Cellular and proteomics analysis of the endomembrane system from the unicellular Entamoeba histolytica

AbstractEntamoeba histolytica is the protozoan parasite agent of amoebiasis, an infectious disease of the human intestine and liver. Specific active pathogenic factors are secreted toward the external milieu upon interaction of the parasite with human tissue. Trafficking dynamics and secretion of these factors is not known and characterization of the dynamics interplay of subcellular compartments such as the ER or Golgi apparatus is still pending. In this work, we took advantage of cell fractionation and a wide proteomic analysis to search for principal components of the endomembrane system in E. histolytica. Over 1500 proteins were identified and the two top categories contained components of trafficking machinery and GTPases. Trafficking related proteins account for over 100 markers from the ER, Golgi, MVB, and retromers. The lack of important components supporting Golgi polarization was also highlighted. The data further describe principal components of the endosomal traffic highlighting Alix in isolated vesicles and during parasite division.Biological significanceThis work represents the first in-depth proteomics analysis of subcellular compartments in E. histolytica and allows a detailed map of vesicle traffic components in an ancient single-cell organism that lacks a stereotypical ER and Golgi apparatus to be established

Glucose Starvation Boosts Entamoeba histolytica Virulence

The unicellular parasite, Entamoeba histolytica, is exposed to numerous adverse conditions, such as nutrient deprivation, during its life cycle stages in the human host. In the present study, we examined whether the parasite virulence could be influenced by glucose starvation (GS). The migratory behaviour of the parasite and its capability to kill mammalian cells and to lyse erythrocytes is strongly enhanced following GS. In order to gain insights into the mechanism underlying the GS boosting effects on virulence, we analyzed differences in protein expression levels in control and glucose-starved trophozoites, by quantitative proteomic analysis. We observed that upstream regulatory element 3-binding protein (URE3-BP), a transcription factor that modulates E.histolytica virulence, and the lysine-rich protein 1 (KRiP1) which is induced during liver abscess development, are upregulated by GS. We also analyzed E. histolytica membrane fractions and noticed that the Gal/GalNAc lectin light subunit LgL1 is up-regulated by GS. Surprisingly, amoebapore A (Ap-A) and cysteine proteinase A5 (CP-A5), two important E. histolytica virulence factors, were strongly down-regulated by GS. While the boosting effect of GS on E. histolytica virulence was conserved in strains silenced for Ap-A and CP-A5, it was lost in LgL1 and in KRiP1 down-regulated strains. These data emphasize the unexpected role of GS in the modulation of E.histolytica virulence and the involvement of KRiP1 and Lgl1 in this phenomenon

A Proteomic and Cellular Analysis of Uropods in the Pathogen Entamoeba histolytica

Exposure of Entamoeba histolytica to specific ligands induces cell polarization via the activation of signalling pathways and cytoskeletal elements. The process leads to formation of a protruding pseudopod at the front of the cell and a retracting uropod at the rear. In the present study, we show that the uropod forms during the exposure of trophozoites to serum isolated from humans suffering of amoebiasis. To investigate uropod assembly, we used LC-MS/MS technology to identify protein components in isolated uropod fractions. The galactose/N-acetylgalactosamine lectin, the immunodominant antigen M17 (which is specifically recognized by serum from amoeba-infected persons) and a few other cells adhesion-related molecules were primarily involved. Actin-rich cytoskeleton components, GTPases from the Rac and Rab families, filamin, α-actinin and a newly identified ezrin-moesin-radixin protein were the main factors found to potentially interact with capped receptors. A set of specific cysteine proteases and a serine protease were enriched in isolated uropod fractions. However, biological assays indicated that cysteine proteases are not involved in uropod formation in E. histolytica, a fact in contrast to the situation in human motile immune cells. The surface proteins identified here are testable biomarkers which may be either recognized by the immune system and/or released into the circulation during amoebiasis

2′-Fluoro-2′-Deoxycytidine Inhibits Borna Disease Virus Replication and Spread

Borna disease virus (BDV) causes neurological diseases in a variety of warm-blooded animal species, possibly including humans. To date, there is no effective treatment against BDV infection. Recently, we reported on the antiviral activity of 1-β-d-arabinofuranosylcytosine (Ara-C). However, Ara-C's cytotoxic side effects are a major obstacle for its therapeutic use. Herein, we demonstrate that the nucleoside analog 2′-fluoro-2′-deoxycytidine (2′-FdC) exhibits potent antiviral activity against BDV. Importantly, 2′-FdC-associated cytotoxicity is negligible, indicating 2′-FdC as an excellent candidate for the development of antiviral therapy against BDV

PrPC Undergoes Basal to Apical Transcytosis in Polarized Epithelial MDCK Cells.

