Ali Ouaissi

Trypanosoma cruzi elongation factor 1-α: nuclear localization in parasites undergoing apoptosis

The cloning and sequencing of the gene coding for Trypanosoma cruzi elongation factor 1 alpha (TcEF-1 alpha) was performed by screening a T. cruzi genomic library with a probe obtained through the polymerase chain reaction (PCR) amplification of T. cruzi DNA using two oligonucleotides deduced from the sequence of T. brucei EF-1 alpha. Southern blot analysis of T. cruzi digested genomic DNA and Northern blot hybridized with the labeled probe revealed that one copy of TcEF-1 alpha exist in the genome of the parasite. Indirect immunofluorescence technique using anti-EF-1 alpha antibodies and epimastigotes harvested after different days of in vitro culture showed that EF-1 alpha is localised in the cytoplasm of the parasites from the exponential growth phase. Surprisingly, during the stationary phase (ageing parasites), EF-1 alpha was found in the nucleus. Furthermore, treatment of parasites with the antibiotic drug geneticin (G418) which induces the death of epimastigotes by apoptosis showed selective localization of EF-1 alpha in the nucleus of dying parasites. This observation supports the notion already reported in the case of mammalian cells that EF-1 alpha could participate in the transcription processes and possibly in the case of T. cruzi, in the expression regulation of genes involved in the control of cell death. The possible transfection and genomic manipulation of T. cruzi may provide a model to study the role of TcEF-1 alpha in this phenomenon.

In vitro attachment of Pneumocystis carinii from mouse and rat origin

Summary— The attachment of Pneumocystis carinii to lung cells could play a role in the pathophysiology of P carinii pneumonia. The trophozoite attaches to type I alveolar epithelial cells. Physical, chemical, and extracellular matrix factors, involved in the mouseor rat‐derived P carinii attachment to fibroblastic cells in culture, were examined using a new model of in vitro adherence. The development of parasite filopodia penetrating deeply the host cell cytoplasm was observed using transmission electronic microscopy. Killed P carinii organisms were unable to attach to cultured cells. Also, parasites were unable to attach to killed target cells. The P carinii in vitro attachment was partially inhibited by cytochalasin B. In contrast, the parasite attachment was not affected when the target cell cytoskeleton was altered. In our work conditions, sialic acids were not involved in the attachment process. Present results showed that fibronectin (Fn) plays a role in the parasite attachment, and suggest that a specific Fn‐binding receptor is present at the surface of mouse‐derived P carinii organisms.

Cloning and sequencing of a 24-kDa Trypanosoma cruzi specific antigen released in association with membrane vesicles and defined by a monoclonal antibody

Summary— In the present study we have used the Tcr7 monoclonal antibody (mAb) previously characterized as directed against Trypanosoma cruzi 24–25‐kDa specific antigens, both are immunogenic in man and during experimental T cruzi infections. We have demonstrated that mAb Tcr7 was able to recognize two in vitro translation products of molecular weights of 24 and 25 kDa. This suggested the holoproteic nature of these two related antigens bearing at least a common epitope and allowed us to use Tcr7 for an immunoscreening of a lambda ZAPII T cruzi cDNA library. Indeed, we have obtained several positive clones and completly sequenced the largest one which encoded theoretically for a protein of 23.7 kDa. The sequence analysis revealed a nearly perfect homology between this clone and one already described by other investigators and was shown to express a major flagellar protein of T cruzi able to bind calcium. Using different overlapping peptides derived from the sequence of the cDNA clone, we have localized the immunoreactivity of mAb Tcr7 mainly on several primary sequences present in the N‐terminal part of the sequence, suggesting that the mAb could recognize a discontinuous epitope. Moreover, the immunoelectron microscopy allowed us to show that the antigen(s) carrying the epitope reacting with mAb Tcr7 is (are) released in association with membrane vesicles which protruded from the parasite surface and the flagellar pocket. This new mechanism of antigen shedding is likely to be independent of phospholipase C‐mediated release of GPI‐anchored molecules.

