartment Is Not Required for Neuroinvasion in Scrapie Mouse Model

Fatal neurodegenerative prion diseases are caused by the transmissible PrPSc prion agent whose initial replication after peripheral inoculation takes place in follicular dendritic cells present in germinal centers of lymphoid organs. However, prion replication also occurs in lymphoid cells. To assess the role of the hematopoietic compartment in neuroinvasion and prion replication, we generated chimeric mice, on a uniform congenic C57/BL6J background, by bone marrow replacement with hematopoietic cells expressing different levels of PrP protein. Nine different types of chimeric mice were inoculated intraperitoneally either with the lymphotropic Rocky Mountain Laboratory (RML) strain or the non lymphotropic ME-7 scrapie strain, at different doses. Here, we clearly demonstrate that overexpression of PrP by the hematopoietic system, or the lack of PrP expression by the bone marrow derived cells, does not change the incubation time period of the disease, even when the mice are infected at limiting doses. We conclude that the hematopoietic compartment is more or less permissive to prion replication, both for RML and ME-7, but does not play a role in neuroinvasion

Genomic determinants of the efficiency of internal ribosomal entry sites of viral and cellular origin

Variation in cellular gene expression levels has been shown to be inherited. Expression is controlled at transcriptional and post-transcriptional levels. Internal ribosome entry sites (IRES) are used by viruses to bypass inhibition of cap-dependent translation, and by eukaryotic cells to control translation under conditions when protein synthesis is inhibited. We aimed at identifying genomic determinants of variability in IRES-mediated translation of viral [Encephalomyocarditis virus (EMCV)] and cellular IRES [X-linked inhibitor-of-apoptosis (XIAP) and c-myc]. Bicistronic lentiviral constructs expressing two fluorescent reporters were used to transduce laboratory and B lymphoblastoid cell lines [15 CEPH pedigrees (n = 205) and 50 unrelated individuals]. IRES efficiency varied according to cell type and among individuals. Control of IRES activity has a significant genetic component (h2 of 0.47 and 0.36 for EMCV and XIAP, respectively). Quantitative linkage analysis identified a suggestive locus (LOD 2.35) on chromosome 18q21.2, and genome-wide association analysis revealed of a cluster of SNPs on chromosome 3, intronic to the FHIT gene, marginally associated (P = 5.9E-7) with XIAP IRES function. This study illustrates the in vitro generation of intermediate phenotypes by using cell lines for the evaluation of genetic determinants of control of elements such as IRE

A Histone Deacetylase (HDAC) inhibitor with pleiotropic in vitro anti-toxoplasma and anti-plasmodium activities controls acute and chronic toxoplasma infection in mice

© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).Toxoplasmosis is a highly prevalent human disease, and virulent strains of this parasite emerge from wild biotopes. Here, we report on the potential of a histone deacetylase (HDAC) inhibitor we previously synthesized, named JF363, to act in vitro against a large panel of Toxoplasma strains, as well as against the liver and blood stages of Plasmodium parasites, the causative agents of malaria. In vivo administration of the drug significantly increases the survival of mice during the acute phase of infection by T. gondii, thus delaying its spreading. We further provide evidence of the compound's efficiency in controlling the formation of cysts in the brain of T. gondii-infected mice. A convincing docking of the JF363 compound in the active site of the five annotated ME49 T. gondii HDACs was performed by extensive sequence-structure comparison modeling. The resulting complexes show a similar mode of binding in the five paralogous structures and a quite similar prediction of affinities in the micromolar range. Altogether, these results pave the way for further development of this compound to treat acute and chronic toxoplasmosis. It also shows promise for the future development of anti-Plasmodium therapeutic interventions.This research was funded by IDEX Innovation Grant, UGA, 2017 and The GIS ChemBioFranceinfo:eu-repo/semantics/publishedVersio

Toxoplasma Hypervirulence in the Rat Model Parallels Human Infection and Is Modulated by the Toxo1 Locus

