Biomedical subjects

Marc Ouellette

Publications and source records attributed to Marc Ouellette.

At least 19 recordsLinked to original sources

PubMed2006

Stage specific gene expression and cellular localization of two isoforms of the serine hydroxymethyltransferase in the protozoan parasite Leishmania.

Serine hydroxymethyltransferase (SHMT) catalyses the reversible conversion of serine and tetrahydrofolate to glycine and methylene-tetrahydrofolate. The recent completion of the genome sequence of Leishmania major revealed the presence of two genes coding for two isoforms of this protein. In silico analysis showed that one isoform had an extension at its N-terminus and was predicted to localize to the mitochondrion. The situation is different in other kinetoplastid parasites with only one SHMT encoding gene in Trypanosoma cruzi and no SHMT encoding gene in Trypanosoma brucei. The two L. major SHMT genes were cloned in frame with the green fluorescent protein and the resulting fusion proteins showed differential localization: the short form (SHMT-S) was found in the cytosol while the long one (SHMT-L) was found in an organelle that has hallmarks of the parasite mitochondrion. Indeed, SHMT-L had a similar cellular fractionation pattern as the mitochondrial HSP60 as determined by digitonin fractionation. Both SHMT-S and SHMT-L genes were expressed preferentially in the amastigote stage of the parasite and the RNA levels of SHMT-L could be modulated by glycine, serine, and folate. Overexpression of SHMT-S increased resistance to the antifolate methotrexate and to a lower level to the inhibitor thiosemicarbazide in a rich folate containing medium. These findings suggest that folate metabolism is compartmentalised in Leishmania and that SHMT RNA levels are responsive to environmental conditions.

Amino Acid Sequence↗

PubMed2006

Unresponsiveness to Glucantime treatment in Iranian cutaneous leishmaniasis due to drug-resistant Leishmania tropica parasites.

BACKGROUND: Recent circumstantial evidence suggests that an increasing number of Iranian patients with cutaneous leishmaniasis are unresponsive to meglumine antimoniate (Glucantime), the first line of treatment in Iran. This study was designed to determine whether the clinical responses (healing, or non-healing) were correlated with the susceptibility of Leishmania parasites to Glucantime. METHODS AND FINDINGS: In vitro susceptibility testing was first performed on 185 isolated parasites in the intracellular mouse peritoneal macrophage model. A strong correlation between the clinical outcome and the in vitro effective concentration 50% (EC50) values was observed. Parasites derived from patients with non-healing lesions had EC50 values at least 4-fold higher than parasites derived from lesions of healing patients. A selection of these strains was typed at the molecular level by pulsed-field gels and by sequencing the pteridine reductase 1 (PTR1) gene. These techniques indicated that 28 out of 31 selected strains were Leishmania tropica and that three were Leishmania major. The L. major isolates were part of a distinct pulsed-field group, and the L. tropica isolates could be classified in three related additional pulsed-field groups. For each pulsed-field karyotype, we selected sensitive and resistant parasites in which we transfected the firefly luciferase marker to assess further the in vitro susceptibility of field isolates in the monocyte cell line THP1. These determinations confirmed unequivocally that patients with non-healing lesions were infected with L. tropica parasites resistant to Glucantime. Additional characterization of the resistant isolates showed that resistance is stable and can be reversed by buthionine sulfoximine, an inhibitor of glutathione biosynthesis. CONCLUSIONS: To the authors' knowledge, this is the first report of proven resistant parasites contributing to treatment failure for cutaneous leishmaniasis and shows that primary Glucantime-resistant L. tropica field isolates are now frequent in Iran.

Adult↗

PubMed2006

Resistance to antimony and treatment failure in human Leishmania (Viannia) infection.