A Leishmania major protein with extensive homology to silent information regulator 2 of Saccharomyces cerevisiae

We have isolated a cDNA from the protozoan parasite Leishmania major (Lm) that encodes a protein homologous to the Saccharomyces cerevisiae and Kluyveromyces marxianus silent information regulator 2 (SIR2) proteins. The deduced Lm SIR2-related protein (termed LmSIR2rp) consists of 381 amino acids that share 40.5% identity with yeast SIR2, increasing to 60% when substitutions are included. Moreover, the LmSIR2rp aa sequence contains a single potential zinc-binding domain with a CysXaa2CysXaa20CysXaa2Cys motif, and its C-terminal part is rich in Ser (16 Ser residues over 40 aa) which constitute potential sites for phosphorylation. The characterization of a novel Lm gene product which shows considerable similarity to a yeast mating-type regulatory protein provides a new tool to investigate the parasite differentiation control mechanisms and gene expression regulation.

2-Amino diphenylsulfides as new inhibitors of trypanothione reductase

Trypanothione reductase (TR) is the primary enzyme responsible for the reduction of trypanothione, the analog of glutathione found in trypanosomatidae. We have discovered a series of diphenylsulfides which are potent inhibitors of TR and have no activity on mammalian glutathione reductase. These compounds are also active in vitro on various stages of the parasite. Although structurally related to phenothiazines, which are known to be TR inhibitors, these compounds are devoided of any neuroleptic activity, making them attractive leads to develop specific and non toxic anti-chagasic drugs.

Trypanosoma cruzi: Analysis of cellular and humoral response against a protective recombinant antigen during experimental Chagas' disease

In previous studies we have shown the protective value of T. cruzi excretory/secretory antigens (ESA) as well as a synthetic peptide derived from the primary sequence of a 24-kDa protein present among ESA in mice and rats challenged with a lethal dose of T. cruzi. In the present study, the 24-kDa polypeptide was produced as a fusion protein in the pGEX-2T vector system. Western blot assays revealed that Tc24 is expressed by all T. cruzi strains so far examined (CL, ECH, C23, Tehuantepec, Tulahuen, and Y). The immunization of BALB/c mice with Tc24 fusion protein showed that the protein has the capacity to induce a significant level of protection in BALB/c mice against lethal T. cruzi infection. Moreover, splenic cells from T. cruzi chronically infected mice recognize the recombinant antigen since they proliferate after in vitro stimulation. A typical Th1 pattern of lymphokine secretion (IL-2 and IFN-gamma) was detected in splenic cell culture supernatants from Tc24-immunized mice. In addition, high levels of IFN-gamma were detected in cell culture supernatants from both acute and chronically infected mice after Tc24 antigen stimulation. In contrast, no detectable amounts of IL-4, IL-5 or Th-10 could be detected in those supernatants. Finally, antibodies (IgG isotype) involved in the immune clearance of T. cruzi are elicited by the Tc24 recombinant protein. Taken together, these results demonstrated that the recombinant T. cruzi antigen is able to induce cellular and humoral immune responses which could explain the protection achieved when this protein is used as immunizing agent.

Molecular and immunological characterization of a Trypanosoma cruzi protein homologous to mammalian elongation factor 1 gamma

Summary— In previous studies, we reported the characterization of three Trypanosoma cruzi proteins with molecular masses of 45, 30 and 25 kDa eluted from a glutathione agarose column (these proteins were named TcGBP). Using antibodies against TcGBP native proteins we could isolate from a lambda ZAPII epimastigote cDNA library cDNA clones encoding the 30 and 25 kDa proteins. Comparison of the two sequences with amino acid sequences in several data banks revealed that both protein sequences were highly homologous to human and Artemia salina elongation factor 1β. Thus, the proteins were named TcEF‐1 β25 and TcEF‐1 β30. In the present study we used a double immunoscreening strategy that allowed us to isolate a cDNA clone corresponding to the 45 kDa protein. The protein sequence revealed 31% identity and 61% homology with human and Artemia salina EF1γ and therefore was named TcEF‐1γ. Moreover, three putative phosphorylation sites at position 51 (CSPC), at position 90 (RTPL) and at position 265 (PSPF) were found in the TcEF‐1γ sequence. These sites are compatible with the notion that TcEF‐1γ could be the target of phosphorylation by protein kinase(s). Random primed cDNA hybridized with a single 1.4 kb mRNA found in epimastigote, trypomastigote and amastigote forms. In addition, Southern blot analysis of genomic DNA suggested that the protein is encoded by a single gene. The TcEF‐1γ cDNA was subcloned into the pGEX‐4T‐3 vector for expression in Escherichia coli. Antibodies against the fusion peptide allowed us to identify the weight sizes of the native protein (48 kDa) and its major degradation product (24.4 kDa) which are in close agreement with those of EF1‐γ from Artemia salina and Schizosaccharomyces pombe. These antibodies reacted against macrophage cell line J774 extracts which indicates that EF‐1γ epitopes were conserved throughout evolution.

