Mourad Barhoumi

Leishmania infantum LeIF protein is an ATP-dependent RNA helicase and an eIF4A-like factor that inhibits translation in yeast

LeIF, a Leishmania protein similar to the eukaryotic initiation factor eIF4A, which is a prototype of the DEAD box protein family, was originally described as a Th1‐type natural adjuvant and as an antigen that induces an IL12‐mediated Th1 response in the peripheral blood mononuclear cells of leishmaniasis patients. This study aims to characterize this protein by comparative biochemical and genetic analysis with eIF4A in order to assess its potential as a target for drug development. We show that a His‐tagged, recombinant, LeIF protein of Leishmania infantum, which was purified from Escherichia coli, is both an RNA‐dependent ATPase and an ATP‐dependent RNA helicase in vitro, as described previously for other members of the DEAD box helicase protein family. In vivo experiments show that the LeIF gene cannot complement the deletion of the essential TIF1 and TIF2 genes in the yeast Saccharomyces cerevisiae that encode eIF4A. In contrast, expression of LeIF inhibits yeast growth when endogenous eIF4A is expressed off only one of its two encoding genes. Furthermore, in vitro binding assays show that LeIF interacts with yeast eIF4G. These results show an unproductive interaction of LeIF with translation initiation factors in yeast. Furthermore, the 25 amino terminal residues were shown to enhance the ability of LeIF to interfere with the translation machinery in yeast.

Leishmania Eukaryotic Initiation Factor (LeIF) Inhibits Parasite Growth in Murine Macrophages

The leishmaniases constitute neglected global public health problems that require adequate control measures, prophylactic clinical vaccines and effective and non-toxic drug treatments. In this study, we explored the potential of Leishmania infantum eukaryotic initiation factor (LieIF), an exosomal protein, as a novel anti-infective therapeutic molecule. More specifically, we assessed the efficacy of recombinant LieIF, in combination with recombinant IFN-γ, in eliminating intracellular L. donovani parasites in an in vitro macrophage model. J774A.1 macrophages were initially treated with LieIF/IFN-γ prior to in vitro infection with L. donovani stationary phase promastigotes (pre-infection treatment), and resistance to infection was observed 72 h after infection. J774A.1 macrophages were also treated with LieIF/IFN-γ after L. donovani infection (post-infection treatment), and resistance to infection was also observed at both time points tested (19 h and 72 h) after infection. To elucidate the LieIF/IFN-γ-induced mechanism(s) that mediate the reduction of intracellular parasite growth, we examined the generation of potent microbicidal molecules, such as nitric oxide (NO) and reactive oxygen species (ROS), within infected macrophages. Furthermore, macrophages pre-treated with LieIF/IFN-γ showed a clear up-regulation in macrophage inflammatory protein 1α (MIP-1α) as well as tumor necrosis factor alpha (TNF-α) expression. However, significant different protein levels were not detected. In addition, macrophages pre-treated with LieIF/IFN-γ combined with anti-TNF-α monoclonal antibody produced significantly lower amounts of ROS. These data suggest that during the pre-treatment state, LieIF induces intramacrophage parasite growth inhibition through the production of TNF-α, which induces microbicidal activity by stimulating NO and ROS production. The mechanisms of NO and ROS production when macrophages are treated with LieIF after infection are probably different. Overall, these results indicate that LieIF is a good candidate for use as an anti-leishmanial molecule.

Leishmania infantum LeIF and its recombinant polypeptides modulate interleukin IL-12p70, IL-10 and tumour necrosis factor-α production by human monocytes