BACKGROUND: Failure of antimonial therapy has been increasingly reported in anthroponotic visceral leishmaniasis and in cutaneous disease. The role of drug resistance in treatment failure has been difficult to ascertain because therapeutic response is multifactorial, and the efficacy of antimonial drugs depends on an effective immune response. In this study, we sought to determine whether standard treatment selects for resistant organisms and whether drug resistance contributes to treatment failure. METHODS: We evaluated the susceptibility to antimony of 19 strains isolated before treatment with meglumine antimoniate and 21 strains isolated at treatment failure from 20 patients. The 50% effective dose (ED50) of antimony in the form of additive-free meglumine antimoniate was determined for intracellular amastigotes in human promonocytic U-937 cells. RESULTS: Before treatment, 16% of strains (3/19) showed primary resistance (ED50 of >128 microg Sb/mL), whereas 84% (16/19) were susceptible (ED50 of <20 microg Sb/mL). However, 88% of susceptible strains (14/16) had ED90 values of >128 microg Sb/mL. At treatment failure, 40% of strains (8/20) were resistant. Secondary resistance was documented in 4 patients. CONCLUSIONS: Primary and secondary resistance to antimony can contribute to treatment failure in American cutaneous leishmaniasis. Selection for resistance to antimony occurs during standard treatment with antimonial drugs, and primary resistance to antimony supports the plausibility of anthroponotic transmission.

Adolescent↗

PubMed2006

Identification of developmentally-regulated proteins in Leishmania panamensis by proteome profiling of promastigotes and axenic amastigotes.

We have employed proteomics to identify proteins upregulated in the amastigote life-stage of Leishmaniapanamensis, using axenically-differentiated forms as models of authentic intracellular parasites. Resolution of the soluble proteomes of axenic amastigotes and promastigotes by two-dimensional electrophoresis (2DE) in the neutral pI range (5-7) revealed equivalent numbers of protein spots in both life-stages (644-682 using Coomassie Blue and 851-863 by silver staining). Although representing a relatively low proportion (8.1-10.8%) of the predicted 8000 gene products of Leishmania, these proteome maps enabled the reproducible detection of 75 differentially-regulated protein spots in amastigotes, comprising 24 spots "uniquely" expressed in this life-stage and 51 over-expressed by 1.2-5.7-fold compared to promastigotes. Of the 11 amastigote-specific spots analysed by mass spectrometry (MS), 5 yielded peptide sequences with no orthologues in Leishmania major, and the remaining 6 were identified as 7 distinct proteins (some of which were truncated isoforms) representing several functional classes: carbohydrate/energy metabolism (fructose 1,6-bisphosphate aldolase, glucose 6-phosphate dehydrogenase, pyruvate dehydrogenase), stress response (heat shock protein [HSP] 83), cell membrane/cytoskeleton (beta-tubulin), amino acid metabolism (cysteine synthase) and cell-cycle (ran-binding protein). Four additional over-expressed spots were tentatively identified as HSPs 60 and 70 and HSP 70-related proteins -1 and -4 by positional analogy with these landmark proteins in the Leishmania guyanensis proteome. Our data demonstrate the feasibility of proteomics as an approach to identify novel developmentally-regulated proteins linked to Leishmania differentiation and intracellular survival, while simultaneously pinpointing therapeutic targets. In particular, the amastigote-specific expression of cysteine synthase underlines the importance of de novo cysteine synthesis both as a potential parasite virulence factor and as a major metabolic difference from mammalian host cells.

Amino Acid Sequence↗

PubMed2006

A combined proteomic and transcriptomic approach to the study of stage differentiation in Leishmania infantum.

Protozoan parasites of the genus Leishmania are found as promastigotes in the sandfly vector and as amastigotes in mammalian macrophages. Mechanisms controlling stage-regulated gene expression in these organisms are poorly understood. Here, we applied a comprehensive approach consisting of protein prefractionation, global proteomics and targeted DNA microarray analysis to the study of stage differentiation in Leishmania. By excluding some abundant structural proteins and reducing complexity, we detected and identified numerous novel differentially expressed protein isoforms in L. infantum. Using 2-D gels, over 2200 protein isoforms were visualized in each developmental stage. Of these, 6.1% were strongly increased or appeared unique in the promastigote stage, while the relative amounts of 12.4% were increased in amastigotes. Amastigote-specific protein isoform and mRNA expression trends correlated modestly (53%), while no correlation was found for promastigote-specific spots. Even where direction of regulation was similar, fold-changes were more modest at the RNA than protein level. Many proteins were present in multiple spots, suggesting that PTM is extensive in this organism. In several cases, different isoforms appeared to be specific to different life stages. Our results suggest that post-transcriptional controls at translational and post-translational levels could play major roles in differentiation in Leishmania parasites.

