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Biomedical subjects

Marc Ouellette

Publications and source records attributed to Marc Ouellette.

44 records · Page 3Linked to original sources
PubMed2002

A new type of high affinity folic acid transporter in the protozoan parasite Leishmania and deletion of its gene in methotrexate-resistant cells.

The protozoan parasite Leishmania is a folate auxotroph and thus depends on the uptake of folate from the environment to meet its folate requirement. We show here that Leishmania contains several putative pteridine transporter genes. Some of these genes are deleted in methotrexate-resistant Leishmania cells where there is no measurable uptake of methotrexate. Transport studies suggest that Leishmania has more than one active folate transporter, and one of these, named FT5, corresponds to a very high affinity folate transporter (K(m) 84 nm). The uptake of both folate and methotrexate was impaired in an FT5 null mutant at low substrate concentrations (50 nm), although transport properties at higher concentrations (1000 nm) were not statistically different between wild-type and the FT5 null mutant. Modulation of the expression of FT5 also changes the susceptibility of Leishmania cells to methotrexate. These results have permitted the characterization of a novel class of folate transporters and suggest that the parasite Leishmania has several gene products possibly transporting folates and related molecules under varying conditions.

Amino Acid Sequence↗
PubMed2002

Involvement of zinc in the binding of Mycoplasma arthritidis-derived mitogen to the proximity of the HLA-DR binding groove regardless of histidine 81 of the beta chain.

Although our recent studies have provided the first evidence demonstrating the direct binding of Mycoplasma arthritidis-derived mitogen (MAM) to MHC class II molecules, it is not yet established how MAM interacts with these molecules. Herein, we demonstrate that MAM binds preferentially and with high affinity to HLA-DR molecules in a zinc-dependent manner. MAM's affinity (25 nM) for HLA-DR molecules is comparable to that of staphylococcal superantigens, and is slightly higher than that for murine MHC class II molecules expressed on the A20 B cell line (111 nM). The amino acid residues located between 14 - 31 and 76 - 90 of the MAM N-terminus play a critical role in MAM / HLA-DR interactions. Histidine at position 81 of the HLA-DR beta-chain, known to be critical for binding of zinc-coordinated superantigens, is not necessary for MAM / HLA-DR interactions. The HLA-DR residues involved in MAM binding are located in the proximal binding groove of the HLA-DR molecule, where the nature of the peptide of the binding groove plays an important role in MAM / HLA-DR interaction. This is the first detailed characterization of MAM's interactions with MHC class II molecules showing a mode of interaction with HLA-DR distinct from that of other superantigens.

Amino Acid Sequence↗
PubMed2002

A proteomic approach to identify developmentally regulated proteins in Leishmania infantum.

We used comparative two-dimensional gel electrophoresis (2-DE) and mass spectrometry methodologies to highlight and identify proteins that are differentially expressed in the intracellular stage of the parasite Leishmania donovani infantum, a causative agent of visceral leishmaniasis. During its digenetic life cycle, Leishmania alternates between the alimentary tract of the sandfly vector as an extracellular promastigote and the acidic phagolysosomes of macrophage cells as an intracellular amastigote. Proteins differentially expressed in the intracellular form of the parasite are thought to be important for intracellular survival and pathogenesis. We used narrow pH range strips for isoelectric focusing to resolve soluble proteins of both developmental stages of L. infantum. More than 62 proteins differentially expressed in amastigotes were detected among approximately 2000 protein spots resolved by 2-DE. A quadrupole time-of-flight analysis of few selected protein spots, specifically expressed in the amastigote stage, permitted the identification of two proteins, part of the energetic metabolism pathways, the isocitrate dehydrogenase and the glycolytic enzyme triosephosphate isomerase. The kinetic parameters of these two enzymes were measured in both developmental stages of the parasite and their activity was indeed found to be higher in amastigotes. These findings bring a new insight in our understanding of metabolic and energy requirements of the intracellular form of Leishmania. Comparative analysis of the proteome of both developmental stages of the protozoan parasite Leishmania should permit the identification of protein candidates for the development of vaccines and new drugs.

