Deficiency of a Niemann-Pick, Type C1-related Protein in Toxoplasma Is Associated with Multiple Lipidoses and Increased Pathogenicity

Several proteins that play key roles in cholesterol synthesis, regulation, trafficking and signaling are united by sharing the phylogenetically conserved ‘sterol-sensing domain’ (SSD). The intracellular parasite Toxoplasma possesses at least one gene coding for a protein containing the canonical SSD. We investigated the role of this protein to provide information on lipid regulatory mechanisms in the parasite. The protein sequence predicts an uncharacterized Niemann-Pick, type C1-related protein (NPC1) with significant identity to human NPC1, and it contains many residues implicated in human NPC disease. We named this NPC1-related protein, TgNCR1. Mammalian NPC1 localizes to endo-lysosomes and promotes the movement of sterols and sphingolipids across the membranes of these organelles. Miscoding patient mutations in NPC1 cause overloading of these lipids in endo-lysosomes. TgNCR1, however, lacks endosomal targeting signals, and localizes to flattened vesicles beneath the plasma membrane of Toxoplasma. When expressed in mammalian NPC1 mutant cells and properly addressed to endo-lysosomes, TgNCR1 restores cholesterol and GM1 clearance from these organelles. To clarify the role of TgNCR1 in the parasite, we genetically disrupted NCR1; mutant parasites were viable. Quantitative lipidomic analyses on the ΔNCR1 strain reveal normal cholesterol levels but an overaccumulation of several species of cholesteryl esters, sphingomyelins and ceramides. ΔNCR1 parasites are also characterized by abundant storage lipid bodies and long membranous tubules derived from their parasitophorous vacuoles. Interestingly, these mutants can generate multiple daughters per single mother cell at high frequencies, allowing fast replication in vitro, and they are slightly more virulent in mice than the parental strain. These data suggest that the ΔNCR1 strain has lost the ability to control the intracellular levels of several lipids, which subsequently results in the stimulation of lipid storage, membrane biosynthesis and parasite division. Based on these observations, we ascribe a role for TgNCR1 in lipid homeostasis in Toxoplasma

T-cell mediated control of Epstein-Barr virus infection : viral mechanisms of immune escape

Mechanisms regulating the immunogenicity of Epstein-Barr Virus (EBV)-encoded CTL peptide epitopes and their contribution to the escape of virus infected cells from CTL-mediated immunosurveillance were the subject of these studies. It was demonstrated that two HLA A11-restricted CTL epitopes of EBV nuclear antigen (EBNA)-4 (designated IVT and AVF) possessed different immunogenicity that appeared to be due to a rapid surface turnover of All molecules containing the AVF peptide. This difference in persistence of IVT and AVF-containing complexes is likely to determine the representation of individual class I-restricted CTL epitopes within the cell surface pool of molecules and may be an important factor contributing to their immunogenicity. EBV isolates from human populations with a high frequency of HLA A11 evade recognition by CTLs specific for the IVT epitope. The epitope variants containing the substitutions in the anchor residues were able to bind to HLA A11 but formed complexes of significantly lower stability in comparison to the immunogenic epitope. In spite of the efficient production by endogenous processing and efficient association with All-molecules these complexes failed to accumulate at the cell surface, which suggests that complexes containing the non-immunogenic epitope variants are formed but are then destroyed intracellulary. Thus, a specialised sorting mechanism seems to contribute to shaping the repertoire of peptides presented to T-lymphocytes. EBV infected B-lymphocytes persist for life in healthy virus carriers. These infected cells express the EBV encoded nuclear antigen (EBNA)l but down-regulate other highly immunogenic viral products. We have shown that the glycine-alanine domain of EBNA1 generate a cis-acting inhibitory signal which interferes with antigen processing and MHC class I restricted presentation. In vitro processing assays suggest that the repeat may affect MHC class I-restricted responses by inhibiting antigen processing via the ubiquitin/ proteasome pathway. Presense of the Gly-Ala repeat was also shown to prolong half-life of EBNAl in vivo

Inhibition of Heavy Chain and β(2)-Microglobulin Synthesis as a Mechanism of Major Histocompatibility Complex Class I Downregulation during Epstein-Barr Virus Replication