Animals↗

PubMed2006

Prefractionation by digitonin extraction increases representation of the cytosolic and intracellular proteome of Leishmania infantum.

Proteome coverage is limited by the dynamic range of proteins present in a sample and often is confined to the analysis of abundant proteins. We have developed a protein prefractionation protocol, based on the differential solubilization of membranes using digitonin, that has allowed an increase in the resolution and depth of comparative proteomic studies. This prefractionation protocol can also be used to infer the subcellular localization of hypothetical proteins as tested experimentally using green fluorescent fusion proteins. The abundant tubulins and associated proteins of the cytoskeleton were removed from the sample using digitonin extraction, hence facilitating the visualization of lower abundance proteins. The digitonin prefractionation protocol was applied for a comparative proteomic analysis of the promastigote and amastigote life cycle stages of Leishmania infantum and has allowed the identification of novel proteins expressed in a stage-specific manner.

Animals↗

PubMed2006

In primary human monocyte-derived macrophages exposed to Human immunodeficiency virus type 1, does the increased intracellular growth of Leishmania infantum rely on its enhanced uptake?

Concurrent uncontrolled development of human immunodeficiency virus type 1 (HIV-1) and Leishmania spp. is regarded as an emerging pathogenic combination in countries where human beings are exposed to these two micro-organisms. The present study was aimed at exploring whether HIV-1 development within a culture of human monocyte-derived macrophages (MDMs) affected the further development of luciferase-encoding Leishmania infantum using the luciferase activity as a readout assay. It was demonstrated that, in cultures of HIV-1-loaded MDMs exposed to axenic amastigotes, the luciferase activity was higher than in HIV-1-free MDMs. As a preliminary approach to deciphering the possible mechanism through which HIV-1 can affect Leishmania infantum, attention was focused on the very early processes that could underlie this increased luciferase activity. Using GFP-labelled parasites, it was possible to establish that, in HIV-1-infected MDMs, the percentage of GFP-expressing MDMs was higher (10-20 %) than in cell cultures not exposed to HIV-1 (5 %). Two-colour immunofluorescence staining suggested that HIV-1 indirectly affects the uptake of parasites inside MDMs. Thus, the observed phenomenon seems to be linked with a higher uptake of parasites within MDMs. Taken together, the data reported here may contribute to our understanding of disseminated Leishmania infection in HIV-1-infected individuals.

Animals↗

PubMed2005

Microfluidic device for rapid (<15 min) automated microarray hybridization.

BACKGROUND: Current hybridization protocols on microarrays are slow and need skilled personnel. Microfluidics is an emerging science that enables the processing of minute volumes of liquids to perform chemical, biochemical, or enzymatic analyzes. The merging of microfluidics and microarray technologies constitutes an elegant solution that will automate and speed up microarray hybridization. METHODS: We developed a microfluidic flow cell consisting of a network of chambers and channels molded into a polydimethylsiloxane substrate. The substrate was aligned and reversibly bound to the microarray printed on a standard glass slide to form a functional microfluidic unit. The microfluidic units were placed on an engraved, disc-shaped support fixed on a rotational device. Centrifugal forces drove the sample and buffers directly onto the microarray surface. RESULTS: This microfluidic system increased the hybridization signal by approximately 10fold compared with a passive system that made use of 10 times more sample. By means of a 15-min automated hybridization process, performed at room temperature, we demonstrated the discrimination of 4 clinically relevant Staphylococcus species that differ by as little as a single-nucleotide polymorphism. This process included hybridization, washing, rinsing, and drying steps and did not require any purification of target nucleic acids. This platform was sensitive enough to detect 10 PCR-amplified bacterial genomes. CONCLUSION: This removable microfluidic system for performing microarray hybridization on glass slides is promising for molecular diagnostics and gene profiling.

DNA↗

PubMed2005

Phylogeny of the Enterobacteriaceae based on genes encoding elongation factor Tu and F-ATPase beta-subunit.

