Sabrina Sonda

Differential expression of cell surface- and dense granule-associated Neospora caninum proteins in tachyzoites and bradyzoites.

Morphologically, the tachyzoites and the tissue cysts of Neospora caninum are difficult to distinguish from those of other cyst-forming apicomplexan parasites such as Toxoplasma gondii. Several stage-specific antigens have been identified in T. gondii tachyzoites and bradyzoites, and respective antibodies are useful tools for discriminating between the 2 stages during tachyzoite-bradyzoite interconversion in T. gondii infections. Whereas several cell surface- and dense granule-associated proteins have been identified and characterized in N. caninum tachyzoites, not much is known about antigenic components expressed in N. caninum bradyzoites. In this study, the differential expression of the 2 N. caninum surface proteins Nc-p43 and Nc-p36 and the dense granule protein Nc-p33 (NCDG1) within tachyzoites and bradyzoites of N. caninum has been investigated.

Identification and partial characterization of a 36 kDa surface protein on Neospora caninum tachyzoites.

Neospora caninum, the causative agent of neosporosis, is a recently identified apicomplexan parasite which is structurally and biologically closely related to, but antigenically distinct from, Toxoplasma gondii. Molecules associated with the surfaces of N. caninum tachyzoites are likely to participate in the host cell entry process, could be involved in the interaction of the parasite with the immune system, and they could influence the pathogenesis of neosporosis. Isolated N. caninum tachyzoites were extracted with the non-ionic detergent Triton X-114 and were further analysed using a polyclonal anti-N. caninum antiserum. Immunoblots revealed several reactive bands, 1 of which represented a glycoprotein of approximately 36 kDa (Nc-p36). This molecule was present in 2 isolates of Neospora (NC-1 and Liverpool), but was absent in Toxoplasma (RH-strain) tachyzoites. Immunofluorescence and pre-embedding immunogold transmission electron microscopy employing affinity-purified anti-Nc-p36 antibodies showed that the Nc-p36 is a cell surface-associated protein. Immunogold on-section labelling of LR-White-embedded parasites, fixed prior and at defined time-points after host cell entry, demonstrated the presence of this molecule on the surface as well as within the dense granules of N. caninum tachyzoites.

Molecular characterization of a novel microneme antigen in Neospora caninum.

The apical complex of the parasites belonging to the phylum Sporozoa is believed to be critically involved in the events leading to host cell invasion. The characterization of the components of this subcellular structure is therefore an important step towards understanding how these parasites achieve host cell entry. Affinity-purification of an anti-Neospora caninum antiserum on a reactive protein band of approximately 40 kDa following Triton-X-114 extraction of parasite proteins, SDS-PAGE and Western blotting, yielded an immunoglobulin fraction which, by immunofluorescence, stained predominantly the apical portion of N. caninum tachyzoites. Following immunoscreening of a N. caninum tachyzoite lambdagt22 cDNA expression library, the respective full length cDNA sequence was determined. This sequence was found to encode a protein of 362 amino acids, with a calculated Mr of 38086. This protein is encoded by a single copy gene which produces a transcript of 2.4 kb. Sequence analysis showed that it contains a N-terminal putative signal peptide sequence and two potential membrane spanning regions. Four consecutive epidermal growth factor like domains were identified, as well a conserved sequence motif for binding of ATP/GTP (P-loop). The full length cDNA was expressed as a recombinant poly-histidine fusion protein in Escherichia coli, and antibodies affinity purified on this protein labelled exclusively a 38 kDa band on immunoblots of N. caninum extracts. In addition, specific labeling of a 45 kDa band in Toxoplasma gondii tachyzoite extracts was observed. By immunofluorescence, these antibodies stained predominantly the apical portion of both N. caninum and T. gondii tachyzoites, but the protein was absent from the parasite surface. Immunogold localization in LR-White embedded N. caninum tachyzoites demonstrated staining of predominantly the apically located micronemes, as well as of dense granules located at the posterior end of the tachyzoites. As evidenced by immunohistochemistry, this Neospora microneme antigen and its immunoreactive counterpart in Toxoplasma appeared to be expressed in both tachyzoite and bradyzoite stages.