Toxoplasmosis is considered as an opportunistic parasitic disease. If post-natally acquired in children or adults, it may pass unnoticed, at least with strains of European origin. However, in the wild biotopes especially in South America, Toxoplasma gondii strains display a greater genetic diversity, which correlates to higher virulence for humans, particularly along the Amazon River and its tributaries. In French Guiana, several atypical strains have been associated with severe clinical forms: ocular toxoplasmosis and acute respiratory distress syndrome both of which can result in death. Among these, the GUY008-ABE strain was responsible for an epidemic of severe disseminated toxoplasmosis in Suriname, which led to the death of one immunocompetent individual. To better understand the mechanism underlying the hypervirulence of the GUY008-ABE strain, we have tested the rat model which compared to the mouse, better reflects the immune resistance of humans to Toxoplasma infection. Here we compare the outcome of toxoplasmosis in F344 rats infected either by the GUY008-ABE strain or the type II Prugniaud strain. We show that the GUY008-ABE strain displays a higher virulence phenotype leading to the death of all infected rats observed in this study. GUY008-ABE infection was characterized by an increase of the parasite load in several organs, especially the heart and lung, and was mainly associated with severe histological changes in lungs. Moreover, correlating with its hypervirulence trait, the GUY008-ABE strain was able to form cysts in the LEW rat model otherwise known to be refractory to infection by other Toxoplasma strains. Together, these results show that the rat is a discriminating experimental model to study Toxoplasma virulence factors relevant to the pathogenesis of human infection and that the degree of virulence is linked to the Toxo1 locus

Genomic determinants of the efficiency of internal ribosomal entry sites of viral and cellular origin

Variation in cellular gene expression levels has been shown to be inherited. Expression is controlled at transcriptional and post-transcriptional levels. Internal ribosome entry sites (IRES) are used by viruses to bypass inhibition of cap-dependent translation, and by eukaryotic cells to control translation under conditions when protein synthesis is inhibited. We aimed at identifying genomic determinants of variability in IRES-mediated translation of viral [Encephalomyocarditis virus (EMCV)] and cellular IRES [X-linked inhibitor-of-apoptosis (XIAP) and c-myc]. Bicistronic lentiviral constructs expressing two fluorescent reporters were used to transduce laboratory and B lymphoblastoid cell lines [15 CEPH pedigrees (n = 205) and 50 unrelated individuals]. IRES efficiency varied according to cell type and among individuals. Control of IRES activity has a significant genetic component (h2 of 0.47 and 0.36 for EMCV and XIAP, respectively). Quantitative linkage analysis identified a suggestive locus (LOD 2.35) on chromosome 18q21.2, and genome-wide association analysis revealed of a cluster of SNPs on chromosome 3, intronic to the FHIT gene, marginally associated (P = 5.9E-7) with XIAP IRES function. This study illustrates the in vitro generation of intermediate phenotypes by using cell lines for the evaluation of genetic determinants of control of elements such as IRES

Study of Leishmania pathogenesis in mice : experimental considerations

Although leishmaniases are endemic in 98 countries, they are still considered neglected tropical diseases. Leishmaniases are characterized by the emergence of new virulent and asymptomatic strains of Leishmania spp. and, as a consequence, by a very diverse clinical spectrum. To fight more efficiently these parasites, the mechanisms of host defense and of parasite virulence need to be thoroughly investigated. To this aim, animal models are widely used. However, the results obtained with these models are influenced by several experimental parameters, such as the mouse genetic background, parasite genotype, inoculation route/infection site, parasite dose and phlebotome saliva. In this review, we propose an update on their influence in the two main clinical forms of the disease: cutaneous and visceral leishmaniases

A Tiny Change Makes a Big Difference in the Anti-Parasitic Activities of an HDAC Inhibitor

International audienceWe previously synthesized an hydroxamate derivative (N-hydroxy-4-[2-(3- methoxyphenyl)acetamido]benzamide) named 363 with potent anti-Toxoplasma gondii activity and histone deacetylase inhibitor (HDACi) effects. Here we show that 1-N-hydroxy-4-N- [(2-methoxyphenyl)methyl]benzene-1,4-dicarboxamide, a 363 isomer, does not have antiparasitic potency and has a 13-fold decrease in HDACi activity. The in silico modeling of T. gondii HDACs of the type II strain discloses identity varying from 25% to 62% on more than 250 residues for S8EP32_TOXG and A0A125YPH4_TOXGM. We observed a high conservation degree with the human HDAC2 (53% and 64% identity, respectively) and a moderate one with the human HDAC8 (30-40%). Two other TgHDACs, S8F6L4_TOXGM and S8GEI3_TOXGM, were identified as displaying a higher similarity with some bacterial orthologs (~35%) than with the human enzymes (~25%). The docking in parallel of the two compounds on the models generated allowed us to gain insights on the docking of these hydroxamate derivatives that guide their specificity and potency against T. gondii histone deacetylase. This information would constitute the rationale from which more specific derivatives can be synthetized