Amino Acid Sequence↗
PubMed2002

Pterin transport and metabolism in Leishmania and related trypanosomatid parasites.

The folate metabolic pathway has been exploited successfully for the development of antimicrobial and antineoplasic agents. Inhibitors of this pathway, however, are not useful against Leishmania and other trypanosomatids. Work on the mechanism of methotrexate resistance in Leishmania has dramatically increased our understanding of folate and pterin metabolism in this organism. The metabolic and cellular functions of the reduced form of folates and pterins are beginning to be established and this work has led to several unexpected findings. Moreover, the currently ongoing sequencing efforts on trypanosomatid genomes are suggesting the presence of several gene products that are likely to require folates and pterins. A number of the properties of folate and pterin metabolism are unique suggesting that these pathways are valid and worthwhile targets for drug development.

Animals↗
PubMed2002

Reduced infectivity of a Leishmania donovani biopterin transporter genetic mutant and its use as an attenuated strain for vaccination.

Pterins are essential for the growth of Leishmania species, and recent work has led to the isolation of the biopterin transporter BT1. In this study, we inactivated the Leishmania donovani biopterin transporter BT1 by gene disruption mediated by homologous recombination. No transport of biopterin was detected in this mutant. The L. donovani BT1 null mutant showed a much lesser capacity for inducing infection in mice than wild-type parasites and could elicit protective immunity in mice susceptible to infection against a L. donovani challenge. Splenocytes isolated from mice immunized with the BT1 null mutant parasites produced significant amounts of interferon gamma following stimulation with L. donovani promastigotes as measured by enzyme-linked immunosorbent assay and enzyme-linked immunospot assays. Overall, these results show that by genetically manipulating the pterin transport in L. donovani, it is possible to generate an attenuated organism that could be part of a vaccination strategy.

Animals↗
PubMedDate not supplied

Leishmaniasis: drugs in the clinic, resistance and new developments.

The control of Leishmania infections relies primarily on chemotherapy. The arsenal of drugs available for treating Leishmania infections is limited and includes pentavalent antimonials, pentamidine, amphotericin B, miltefosine, fluconazole and few other drugs at various stages of their development process. In this review, we will discuss the latest results regarding resistance mechanisms to drugs used in the clinic against Leishmania infections.

Animals↗
PubMedDate not supplied

Characterization of the folylpolyglutamate synthetase gene and polyglutamylation of folates in the protozoan parasite Leishmania.

Folates are polyglutamylated in most organisms by the enzyme folylpolyglutamate synthetase (FPGS). The Leishmania tarentolae FPGS gene was isolated. Its predicted product contains 538 amino acids and shows 33 and 30% identity with the human and yeast FPGS proteins, respectively. The level of folate polygtutamylation was studied in L. tarentolae promastigotes and in Leishmania infantum promastigotes and axenic amastigotes. In all species examined, folates were found predominantly as pentaglutamates, although monoglutamates were found in higher proportion in L. infantum axenic amastigote cells. Leishmania cells transfected with a FPGS containing plasmid (FPGS transfectant) exhibited a 6-fold increase in FPGS activity (32.7 pmol mg(-1) h(-1)) compared with wild-type cells (4.7 pmol mg(-1) h(-1)). HPLC analysis of the polyglutamylated forms of folates indicated a 2-fold increase of hexaglutamates in the FPGS transfectant compared with wild-type cells, while cells with one FPGS allele interrupted showed a higher proportion of short chain glutamates. The long-term accumulation of folates was greatly increased in the FPGS transfectant. Overall, this work indicates that FPGS activity is expressed in all forms of the parasite, and modulates the retention of folate, thereby possibly playing an important role in physiology.

Amino Acid Sequence↗