Toxoplasma gondii salvages sphingolipids from the host Golgi through the rerouting of selected Rab vesicles to the parasitophorous vacuole

The intracellular parasite Toxoplasma scavenges sphingolipids from its host mammalian cell by establishing a close relationship with the host Golgi. It subverts the Golgis structure, hijacks selected Rab Golgi-derived vesicles within its parasitophorous vacuole, and retrieves sphingolipids from these vesicles.

Epigenetic mechanisms regulate stage differentiation in the minimized protozoan Giardia lamblia.

Histone modification is an important mechanism regulating both gene expression and the establishment and maintenance of cellular phenotypes during development. Regulation of histone acetylation via histone acetylases and deacetylases (HDACs) appears to be particularly crucial in determining gene expression patterns. In this study we explored the effect of HDAC inhibition on the life cycle of the human pathogen Giardia lamblia, a highly reduced parasitic protozoan characterized by minimized cellular processes. We found that the HDAC inhibitor FR235222 increased the level of histone acetylation and induced transcriptional regulation of ∼2% of genes in proliferating and encysting parasites. In addition, our analyses showed that the levels of histone acetylation decreased during differentiation into cysts, the infective stage of the parasite. Importantly, FR235222 treatment during encystation reversed this histone hypo‐acetylation and potently blocked the formation of cysts. These results provide the first direct evidence for epigenetic regulation of gene expression in this simple eukaryote. This suggests that regulation of histone acetylation is involved in the control of Giardia stage differentiation, and identifies epigenetic mechanisms as a promising target to prevent Giardia transmission.

The major 36 kDa Neospora caninum tachyzoite surface protein is closely related to the major Toxoplasma gondii surface antigen

The tachyzoites and the tissue cysts containing bradyzoites of Neospora caninum and Toxoplasma gondii, respectively, are difficult to distinguish morphologically. Specific antigens have been identified in T. gondii tachyzoites and bradyzoites, some of which are stage-specifically expressed, and different functions have been attributed to some of them. A tachyzoite stage-specifically expressed surface protein is the major surface antigen 1 (SAG1) which has been shown to be involved in host cell attachment and invasion. Previously we have identified a cell surface-associated glycoprotein (p36) in N. caninum tachyzoites. The full length coding sequence of the cDNA coding for p36 was determined, and analysis of the deduced amino acid sequence demonstrated that p36 is closely related to SAG1. p36 is encoded by a single copy gene which produces a transcript of 1.4 kb. Immunogold labeling of resin-embedded parasites using polyclonal antibodies affinity-purified on a recombinant p36 fusion protein expressed in Escherichia coli showed that this protein is located exclusively on the tachyzoite cell surface. As SAG1 in T. gondii, p36 is expressed in the tachyzoite stage, but is absent from bradyzoites. p36 is recognized by antibodies present in sera of cows experimentally infected with N. caninum tachyzoites.

Inhibitory Effect of Aureobasidin A on Toxoplasma gondii

ABSTRACT The apicomplexan parasite Toxoplasma gondii is a leading opportunistic pathogen associated with AIDS and congenital birth defects. Due to the need for identifying new parasite-specific treatments, the possibility of targeting sphingolipid biosynthesis in the parasite was investigated. Aureobasidin A, an inhibitor of the enzyme synthesizing the sphingolipid inositol phosphorylceramide, which is present in fungi, plants, and some protozoa but absent in mammalian cells, was found to block in vitro T. gondii replication without affecting host cell metabolism. Aureobasidin A treatment did not induce tachyzoite to bradyzoite stage conversion in T. gondii but resulted in a loss of intracellular structures and vacuolization within the parasite. In addition, aureobasidin A inhibited sphingolipid synthesis in T. gondii. Sphingolipid biosynthetic pathways may therefore be considered targets for the development of anti-T. gondii agents.