The phylogeny of enterobacterial species commonly found in clinical samples was analysed by comparing partial sequences of their elongation factor Tu gene (tuf) and of their F-ATPase beta-subunit gene (atpD). An 884 bp fragment for tuf and an 884 or 871 bp fragment for atpD were sequenced for 96 strains representing 78 species from 31 enterobacterial genera. The atpD sequence analysis exhibited an indel specific to Pantoea and Tatumella species, showing, for the first time, a tight phylogenetic affiliation between these two genera. Comprehensive tuf and atpD phylogenetic trees were constructed and are in agreement with each other. Monophyletic genera are Cedecea, Edwardsiella, Proteus, Providencia, Salmonella, Serratia, Raoultella and Yersinia. Analogous trees based on 16S rRNA gene sequences available from databases were also reconstructed. The tuf and atpD phylogenies are in agreement with the 16S rRNA gene sequence analysis, and distance comparisons revealed that the tuf and atpD genes provide better discrimination for pairs of species belonging to the family Enterobacteriaceae. In conclusion, phylogeny based on tuf and atpD conserved genes allows discrimination between species of the Enterobacteriaceae.

Amino Acid Sequence↗

PubMed2005

Modulation in aquaglyceroporin AQP1 gene transcript levels in drug-resistant Leishmania.

Antimonial-containing drugs are the first line of treatment against the parasite Leishmania. Resistance to antimonials has been correlated to its reduced accumulation. We used a dominant negative functional cloning strategy where a Leishmania mexicana expression cosmid bank was transfected in cells resistant to trivalent antimony (SbIII). Cells were selected for increased sensitivity to SbIII. One cosmid was isolated that could bestow SbIII sensitivity to resistant cells. The gene part of this cosmid that is responsible for increased SbIII sensitivity corresponds to AQP1, an aquaglyceroporin. AQP1 was recently shown to be a route by which SbIII can accumulate in Leishmania cells. Transport studies have shown that the L. mexicana AQP1 can restore SbIII transport in resistant cells. Southern blot analysis indicated that the copy number of neither the AQP1 gene nor the other AQP homologues was changed in antimony-resistant mutants of several Leishmania species. The AQP1 gene sequence was also unchanged in mutants. However, the AQP1 RNA levels were downregulated in several Leishmania promastigote species resistant to antimonials. In general, but not always, the level of AQP1 transcript levels correlated well with the accumulation of SbIII and resistance levels in Leishmania cells. AQP1 thus appears to be a key determinant of antimonials accumulation and susceptibility in Leishmania.

Amino Acid Sequence↗

PubMed2005

A proteomic analysis of penicillin resistance in Streptococcus pneumoniae reveals a novel role for PstS, a subunit of the phosphate ABC transporter.

Resistance to penicillin is widespread in the Gram-positive bacterium Streptococcus pneumoniae, and while several mutations are known to be implicated in resistance other mechanisms are likely to occur. We used a proteomic screen of two independent mutants in which resistance was selected in vitro. We found a number of differentially expressed proteins including PstS, a subunit of the phosphate ABC transporter of S. pneumoniae. This protein was increased in both mutants, a phenotype correlated to increased RNA expression of the entire phosphate ABC transporter operon. Inactivation of the pstS gene led to increased susceptibility to penicillin in the wild-type strain. To further link the expression of the ABC phosphate transporter with penicillin resistance, we looked at pstS mRNA levels in 12 independent clinical isolates sensitive and resistant to penicillin and found an excellent correlation between resistance and increased expression of pstS. Inactivation of pstS in one of the clinical isolates significantly reduced penicillin resistance. Global approaches are ideally suited for the discovery of novel factors in the biology of resistance.

ATP-Binding Cassette Transporters↗

PubMed2005

Role of the ABC transporter MRPA (PGPA) in antimony resistance in Leishmania infantum axenic and intracellular amastigotes.