Leishmania eukaryotic initiation factor (LeIF) antigen, a Leishmania protein, was shown to induce IL‐12, IL‐10 and tumour necrosis factor‐α (TNF‐α) production by human monocytes‐derived macrophages and dendritic cells from healthy individuals. This cytokine‐inducing activity was previously found to be located in the amino‐terminal region of LeIF protein. This study aimed at characterizing the cytokine‐inducing activity of Leishmania infantum LeIF [Leishmania (L.) infantum (LieIF)] and at defining the fragments necessary for inducing cytokine secretion. Eleven rationally designed recombinant polypeptides, corresponding to the entire LeIF protein or parts of it, were expressed and used to stimulate monocytes from healthy individuals. Leishmania (L.) infantum was able to induce IL‐12p70, IL‐10 and TNF‐α secretion in human monocytes. In addition, both amino‐ (1–226) and carboxyl‐terminal (196–403) parts of the protein were shown to induce significant levels of the three cytokines analysed. However, IL‐12p70‐inducing activity was not significant when monocytes were stimulated with the fragments 129–226 and 129–261, inferring that IL‐12p70‐inducing activity was primarily located within amino acids 1–129 and 261–403. Although the full‐length LieIF protein was a more potent inducer than the tested fragments, a significant cytokine‐inducing activity was maintained in smaller amino acid regions. This work suggests that cytokine‐inducing activity of LieIF or its parts could be exploited in vaccination or immunotherapy protocols.

Bioinformatic characterizations and prediction of K+ and Na+ ion channels effector toxins.

Background K+ and Na+ channel toxins constitute a large set of polypeptides, which interact with their ion channel targets. These polypeptides are classified in two different structural groups. Recently a new structural group called birtoxin-like appeared to contain both types of toxins has been described. We hypothesized that peptides of this group may contain two conserved structural motifs in K+ and/or Na+ channels scorpion toxins, allowing these birtoxin-like peptides to be active on K+ and/or Na+ channels. Results Four multilevel motifs, overrepresented and specific to each group of K+ and/or Na+ ion channel toxins have been identified, using GIBBS and MEME and based on a training dataset of 79 sequences judged as representative of K+ and Na+ toxins. Unexpectedly birtoxin-like peptides appeared to present a new structural motif distinct from those present in K+ and Na+ channels Toxins. This result, supported by previous experimental data, suggests that birtoxin-like peptides may exert their activity on different sites than those targeted by classic K+ or Na+ toxins. Searching, the nr database with these newly identified motifs using MAST, retrieved several sequences (116 with e-value < 1) from various scorpion species (test dataset). The filtering process left 30 new and highly likely ion channel effectors. Phylogenetic analysis was used to classify the newly found sequences. Alternatively, classification tree analysis, using CART algorithm adjusted with the training dataset, using the motifs and their 2D structure as explanatory variables, provided a model for prediction of the activity of the new sequences. Conclusion The phylogenetic results were in perfect agreement with those obtained by the CART algorithm. Our results may be used as criteria for a new classification of scorpion toxins based on functional motifs.BackgroundK+ and Na+ channel toxins constitute a large set of polypeptides, which interact with their ion channel targets. These polypeptides are classified in two different structural groups. Recently a new structural group called birtoxin-like appeared to contain both types of toxins has been described. We hypothesized that peptides of this group may contain two conserved structural motifs in K+ and/or Na+ channels scorpion toxins, allowing these birtoxin-like peptides to be active on K+ and/or Na+ channels.ResultsFour multilevel motifs, overrepresented and specific to each group of K+ and/or Na+ ion channel toxins have been identified, using GIBBS and MEME and based on a training dataset of 79 sequences judged as representative of K+ and Na+ toxins.Unexpectedly birtoxin-like peptides appeared to present a new structural motif distinct from those present in K+ and Na+ channels Toxins. This result, supported by previous experimental data, suggests that birtoxin-like peptides may exert their activity on different sites than those targeted by classic K+ or Na+ toxins.Searching, the nr database with these newly identified motifs using MAST, retrieved several sequences (116 with e-value < 1) from various scorpion species (test dataset). The filtering process left 30 new and highly likely ion channel effectors.Phylogenetic analysis was used to classify the newly found sequences. Alternatively, classification tree analysis, using CART algorithm adjusted with the training dataset, using the motifs and their 2D structure as explanatory variables, provided a model for prediction of the activity of the new sequences.ConclusionThe phylogenetic results were in perfect agreement with those obtained by the CART algorithm.Our results may be used as criteria for a new classification of scorpion toxins based on functional motifs.

DEAD-box proteins, like Leishmania eIF4A, modulate interleukin (IL)-12, IL-10 and tumour necrosis factor-alpha production by human monocytes.