Deoxysphingolipids, Novel Biomarkers for Type 2 Diabetes, Are Cytotoxic for Insulin- Producing Cells

Irreversible failure of pancreatic β-cells is the main culprit in the pathophysiology of diabetes, a disease that is now a global epidemic. Recently, elevated plasma levels of deoxysphingolipids, including 1-deoxysphinganine, have been identified as a novel biomarker for the disease. In this study, we analyzed whether deoxysphingolipids directly compromise the functionality of insulin-producing Ins-1 cells and primary islets. Treatment with 1-deoxysphinganine induced dose-dependent cytotoxicity with senescent, necrotic, and apoptotic characteristics and compromised glucose-stimulated insulin secretion. In addition, 1-deoxysphinganine altered cytoskeleton dynamics, resulting in intracellular accumulation of filamentous actin and activation of the Rho family GTPase Rac1. Moreover, 1-deoxysphinganine selectively upregulated ceramide synthase 5 expression and was converted to 1-deoxy-dihydroceramides without altering normal ceramide levels. Inhibition of intracellular 1-deoxysphinganine trafficking and ceramide synthesis improved the viability of the cells, indicating that the intracellular metabolites of 1-deoxysphinganine contribute to its cytotoxicity. Analyses of signaling pathways identified Jun N-terminal kinase and p38 mitogen-activated protein kinase as antagonistic effectors of cellular senescence. The results revealed that 1-deoxysphinganine is a cytotoxic lipid for insulin-producing cells, suggesting that the increased levels of this sphingolipid observed in diabetic patients may contribute to the reduced functionality of pancreatic β-cells. Thus, targeting deoxysphingolipid synthesis may complement the currently available therapies for diabetes.

Detection of surface-associated and intracellular glycoconjugates and glycoproteins in Neospora caninum tachyzoites.

The surface-associated molecules of the invasive stages of apicomplexan parasites such as Neospora caninum and Toxoplasma gondii are most likely crucially involved in mediating the interaction between the parasite and its host cell. In N. caninum, several antigens have recently been identified which could participate in host cell adhesion and/or invasion. These are antigens which are either constitutively expressed on the outer plasma membrane, or antigens which are only transiently localised on the surface as they are expulsed from the secretory vesicles either prior, or after host cell invasion. Some of these proteins have been characterised at the molecular level, and it has been shown that they are, with respect to protein sequences, closely related to homologous counterparts in T. gondii. Nevertheless, there is only a low degree of cross-antigenicity between the two species. In microbial interactions it has been shown that carbohydrates could also play a crucial role in host cell recognition and immunological host parasite interactions. In this study we present data which strongly suggest that the surface of N. caninum tachyzoites is glycosylated. In SDS-PAGE, glycoproteins comigrated largely with glycosylphosphatidylinositol-anchored proteins which were identified using in vivo [3H]ethanolamine labelling followed by autoradiography. The lectin Con A reacted strongly with the surface of these parasites, binding of which is indicative for the presence of N-glycans. Additional surface binding was observed, although only in a subpopulation of all tachyzoites, for wheat germ agglutinin and Jacalin. Intracellular binding sites for Con A were mainly associated with the parasite dense granules. By lectin labelling of Western blots of N. caninum protein extracts, glycoproteins were identified which reacted specifically with the lectins Con A, wheat germ agglutinin, Jacalin and soy bean agglutinin.

Sphingolipid synthesis and scavenging in the intracellular apicomplexan parasite, Toxoplasma gondii

Graphical abstract Highlights ► Identification and characterisation of Toxoplasma sphingolipid synthase (TgSLS). ► Demonstration of TgSLS inositol phosphorylceramide synthase activity. ► Identification of inositol phosphorylceramide in Toxoplasma extracts. ► Delineation of role of host sphingolipid biosynthesis in Toxoplasma proliferation. ► Host biosynthesis non-essential for proliferation, de novo synthesis could be key.