Antimonial compounds are the mainstay for the treatment of infections with the protozoan parasite Leishmania. We present our studies on Leishmania infantum amastigote parasites selected for resistance to potassium antimonyl tartrate [Sb(III)]. Inside macrophages, the Sb(III)-selected cells are cross-resistant to sodium stibogluconate (Pentostam), the main drug used against Leishmania. Putative alterations in the level of expression of more than 40 genes were compared between susceptible and resistant axenic amastigotes using customized DNA microarrays. The expression of three genes coding for the ABC transporter MRPA (PGPA), S-adenosylhomocysteine hydrolase, and folylpolyglutamate synthase was found to be consistently increased. The levels of cysteine were found to be increased in the mutant. Transfection of the MRPA gene was shown to confer sodium stibogluconate resistance in intracellular parasites. This MRPA-mediated resistance could be reverted by using the glutathione biosynthesis-specific inhibitor buthionine sulfoximine. These results highlight for the first time the role of MRPA in antimony resistance in the amastigote stage of the parasite and suggest a strategy for reversing resistance.

ATP-Binding Cassette Transporters↗

PubMed2005

Live nonpathogenic parasitic vector as a candidate vaccine against visceral leishmaniasis.

To date, there are no proven vaccines against any form of leishmaniasis. The development of live attenuated vectors shows promise in the field of Leishmania vaccination because these organisms mimic more effectively the course of real infections and can elicit potent activation of the immune system. In the present study, we investigated the potential of a parasitic protozoan that is nonpathogenic to humans, Leishmania tarentolae, as a live candidate vaccine that efficiently targets dendritic cells and lymphoid organs, thus enhancing antigen presentation and consequently influencing the magnitude and quality of T-cell immune responses. We demonstrated that L. tarentolae activates the dendritic cell maturation process and induces T-cell proliferation and the production of gamma interferon, thus skewing CD4(+) T cells toward a Th1 cell phenotype. More importantly, we found that a single intraperitoneal injection of L. tarentolae could elicit a protective immune response against infectious challenge with Leishmania donovani in susceptible BALB/c mice. These results suggest that the use of L. tarentolae as a live vaccine vector may represent a promising approach for improving the effectiveness and safety of candidate live vaccines against Leishmania infections and possibly other intracellular pathogens for which T-cell mediated responses are critical for the development of protective immunity.

Animals↗

PubMed2005

Correlation between microarray DNA hybridization efficiency and the position of short capture probe on the target nucleic acid.

The hybridization behavior of small oligonucleotides arrayed on glass slides is currently unpredictable. In order to examine the hybridization efficiency of capture probes along target nucleic acid, 20-mer oligonucleotide probes were designed to hybridize at different distances from the 5' end of two overlapping 402- and 432-bp ermB products amplified from the target DNA. These probes were immobilized via their 5' end onto glass slides and hybridized with the two labeled products. Evaluation of the hybridization signal for each probe revealed an inverse correlation with the length of the 5' overhanging end of the captured strand and the hybridization signal intensity. Further experiments demonstrated that this phenomenon is dependent on the reassociation kinetics of the free overhanging tail of the captured DNA strand with its complementary strand. This study delineates key predictable parameters that govern the hybridization efficiency of short capture probes arrayed on glass slides. This should be most useful for designing arrays for detection of PCR products and nucleotide polymorphisms.

Benchmarking↗

PubMed2004

Growth phase regulation of the main folate transporter of Leishmania infantum and its role in methotrexate resistance.

The protozoan parasite Leishmania relies on the uptake of folate and pterin from the environment to meet its nutritional requirements. We show here that a novel gene (folate transporter 1 (FT1)) deleted in a Leishmania infantum methotrexate-resistant mutant corresponds to the main folate transporter (K(m), 410 nM). FT1 was established as the main folate transporter by both gene transfection and by targeted gene deletion. Modulation of the expression of FT1 by these manipulations altered the susceptibility of Leishmania cells to methotrexate. Folate transport was stage-regulated with higher activity in the logarithmic phase and less in the stationary phase. FT1 fused to green fluorescent protein led to the observation that FT1 was located in the plasma membrane in the logarithmic phase but was retargeted to an intracellular organelle followed by a degradation of the protein in stationary phase. Leishmania has several folate transporters with different characteristics, and the growth stage-related activity of at least one transporter is regulated post-translationally.

