Jean-François Dubremetz

Targeting of soluble proteins to the rhoptries and micronemes in Toxoplasma gondii

Rhoptry and microneme organelles of the protozoan parasite Toxoplasma gondii are closely associated with host cell adhesion/invasion and establishment of the intracellular parasitophorous vacuole. In order to study the targeting of proteins to these specialized secretory organelles, we have engineered green fluorescent protein (GFP) fusions to the rhoptry protein ROP1 and the microneme protein MIC3. Both chimeras are correctly targeted to the appropriate organelles, permitting deletion analysis to map protein subdomains critical for targeting. The propeptide and a central 146 amino acid region of ROP1 are sufficient to target GFP to the rhoptries. More extensive deletions result in a loss of rhoptry targeting; the GFP reporter is diverted into the parasitophorous vacuole via dense granules. Certain MIC3 deletion mutants were also secreted into the parasitophorous vacuole via dense granules, supporting the view that this route constitutes the default pathway in T. gondii, and that specific signals are required for sorting to rhoptries and micronemes. Deletions within the cysteine-rich central region of MIC3 cause this protein to be arrested at various locations within the secretory pathway, presumably due to improper folding. Although correctly targeted to the appropriate organelles in living parasites, ROP1-GFP and MIC3-GFP fusion proteins were not secreted during invasion. GFP fusion proteins were readily secreted from dense granules, however, suggesting that protein secretion from rhoptries and micronemes might involve more than a simple release of organellar contents.

Potent and specific inhibitors of trypanothione reductase from Trypanosoma cruzi: bis(2-aminodiphenylsulfides) for fluorescent labeling studies

In order to optimise the activity of bis(2-aminodiphenylsulfides) upon trypanothione reductase (TR) from Trypanosoma cruzi, a new series of bis(2-aminodiphenylsulfides) possessing three side chains was synthesized. Various moieties were introduced at the end of the third side chain, including acridinyl or biotinyl moieties for fluorescent labeling studies. TR inhibition was improved: the most potent inhibitor (IC50 = 200 nM) was selective towards TR versus human glutathione reductase and corresponded to a single myristyl group. Compounds were also tested in vitro upon Trypanosoma cruzi and Leishmania infantum amastigotes, upon-Trapanosoma brucei trypomastigotes, and for their cytotoxicity upon human MRC-5 cells. In the presence of serum, acridine derivative was no longer detectable in mass spectrometry and its antitrypanosomal activity no longer observed. This transformation might explain the absence of correlation between the potent TR inhibition and the in vitro and in vivo antiparasitic activity with both of the first generation of 2-aminodiphenylsulfides.

Two glycoforms are present in the GPI-membrane anchor of the surface antigen 1 (P30) of Toxoplasma gondii

SAG1 (P30) is the major surface protein of the Toxoplasma gondii tachyzoite, the life cycle stage associated with the acute phase of infection. The protein is inserted into the parasites plasma membrane by a glycosyl-phosphatidylinositol anchor, a modification that is present on all T. gondii surface proteins characterized so far. Here we describe a detailed structural analysis of this anchor. GPI anchor peptides were isolated from [3H]glucosamine labeled purified P30 by protease digestion and phase partitioning. Neutral glycans were prepared from this material by dephosphorylation and deamination. Two glycoforms were characterized by gel filtration and high performance ion exchange chromatography in combination with exoglycosidase treatment. Both forms were shown to carry an N-acetylgalactosamine bound to the first mannose of the conserved three-mannosyl core. Glycan B carries an additional terminal hexose linked to GalNAc. To identify the nature of this hexose, bulk anchor peptide was prepared and glycans were purified by aminopropyl-HPLC. Highly purified glycans were subjected to MALDI-TOF-MS and, after derivatization, to FAB-MS and methylation linkage analysis. The structures of the two anchors found on SAG1 were determined to be: Man-alpha1,2-Man-alpha1,6-Man-[GalNAc-beta1,4-]-alpha1,4-GlcN-PI and Man-alpha1,2-Man-alpha1,6-Man [Glc-alpha1,4-GalNAc-beta1,4-]-alpha1,4-GlcN-PI. Comparison of these structures with free GPI glycolipid precursors characterized in T. gondii suggests that core modification of the anchor takes place prior to transfer to the protein.

Expression of Apical Organelles Antigens by a Toxoplasma Gondii Genomic Library

A genomic library of Toxoplasma gondii has been constructed in Lambda gt11. The proteins expressed in E. coli. were detected with the serum of a chronically infected rabbit. Specific antibodies were affinity-purified on the proteins expressed by the major reacting clones and used to characterize the corresponding T. gondii antigens. Most identified proteins were located in apical organelles (rhoptries, micronemes and dense granules). Additional clones encoding organelle antigens were isolated by screening with a serum from a rabbit immunized with a purified subcellular fraction containing apical organelles. An antiserum raised to recombinant Rop 2/3 reacted with a 100 kDa Plasmodium falciparum antigen comigrating with RhopH3.