International audienceThe Prion Protein (PrP) is an ubiquitously expressed glycosylated membrane protein attached to the external leaflet of the plasma membrane via a glycosylphosphatidylinositol anchor (GPI). While the misfolded PrPSc scrapie isoform is the infectious agent of prion disease, the cellular isoform (PrPC) is an enigmatic protein with unclear function. Of interest, PrP localization in polarized MDCK cells is controversial and its mechanism of trafficking is not clear. Here we investigated PrP traffic in MDCK cells polarized on filters and in three-dimensional MDCK cysts, a more physiological model of polarized epithelia. We found that, unlike other GPI-anchored proteins (GPI-APs), PrP undergoes basolateral-to-apical transcytosis in fully polarized MDCK cells. Following this event full-length PrP and its cleavage fragments are segregated in different domains of the plasma membrane in polarized cells in both 2D and 3D cultures

Astrocyte-to-neuron intercellular prion transfer is mediated by cell-cell contact.

International audiencePrion diseases are caused by misfolding of the cellular protein PrP(C) to an infectious conformer, PrP(Sc). Intercellular PrP(Sc) transfer propagates conversion and allows infectivity to move from the periphery to the brain. However, how prions spread between cells of the central nervous system is unclear. Astrocytes are specialized non-neuronal cells within the brain that have a number of functions indispensable for brain homeostasis. Interestingly, they are one of the earliest sites of prion accumulation in the brain. A fundamental question arising from this observation is whether these cells are involved in intercellular prion transfer and thereby disease propagation. Using co-culture systems between primary infected astrocytes and granule neurons or neuronal cell lines, we provide direct evidence that prion-infected astrocytes can disseminate prion to neurons. Though astrocytes are capable of secreting PrP, this is an inefficient method of transferring prion infectivity. Efficient transfer required co-culturing and direct cell contact. Astrocytes form numerous intercellular connections including tunneling nanotubes, containing PrP(Sc), often colocalized with endolysosomal vesicles, which may constitute the major mechanism of transfer. Because of their role in intercellular transfer of prions astrocytes may influence progression of the disease

Rab11a–Rab8a cascade regulates the formation of tunneling nanotubes through vesicle recycling

International audienceTunneling nanotubes (TNTs) are actin-enriched membranous channels enabling cells to communicate over long distances. TNT-like structures form between various cell types and mediate the exchange of different cargos, such as ions, vesicles, organelles and pathogens; thus, they may play a role in physiological conditions and diseases (e.g. cancer and infection). TNTs also allow the intercellular passage of protein aggregates related to neurodegenerative diseases, thus propagating protein misfolding. Understanding the mechanism of TNT formation is mandatory in order to reveal the mechanism of disease propagation and to uncover their physiological function. Vesicular transport controlled by the small GTPases Rab11a and Rab8a can promote the formation of different plasma membrane protrusions (filopodia, cilia and neurites). Here, we report that inhibiting membrane recycling reduces the number of TNT-connected cells and that overexpression of Rab11a and Rab8a increases the number of TNT-connected cells and the propagation of vesicles between cells in co-culture. We demonstrate that these two Rab GTPases act in a cascade in which Rab11a activation of Rab8a is independent of Rabin8. We also show that VAMP3 acts downstream of Rab8a to regulate TNT formation

Data set for the proteomics analysis of the endomembrane system from the unicellular Entamoeba histolytica

Entamoeba histolytica is the protozoan parasite agent of amebiasis, an infectious disease of the human intestine and liver. This parasite contact and kills human cells by an active process involving pathogenic factors. Cellular traffic and secretion activities are poorly characterized in E. histolytica. In this work, we took advantage of a wide proteomic analysis to search for principal components of the endomembrane system in E. histolytica. A total of 5683 peptides matching with 1531 proteins (FDR of 1%) were identified which corresponds to roughly 20% of the total amebic proteome. Bioinformatics investigations searching for domain homologies (Smart and InterProScan programs) and functional descriptions (KEGG and GO terms) allowed this data to be organized into distinct categories. This data represents the first in-depth proteomics analysis of subcellular compartments in E. histolytica and allows a detailed map of vesicle traffic components in an ancient single-cell organism that lacks a stereotypical ER and Golgi apparatus to be established. The data are related to [1]