Previously we showed that His‐tagged, recombinant, Leishmania infantum eukaryotic initiation factor (LeIF) was both an RNA‐dependent ATPase and an ATP‐dependent RNA helicase in vitro, as described for other members of the DEAD‐box helicase family. In addition, we showed that LeIF induces the production of IL‐12, IL‐10, and TNF‐α by human monocytes. This study aims to characterize the cytokine‐inducing activity in human monocytes of several proteins belonging to the DEAD‐box family from mammals and yeast. All tested proteins contained the 11 conserved motifs (Q, I, Ia, GG Ib, II, III, IV, QxxR, V and VI) characteristic of DEAD‐box proteins, but they have different biological functions and different percentages of identities with LeIF. We show that these mammalian or yeast recombinant proteins also are able to induce IL‐12, IL‐10 and TNF‐α secretion by monocytes of healthy human subjects. This cytokine‐inducing activity is proteinase K sensitive and polymyxin B resistant. Our results show that the induction of cytokines in human monocytes is not unique to the protein LeIF of Leishmania, and it suggests that the activity of certain DEAD‐box proteins can be exploited as adjuvant and/or to direct immune responses towards a Th1 profile in vaccination or immunotherapy protocols.

LiEIF and its recombinant polypeptides enhance the maturation of mouse dendritic cells and the production of the protective IL-12 cytokine

Dendritic cells (DCs) maturation is associated with upregulation of costimulatory molecules (CD80, CD86, CD40) and secretion of cytokines including IL-12 which is important for generation of effective T cells. Proteins of Leishmania parasite that stimulate the production of IL-12 could be of significant interest either as immunotherapeutic components for leishmaniasis or as adjuvants. LeIF (Leishmania eukaryotic initiation factor) belongs to this group of proteins since it induces the production of IL-12, IL-10 and TNF-α by human monocytes of healthy volunteers. In particular, the induction of cytokines appears to be located in the N-terminal part (1-226) of the protein. In the present study we evaluated the ability of the recombinant protein L. infantum eIF (LieIF) and its constructs to induce in vitro the maturation of myeloid DCs (mDCs) and the production of cytokines supporting the polarization of Th1 type immune response. For this purpose, we used five synthetic peptides (16-18 aa) belonging in the N-terminal region, eight overlapped recombinant polypeptides covering the full protein sequence and the full-length protein. Enriched mDCs were obtained by ex vivo expansion of bone marrow cells, from BALB/c mice, cultured with the hematopoietic factor GM-CSF. The incubation of mDCs with the recombinant polypeptides, led to their maturation since it was observed a significant augmentation of the percentage of mDCs that express the molecules CD40, CD80 and CD86. On the contrary, the synthetic peptides did not enhance CD40 and CD86 expression and drove only to significant augmentation of CD80. In addition, we evaluated the ability of LieIF and its polypeptides to induce the production of IL-12, IL-10 and the expression of iNOS by mouse mDCs. We determined an augmentation of the percentage of mDCs that produce IL-12 upon their incubation with LieIF as well as with all the recombinant polypeptides and with three synthetic peptides whereas negligible amount of IL-10 was obtained. Augmentation of iNOS was found after incubation with the entire molecule of LieIF and with the recombinant polypeptides. In conclusion, LieIF and some of its recombinant polypeptides seem to have immunomodulatory properties demonstrating their potential use as therapeutic and prophylactic vaccine antigens.

In silico analysis and in vitro evaluation of immunogenic and immunomodulatory properties of promiscuous peptides derived from Leishmania infantum eukaryotic initiation factor

It is generally considered as imperative the ability to control leishmaniasis through the development of a protective vaccine capable of inducing long-lasting and protective cell-mediated immune responses. In this current study, we demonstrated potential epitopes that bind to H2 MHC class I and II molecules by conducting the in silico analysis of Leishmania infantum eukaryotic Initiation Factor (LieIF) protein, using online available algorithms. Moreover, we synthesized five peptides (16-18 amino acids long) which are part of the N-terminal portion of LieIF and contain promising MHC class I and II-restricted epitopes and afterwards, their predicted immunogenicity was evaluated in vitro by monitoring peptide-specific T-cell responses. Additionally, the immunomodulatory properties of these peptides were investigated in vitro by exploring their potential of inducing phenotypic maturation and functional differentiation of murine Bone-Marrow derived Dendritic Cells (BM-DCs). It was revealed by our data that all the synthetic peptides predicted for H2 alleles; present the property of immunogenicity. Among the synthetic peptides which contained T-cell epitopes, the peptide 52-68 aa (LieIF_2) exhibited immunomodulatory properties with the larger potential. LieIF_2-pulsed BM-DCs up-regulated the expression of the co-stimulatory surface molecules CD80 and CD86, as well as the production of the proinflammatory cytokine TNF-α and of the Th1-polarizing cytokines IL-12 and IFN-γ. The aforementioned data suggest that selected parts of LieIF could be used to develop innovative subunit protective vaccines able to induce effective immunity mediated by MHC class I-restricted as well as class II-restricted T-cell responses.

