Intra-host Trypanosoma cruzi strain dynamics shape disease progression: the missing link in Chagas disease pathogenesis.

Chronic Chagasic cardiomyopathy develops years after infection in 20-40% of patients, but disease progression is poorly understood. Here, we assessed Trypanosoma cruzi parasite dynamics and pathogenesis over a 2.5-year period in naturally infected rhesus macaques. Individuals with better control of parasitemia were infected with a greater diversity of parasite strains compared to those with increasing parasitemia over time. Also, the in vivo parasite multiplication rate decreased with increasing parasite diversity, suggesting competition among strains or a stronger immune response in multiple infections. Significant differences in electrocardiographic (ECG) profiles were observed in Chagasic macaques compared to uninfected controls, suggesting early conduction defects, and changes in ECG patterns over time were observed only in macaques with increasing parasitemia and lower parasite diversity. Disease progression was also associated with plasma fibronectin degradation, which may serve as a biomarker. These data provide a novel framework for the understanding of Chagas disease pathogenesis, with parasite diversity shaping disease progression.IMPORTANCEChagas disease progression remains poorly understood, and patients at increased risk of developing severe cardiac disease cannot be distinguished from those who may remain asymptomatic. Monitoring of Trypanosoma cruzi strain dynamics and pathogenesis over 2-3 years in naturally infected macaques shows that increasing parasite diversity in hosts is detrimental to parasite multiplication and Chagasic cardiomyopathy disease progression. This provides a novel framework for the understanding of Chagas disease pathogenesis.

Fibronectin degradation as biomarker for Trypanosoma cruzi infection and treatment monitoring in mice.

Biomarkers (coming from host or parasite) to monitor Chagas disease (CD) progression as well as the therapeutic response in chronic CD are critically needed, since seronegativization, which may be considered the best indicator of therapeutic cure, takes several years to be observed in adults. Several molecules have been suggested as biomarkers for CD, however, they have to be validated. Taking advantage of mouse models of Trypanosoma cruzi infection, we investigated changes in the degradation profile of fibronectin in plasma. The degradation profile of fibronectin was different in the acute phase compared to the chronic phase of the infection. Fibronectin fragments of approximately 150, 100, 40 and 30 kDa were identified. Furthermore, those degradation profiles correlated with acute parasitaemia as well as with cardiac parasite burden and tissue damage during the infection. The usefulness of fibronectin degradation as a biomarker for therapeutic response following drug treatment and immunotherapeutic vaccination also was evaluated and a decreased fibronectin degradation profile was observed upon benznidazole or a vaccine candidate treatment.

Acetylation is the most abundant actin modification in Entamoeba histolytica and modifications of actin's amino-terminal domain change cytoskeleton activities.

Actin is one of the most conserved, abundant, and ubiquitous proteins in all eukaryotes characterised to date. Posttranslation modifications of actin modify the organisation of the actin-rich cytoskeleton. In particular, chemical modifications of actin's amino-terminal region determine how filamentous actin is organised into scaffolds. After assuming that protein modifications account for the multiple functional activities exerted by the single actin in Entamoeba histolytica, we profiled posttranslational modifications of this protein. Acetylation (on 21 different amino acids) was the most abundant modification, followed by phosphorylation. Furthermore, the glycine residue at Position 2 in E. histolytica's actin (Gly2, not found in most other eukaryotic actins) was found to be acetylated. The impact of Gly2 on the amoeba's life cycle and pathogenicity was then assessed in mutagenesis experiments. We found that Gly2 was necessary for cell morphology and division, parasite-host cell adhesion, and host invasion in an in vitro model of amoebic human infection.

Morphodynamics of the Actin-Rich Cytoskeleton in Entamoeba histolytica.

Entamoeba histolytica is the anaerobic protozoan parasite responsible for human amoebiasis, the third most deadly parasitic disease worldwide. This highly motile eukaryotic cell invades human tissues and constitutes an excellent experimental model of cell motility and cell shape deformation. The absence of extranuclear microtubules in Entamoeba histolytica means that the actin-rich cytoskeleton takes on a crucial role in not only amoebic motility but also other processes sustaining pathogenesis, such as the phagocytosis of human cells and the parasite's resistance of host immune responses. Actin is highly conserved among eukaryotes, although diverse isoforms exist in almost all organisms studied to date. However, E. histolytica has a single actin protein, the structure of which differs significantly from those of its human homologs. Here, we studied the expression, structure and dynamics of actin in E. histolytica. We used molecular and cellular approaches to evaluate actin gene expression during intestinal invasion by E. histolytica trophozoites. Based on a three-dimensional structural bioinformatics analysis, we characterized protein domains differences between amoebic actin and human actin. Fine-tuned molecular dynamics simulations enabled us to examine protein motion and refine the three-dimensional structures of both actins, including elements potentially accounting for differences changes in the affinity properties of amoebic actin and deoxyribonuclease I. The dynamic, multifunctional nature of the amoebic cytoskeleton prompted us to examine the pleiotropic forms of actin structures within live E. histolytica cells; we observed the cortical cytoskeleton, stress fibers, "dot-like" structures, adhesion plates, and macropinosomes. In line with these data, a proteomics study of actin-binding proteins highlighted the Arp2/3 protein complex as a crucial element for the development of macropinosomes and adhesion plaques.