Amino Acid Sequence↗

PubMed2004

Leishmania major LmACR2 is a pentavalent antimony reductase that confers sensitivity to the drug pentostam.

Arsenicals and antimonials are first line drugs for the treatment of trypanosomal and leishmanial diseases. To create the active form of the drug, Sb(V) must be reduced to Sb(III). Because arsenic and antimony are related metalloids, and arsenical resistant Leishmania strains are frequently cross-resistant to antimonials, we considered the possibility that Sb(V) is reduced by a leishmanial As(V) reductase. The sequence for the arsenate reductase of Saccharomyces cerevisiae, ScAcr2p, was used to clone the gene for a homologue, LmACR2, from Leishmania major. LmACR2 was able to complement the arsenate-sensitive phenotype of an arsC deletion strain of Escherichia coli or an ScACR2 deletion strain of Saccharomyces cerevisiae. Transfection of Leishmania infantum with LmACR2 augmented Pentostam sensitivity in intracellular amastigotes. LmACR2 was purified and shown to reduce both As(V) and Sb(V). This is the first report of an enzyme that confers Pentostam sensitivity in intracellular amastigotes of Leishmania. We propose that LmACR2 is responsible for reduction of the pentavalent antimony in Pentostam to the active trivalent form of the drug in Leishmania.

Amino Acid Sequence↗

PubMed2004

Differential protein expression analysis of Leishmania major reveals novel roles for methionine adenosyltransferase and S-adenosylmethionine in methotrexate resistance.

Leishmania is a trypanosomatid parasite causing serious disease and displaying resistance to various drugs. Here, we present comparative proteomic analyses of Leishmania major parasites that have been either shocked with or selected in vitro for high level resistance to the model antifolate drug methotrexate. Numerous differentially expressed proteins were identified by these experiments. Some were associated with the stress response, whereas others were found to be overexpressed due to genetic linkage to primary resistance mediators present on DNA amplicons. Several proteins not previously associated with resistance were also identified. The role of one of these, methionine adenosyltransferase, was confirmed by gene transfection and metabolite analysis. After a single exposure to low levels of methotrexate, L. major methionine adenosyltransferase transfectants could grow at high concentrations of the drug. Methotrexate resistance was also correlated to increased cellular S-adenosylmethionine levels. The folate and S-adenosylmethionine regeneration pathways are intimately connected, which may provide a basis for this novel resistance phenotype. This thorough comparative proteomic analysis highlights the variety of responses required for drug resistance to be achieved.

Animals↗

PubMed2004

Drug uptake and modulation of drug resistance in Leishmania by an aquaglyceroporin.

Leishmaniasis is a protozoan parasitic disease that affects 12 million people worldwide. The first line choice for the treatment of this disease is antimonial drugs. In the endemic regions, resistance to this class of drugs is a major impediment to treatment. Microbes often become resistant to drugs by mutation or down-regulation of uptake systems, but the uptake system for the antimonial drugs in Leishmania is unknown. In other organisms, aquaglyceroporins have been shown to facilitate uptake of trivalent metalloids. In this study, we report the identification and characterization of aquaglyceroporins from Leishmania major (LmAQP1) and Leishmania tarentolae (LtAQP1), respectively. These Leishmania proteins have the conserved signature motifs of aquaglyceroporins. Transfection of LmAQP1 into three species of Leishmania, L. tarentolae, Leishmania infantum, and L. major, produced hypersensitivity to both As(III) and Sb(III) in all three strains. Increased production of LmAQP1 was detected by immunoblotting. Drug-resistant parasites with various mutations leading to resistance mechanisms became hypersensitive to both metalloids after expression of LmAQP1. Increased rates of uptake of As(III) or Sb(III) correlated with metalloid sensitivity of the wild type and drug-resistant transfectants. Transfection of LmAQP1 in a Pentostam-resistant field isolate also sensitized the parasite in the macrophage-associated amastigote form. One allele of LmAQP1 was disrupted in L. major, and the resulting cells became 10-fold more resistant to Sb(III). This is the first report of the uptake of a metalloid drug by an aquaglyceroporin in Leishmania, suggesting a strategy to reverse resistance in the field.

Amino Acid Sequence↗