The steroid derivative 6-aminocholestanol inhibits the DEAD-box helicase eIF4A (LieIF4A) from the Trypanosomatid parasite Leishmania by perturbing the RNA and ATP binding sites

The antifungal agent 6-aminocholestanol targets the production of ergosterol, which is the principle sterol in many fungi and protozoans; ergosterol serves many of the same roles as cholesterol in animals. We found that it also is an effective inhibitor of the translation-initiation factor eIF4AI from mouse (eIF4AIMus) and the Trypanosomatid parasite Leishmania (LieIF4A). The eIF4A proteins belong to the DEAD-box family of RNA helicases, which are ATP-dependent RNA-binding proteins and RNA-dependent ATPases. DEAD-box proteins contain a commonly-shared core structure consisting of two linked domains with structural homology to that of recombinant protein A (RecA) and that contain conserved motifs that are involved in RNA and ATP binding, and in the enzymatic activity. The compound inhibits both the ATPase and helicase activities by perturbing ATP and RNA binding, and it is capable of binding other proteins containing nucleic acid-binding sites as well. We undertook kinetic analyses and found that the Leishmania LieIF4A protein binds 6-aminocholestanol with a higher apparent affinity than for ATP, although multiple binding sites were probably involved. Competition experiments with the individual RecA-like domains indicate that the primary binding sites are on RecA-like domain 1, and they include a cavity that we previously identified by molecular modeling of LieIF4A that involve conserved RNA-binding motifs. The compound affects the mammalian and Leishmania proteins differently, which indicates the binding sites and affinities are not the same. Thus, it is possible to develop drugs that target DEAD-box proteins from different organisms even when they are implicated in the same biological process.

Leishmania infantum LeIF protein is an eIF4A-like RNA helicase that modulates interleukin IL-12p70, IL-10 and TNF-α production in human monocytes

Leishmania LeIF antigen, homologous to eukaryotic initiation factor eIF4A, was originally described as a Th1-type natural adjuvant and as an antigen that induces an IL-12-mediated Th1 response in the peripheral blood mononuclear cells (PBMC) of leishmaniasis patients. We aimed at addressing the role of this protein and defining the minimum fragment necessary for inducing cytokine secretion. The study necessitated expression cloning of LeIF and 9 domains. Comparative biochemical and genetic analyses of LeIF and yeast eIF4A showed that LieIF is both an RNA-dependent ATPase and ATP-dependent RNA helicase in vitro and highlighted differences with yeast protein. In vivo experiments in yeast showed that LeIF cannot complement the deletion of the essential TIF1 and TIF2 genes in the yeast Saccharomyces cerevisiae that encode eIF4A. However, expression of LeIF results in a dominant negative phenotype, which is abolished by deletion of the most divergent 25 N-terminal residues. LeIF is able to interact with yeast eIF4G; this suggested a role in the translation machinery. The assays measuring production of cytokines IL-12p70, IL-10 and TNF-α by PBMC-derived monocytes of healthy donors exposed to the different proteins showed that LieIF was able to induce the secretion of these cytokines. Unlike previous reports on LeIF from L. braziliensis and L. major, both amino and carboxyl parts of the protein were shown to induce the secretion of cytokines at significant levels. Our results suggest that this activity could be primarily located in amino acids 1-129 and 261-403. Furthermore, the induction of cytokines in monocytes of healthy subjects is not unique to the Leishmania protein. Indeed, 5 homologous proteins DEAD box in mammals and yeast were also able to induce the secretion of cytokines. This study confirms the importance of LeIF protein as a vaccine target and underscores its potential as drug target.