Human Liver Infection in a Dish: Easy-To-Build 3D Liver Models for Studying Microbial Infection.

Human liver infection is a major cause of death worldwide, but fundamental studies on infectious diseases affecting humans have been hampered by the lack of robust experimental models that accurately reproduce pathogen-host interactions in an environment relevant for the human disease. In the case of liver infection, one consequence of this absence of relevant models is a lack of understanding of how pathogens cross the sinusoidal endothelial barrier and parenchyma. To fill that gap we elaborated human 3D liver in vitro models, composed of human liver sinusoidal endothelial cells (LSEC) and Huh-7 hepatoma cells as hepatocyte model, layered in a structure mimicking the hepatic sinusoid, which enable studies of key features of early steps of hepatic infection. Built with established cell lines and scaffold, these models provide a reproducible and easy-to-build cell culture approach of reduced complexity compared to animal models, while preserving higher physiological relevance compared to standard 2D systems. For proof-of-principle we challenged the models with two hepatotropic pathogens: the parasitic amoeba Entamoeba histolytica and hepatitis B virus (HBV). We constructed four distinct setups dedicated to investigating specific aspects of hepatic invasion: 1) pathogen 3D migration towards hepatocytes, 2) hepatocyte barrier crossing, 3) LSEC and subsequent hepatocyte crossing, and 4) quantification of human hepatic virus replication (HBV). Our methods comprise automated quantification of E. histolytica migration and hepatic cells layer crossing in the 3D liver models. Moreover, replication of HBV virus occurs in our virus infection 3D liver model, indicating that routine in vitro assays using HBV or others viruses can be performed in this easy-to-build but more physiological hepatic environment. These results illustrate that our new 3D liver infection models are simple but effective, enabling new investigations on infectious disease mechanisms. The better understanding of these mechanisms in a human-relevant environment could aid the discovery of drugs against pathogenic liver infection.

Maintenance of intracellular hypoxia and adequate heat shock response are essential requirements for pathogenicity and virulence of Entamoeba histolytica.

Adhesion to cells, cytotoxicity and proteolysis are functions required for virulence and pathogenicity of Entamoeba histolytica. However, there was no correlation between these in vitro functions and the early elimination of non-pathogenic E. dispar and non-virulent E. histolytica (nvEh) in experimental amoebic liver abscesses developed in hamsters. Thus, additional functions may be involved in amoebic pathogenicity and virulence. In the present study, an integral experimental assessment, including innovative technologies for analyses of amoebal pathophysiology, cell biology, biochemistry and transcriptomics, was carried out to elucidate whether other cellular processes are involved in amoebal pathogenicity and virulence. In comparison with virulent E. histolytica, the data indicated that the main reasons for the early clearance of nvEh from hamster liver are decreased intracellular H2 O2 detoxification rate and deficient heat shock protein expression, whereas for E. dispar, it is a relatively lower capacity for O2 reduction. Therefore, maintenance of an intracellular hypoxic environment combined with the induction of an adequate parasite response to oxidative stress are essential requirements for Entamoeba survival in the liver, and therefore for pathogenicity.

Gene expression profiling in Entamoeba histolytica identifies key components in iron uptake and metabolism.

Entamoeba histolytica is an ameboid parasite that causes colonic dysentery and liver abscesses in humans. The parasite encounters dramatic changes in iron concentration during its invasion of the host, with relatively low levels in the intestinal lumen and then relatively high levels in the blood and liver. The liver notably contains sources of iron; therefore, the parasite's ability to use these sources might be relevant to its survival in the liver and thus the pathogenesis of liver abscesses. The objective of the present study was to identify factors involved in iron uptake, use and storage in E. histolytica. We compared the respective transcriptomes of E. histolytica trophozoites grown in normal medium (containing around 169 µM iron), low-iron medium (around 123 µM iron), iron-deficient medium (around 91 µM iron), and iron-deficient medium replenished with hemoglobin. The differentially expressed genes included those coding for the ATP-binding cassette transporters and major facilitator transporters (which share homology with bacterial siderophores and heme transporters) and genes involved in heme biosynthesis and degradation. Iron deficiency was associated with increased transcription of genes encoding a subset of cell signaling molecules, some of which have previously been linked to adaptation to the intestinal environment and virulence. The present study is the first to have assessed the transcriptome of E. histolytica grown under various iron concentrations. Our results provide insights into the pathways involved in iron uptake and metabolism in this parasite.