Trypanosoma cruzi glutathione-binding proteins (TcGBP): protection induced by native proteins in an experimental model and analysis of the antibody response.

Three Trypanosoma cruzi glutathione-binding proteins (TcGBP) of 45, 30 and 25 kDa presenting glutathione S transferase activity were characterized from T. cruzi epimastigotes. We show here that immunization of mice using TcGBP and complete Freunds adjuvant (CFA) did not protect the animals against a challenge with bloodstream trypomastigotes. In contrast, immunization of mice using TcGBP in association with Bordetella pertussis plus alum (BpAI) resulted in greatly diminished parasitaemia and significantly protected the animals from lethal infection. Using TcGBP mixed with BpAI and a lower challenge dose, we obtained strongly diminished parasitaemia and 100% protection in terms of survival. Only sera from mice immunized with TcGBP plus BpAI were able to kill trypomastigotes by complement-mediated lysis, whereas sera from mice immunized with TcGBP plus CFA did not. Interestingly, sera from mice immunized with TcGBP plus BpAI showed significant levels of specific IgE, IgG2a and IgG2b antibodies, whereas these isotypes were not detected in sera from mice immunized with TcGBP in CFA. All these levels were increased in sera of protected animals. These results demonstrate that TcGBP antigens can confer resistance to T. cruzi acute infection in mice, and suggest a possible functional role for both IgE and IgG2 isotypes in the induction of protective immunity.

Trypanosoma cruzi: cell type dependent distribution of a tubulin domain in mammalian stages

The distribution of tubulin domains in the mammalian stages of Trypanosoma cruzi was investigated by using monoclonal antibodies elicited against bovine brain tubulin. Western blotting performed on T. brucei trypomastigotes and T. cruzi epimastigotes showed that the monoclonal antibodies 16D3 and 24E3 reacted only with tubulin in these cell types. Indirect immunofluorescence revealed that, whereas 16D3 stained all microtubules, including subpellicular microtubules, the epitope defined by 24E3 was found in only a part of the tubulin pool of amastigotes and intermediate stages infecting murine fibroblasts and of broad trypomastigotes; the staining was limited to the basal bodies and the distal region of the flagellar adhesion zone in these developmental forms. By contrast, slender trypomastigotes did not exhibit any reaction with 24E3. These results are consistent with a transformation of broad trypomastigotes into slender trypomastigotes during which the tubulin domain recognized by 24E3 would undergo modifications leading to its complete masking in slender forms. The morphogenesis of the mammalian stages of T. cruzi would involve modifications of the tubulin molecule.

Detection of IgE antibodies in onchocerciasis using a semi-purified fraction from Dipetalonema viteae total antigen.

A Dipetalonema viteae extract was separated by gel filtration on ACA 34 Ultrogel into four fractions (A, B, C and D). The allergenic activity of the D. viteae extract and its various fractions was assayed by the passive cutaneous anaphylaxis (PCA) test in rats using mouse sera obtained from Balb/c mice transplanted with D. viteae. The PCA reaction showed that fraction B was the most potent allergenic fraction of the D. viteae extract. By the radioallergosorbent test (RAST) the use of fraction B coupled to CNBr-activated paper discs showed elevated binding of IgE antibodies in onchocerciasis human sera. A comparative study demonstrated the efficiency of the above fraction in the RAST technique in distinguishing between Onchocerca volvulus-infected patients and those infected with other human filarial worms or other helminth parasites. The binding of IgE to fraction B was confirmed by the radioimmunoelectrophoresis and radio-double diffusion methods using an 125I anti-human IgE. Since D. viteae antigens are more readily obtainable than those of O. volvulus, a further purification of fraction B to improve its specificity for the detection of IgE antibodies in human onchocerciasis is warranted.