Odile Billaut-Mulot

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.

SS-56, a novel cellular target of autoantibody responses in Sjögren syndrome and systemic lupus erythematosus

Certain autoimmune disorders, including Sjögren syndrome (SS) and systemic lupus erythematosus (SLE), are characterized by autoantibodies against the Ro/SSA and La/SSB cellular antigens. Although the implication of these autoantibodies in disease pathogenesis is still unclear, it is believed that the aberrant responses against autoantigens may extend to other proteins that are not yet well defined. In an attempt to analyze the regulated gene expression in lymphocytes by an HIV-suppressive immunomodulator, we have identified and cloned a novel gene encoding a 56-kDa protein, named SS-56, which is structurally related to the 52-kDa Ro/SSA antigen. The new protein showed primarily perinuclear cytoplasmic localization, and recombinant SS-56 was found to react in ELISA with sera from most patients with SS or SLE. Western blot analysis confirmed the autoantigenic nature of native SS-56 in extracts from HeLa cells. Interestingly, the incidence of antibodies to SS-56 was associated with visceral complications in SLE, and roughly half of the 17 SS or SLE patients with no detectable antibodies to SSA and SSB antigens presented measurable antibodies against recombinant SS-56. Thus, SS-56 represents a new member of the SS family of autoantigens and could become an additional and important diagnostic marker for SS and SLE.

Modulation of cellular and humoral immune responses to a multiepitopic HIV-1 DNA vaccine by interleukin-18 DNA immunization/viral protein boost

In this study, the impact of Th1-inducing cytokine gene co-delivery and antigen boosting on humoral and cellular responses induced by multiepitopic DNA immunization in mice have been investigated. Intramuscular injection of mixed DNA constructs encoding for HIV-1 Gag, Tat and Nef proteins, co-administered with the DNA encoding for interleukin-18 (IL-18) have been used. The effect of boosting with the recombinant proteins was also evaluated on the outcome of the responses in DNA-primed mice. It was demonstrated that at least two DNA immunizations were necessary to generate virus specific Th-1 responses detected by the presence of cytotoxic T lymphocyte (CTL) and by the secretion of IL-2 and IFN-gamma, but not IL-4 and IL-10, in antigen-stimulated splenocyte cultures. Interestingly, co-delivery of Th-1-inducing IL-18 gene was able to shorten by 2 weeks, the CTL induction time, and to increase the antigen-induced secretion of IL-2 and IFN-gamma. Furthermore, IL-18 co-delivery enhanced antigen-specific lymphoproliferative responses, and this was most evident in mice that were primed and boosted with plasmid DNA. However, the induction of detectable antibodies in mice required two DNA vaccinations and a protein boost. In contrast to the effects on cell-mediated immunity, co-administration of IL-18-plasmid resulted in decreased antibody titers against viral proteins.

Interleukin-18 modulates immune responses induced by HIV-1 Nef DNA prime/protein boost vaccine

Many different HIV-1 vaccine strategies have been developed, but as yet none has been completely successful. Promising results from combined DNA prime/protein boost vaccines have been reported. Specific immune responses generated by DNA vaccines can be modulated by the co-delivery of genes coding for cytokines. In this study, we have used the intradermal route by needle injection of a plasmid coding for the HIV-1 Nef accessory protein. We show that DNA prime/protein boost vaccine combinations increase the humoral and cellular immune responses against HIV-1 Nef and that the co-injection of DNA encoding Interleukin-18 (IL-18) modulates the specific immune response towards a Th1 type.

Phenotype of recombinant Trypanosoma cruzi which overexpress elongation factor 1-γ: possible involvement of EF-1γ GST-like domain in the resistance to clomipramine

In previous studies, molecular and immunological approaches have been used to characterize the Trypansosoma cruzi elongation factor 1gamma (TcEF-1gamma). A primary sequence homology search revealed that the TcEF-1gamma N-terminal domain showed significant homology to glutathione S-transferases (GSTs). Although studies have suggested the involvement of EF-1gamma in the protein synthesis machinery, the exact function of this protein, particularly the role of its GST-like domain, is not fully understood. Therefore, we have used the protozoan parasite T. cruzi, as a recipient for a shuttle vector which allows overexpression of TcEF-1gamma in order to gain insight into its biological function. The growth of parasites which overexpress TcEF-1gamma and control cells was equally sensitive to inhibition by nifurtimox and benznidazole, which exert a trypanocidal activity through the production of free radicals. In contrast, a strong resistance of transformed organisms to the tricyclic antidepressant drug, clomipramine, a lipophilic compound, was observed, whereas control cells were highly sensitive. Our findings suggest that TcEF-1gamma participates in the detoxification of lipophilic compounds probably by conjugation with glutathione through its GST-like domain. To our knowledge, this is the first report showing that the eukaryotic EF-1gamma GST conserved enzymatic model could play a role in drug resistance. Furthermore, these results reinforce the notion that the aggressiveness of certain tumours could in part be linked to overexpression of EF-1gamma. They also raise a central question regarding the GST as target for chemotherapeutic drugs in cancer research.

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.