Bioassay-guided fractionation of extracts from Codiaeum variegatum against Entamoeba histolytica discovers compounds that modify expression of ceramide biosynthesis related genes.

Leaves of Codiaeum variegatum ("garden croton") are used against bloody diarrhoea by local populations in Cameroon. This study aims to search for the active components from C. variegatum against Entamoeba histolytica, and thereby initiate the study of their mechanism of action. A bioassay-guided screening of the aqueous extracts from C. variegatum leaves and various fractions was carried out against trophozoites of E. histolytica axenic culture. We found that the anti-amoebic activity of extracts changed with respect to the collection criteria of leaves. Thereby, optimal conditions were defined for leaves' collection to maximise the anti-amoebic activity of the extracts. A fractionation process was performed, and we identified several sub-fractions (or isolated compounds) with significantly higher anti-amoebic activity compared to the unfractionated aqueous extract. Anti-amoebic activity of the most potent fraction was confirmed with the morphological characteristics of induced death in trophozoites, including cell rounding and lysis. Differential gene expression analysis using high-throughput RNA sequencing implies the potential mechanism of its anti-amoebic activity by targeting ceramide, a bioactive lipid involved in disturbance of biochemical processes within the cell membrane including differentiation, proliferation, cell growth arrest and apoptosis. Regulation of ceramide biosynthesis pathway as a target for anti-amoebic compounds is a novel finding which could be an alternative for drug development against E. histolytica.

Plasmodium falciparum heterochromatin protein 1 binds to tri-methylated histone 3 lysine 9 and is linked to mutually exclusive expression of var genes.

Increasing experimental evidence shows a prominent role of histone modifications in the coordinated control of gene expression in the human malaria parasite Plasmodium falciparum. The search for the histone-mark-reading machinery that translates histone modifications into biological processes, such as formation of heterochromatin and antigenic variation is of foremost importance. In this work, we identified the first member of a histone modification specific recognition protein, an orthologue of heterochromatin protein 1 (PfHP1). Analysis of the PfHP1 amino-acid sequence revealed the presence of the two characteristic HP1 domains: a chromodomain (CD) and a chromo shadow domain (CSD). Recombinant CD binds to di- and tri-methylated lysine 9 from histone H3, but not to unmodified or methylated histone H3 in lysine 4. PfHP1 is able to interact with itself to form dimers, underlying its potential role in aggregating nucleosomes to form heterochromatin. Antibodies raised against PfHP1 detect this molecule in foci at the perinuclear region. ChIP analysis using anti-PfHP1 shows that this protein is linked to heterochromatin of subtelomeric non-coding repeat regions and monoallelic expression of the major virulence var gene family. This is the first report implicating an HP1 protein in the control of antigenic variation of a protozoan parasite.

Novel structural and functional findings of the ehFLN protein from Entamoeba histolytica.

The ehFLN protein (previously known as EhABP-120) is the first filamin to be identified in the parasitic protozoan Entamoeba histolytica. Filamins are a family of cross-linking actin-binding proteins that organize filamentous actin in networks and stress fibers. It has been reported that filamins of different organisms directly interact with more than 30 cellular proteins and some PPIs. The biochemical consequences of such interactions may have either positive or negative effects on the cross-linking function. Besides, filamins form a link between cytoskeleton and plasma membrane. In this work, the ehFLN protein was biochemically characterized; amoebae filamin was found to associate with both PA and PI(3)P in vitro, new lipid targets for a member of the filamins. By molecular modeling analysis and protein-lipid overlay assays, K-609, 709, and 710 were determined to be essential for the PA-ehFLN1 complex stability. Also, the integrity of the 4th repeat of ehFLN is essential to keep interaction with the PI(3)P. Transfected trophozoites that overexpressed the d100, d50NH(2), and d50COOH regions of ehFLN1 displayed both increased motility and chemotactic response to TYI-S-33 media. Together, these results suggest that short regions of ehFLN are involved in signaling events that, in cooperation with phosphatidic acid, EhPLD2 and EhPI3K, could promote cell motility.