The mechanisms of major histocompatibility complex (MHC) class I downregulation during Epstein-Barr virus (EBV) replication are not well characterized. Here we show that in several cell lines infected with a recombinant EBV strain encoding green fluorescent protein (GFP), the virus lytic cycle coincides with GFP expression, which thus can be used as a marker of virus replication. EBV replication resulted in downregulation of MHC class II and all classical MHC class I alleles independently of viral DNA synthesis or late gene expression. Although assembled MHC class I complexes, the total pool of heavy chains, and β(2)-microglobulin (β(2)m) were significantly downregulated, free class I heavy chains were stabilized at the surface of cells replicating EBV. Calnexin expression was increased in GFP(+) cells, and calnexin and calreticulin accumulated at the cell surface that could contribute to the stabilization of class I heavy chains. Decreased expression levels of another chaperone, ERp57, and TAP2, a transporter associated with antigen processing and presentation, correlated with delayed kinetics of MHC class I maturation. Levels of both class I heavy chain and β(2)m mRNA were reduced, and metabolic labeling experiments demonstrated a very low rate of class I heavy chain synthesis in lytically infected cells. MHC class I and MHC class II downregulation was mimicked by pharmacological inhibition of protein synthesis in latently infected cells. Our data suggest that although several mechanisms may contribute to MHC class I downregulation in the course of EBV replication, inhibition of MHC class I synthesis plays the primary role in the process

Inhibition of ubiquitin/proteasome-dependent protein degradation by the Gly-Ala repeat domain of the Epstein–Barr virus nuclear antigen 1

The Epstein–Barr virus (EBV) encoded nuclear antigen (EBNA) 1 is expressed in latently infected B lymphocytes that persist for life in healthy virus carriers and is the only viral protein regularly detected in all EBV associated malignancies. The Gly-Ala repeat domain of EBNA1 was shown to inhibit in cis the presentation of major histocompatibility complex (MHC) class I restricted cytotoxic T cell epitopes from EBNA4. It appears that the majority of antigens presented via the MHC I pathway are subject to ATP-dependent ubiquitination and degradation by the proteasome. We have investigated the influence of the repeat on this process by comparing the degradation of EBNA1, EBNA4, and Gly-Ala containing EBNA4 chimeras in a cell-free system. EBNA4 was efficiently degraded in an ATP/ubiquitin/proteasome-dependent fashion whereas EBNA1 was resistant to degradation. Processing of EBNA1 was restored by deletion of the Gly-Ala domain whereas insertion of Gly-Ala repeats of various lengths and in different positions prevented the degradation of EBNA4 without appreciable effect on ubiquitination. Inhibition was also achieved by insertion of a Pro-Ala coding sequence. The results suggest that the repeat may affect MHC I restricted responses by inhibiting antigen processing via the ubiquitin/proteasome pathway. The presence of regularly interspersed Ala residues appears to be important for the effect

Dynamics of the major histocompatibility complex class I processing and presentation pathway in the course of malaria parasite development in human hepatocytes: implications for vaccine development.

Control of parasite replication exerted by MHC class I restricted CD8+ T-cells in the liver is critical for vaccination-induced protection against malaria. While many intracellular pathogens subvert the MHC class I presentation machinery, its functionality in the course of malaria replication in hepatocytes has not been characterized. Using experimental systems based on specific identification, isolation and analysis of human hepatocytes infected with P. berghei ANKA GFP or P. falciparum 3D7 GFP sporozoites we demonstrated that molecular components of the MHC class I pathway exhibit largely unaltered expression in malaria-infected hepatocytes until very late stages of parasite development. Furthermore, infected cells showed no obvious defects in their capacity to upregulate expression of different molecular components of the MHC class I machinery in response to pro-inflammatory lymphokines or trigger direct activation of allo-specific or peptide-specific human CD8+ T-cells. We further demonstrate that ectopic expression of circumsporozoite protein does not alter expression of critical genes of the MHC class I pathway and its response to pro-inflammatory cytokines. In addition, we identified supra-cellular structures, which arose at late stages of parasite replication, possessed the characteristic morphology of merosomes and exhibited nearly complete loss of surface MHC class I expression. These data have multiple implications for our understanding of natural T-cell immunity against malaria and may promote development of novel, efficient anti-malaria vaccines overcoming immune escape of the parasite in the liver

Visualization of parasites in HC-04 cells infected with <i>P</i>. <i>falciparum</i>.

<p>GFP-positive PI-negative events were isolated by flow cytometry-based cell sorting 96 hours postinfection and cytospun. Representative micrographs of EEFs show DNA staining with DAPI, GFP expression and immunofluorescent detection of <i>Pf</i>Hsp70 by anti-Hsp70 monoclonal antibody (Blue = DAPI, Green = GFP, Red = <i>Pf</i>Hsp70. Scale bars indicate 10 μm).</p

Human hepatocyte cell lines can be traversed by <i>P</i>. <i>falciparum</i> sporozoites.

<p>Traversal was assessed by flow cytometry as an ability of cells to uptake and retain fluorescent high molecular weight dextran during sporozoite co-incubation for 6 hours. (A) Representative plots for each cell line are shown, 1.5:1 sporozoite-to-hepatocyte ratio was used. Numbers indicate percentage of dextran-positive cells. (B) Percentage of traversed cells normalized to HC-04 obtained from three sporozoite preparations, each experiment was conducted in triplicate. Mean +/- SD shown.</p