Functional analysis of the murine T lymphocyte immune response to a protozoan parasite, Leishmania tropica

The results presented in this review summarize a seirs of experiments designed to characterize the murine T cell imune response to the protozoan parasite Leishmania tropica. Enriched T cell populations and T cell clones specific for L. tropica antigens were derived from lymph nodes of primed mice and maintained in continous culture in vitro. These T lymphocytes were shown (A) to express the Lyt 1+ 3- cell surface phenotype, (B) to proliferate specifically in vitro in response to parasite antigens, together with a source of irradiated syngeneic macrophages, (C) to transfer antigen-specific delayed-type hypersensitivity (DTH) responses to normal syngeneic mice, (D) to induce specific activation of parasitized macrophages in vitro resulting in the destruction of intracellular parasites, (E) to provide specific helper activity for antibody responses in vitro in a hapten-carrier system. Protection studies using these defiened T cell populations should allow the characterization of parasite antigen(s) implicated in the induction of cellular immune responses beneficial for the host.

Characterization of glycoinositol phospholipids in the amastigote stage of the protozoan parasite Leishmania major.

The major macromolecules on the surface of the parasitic protozoan Leishmania major appear to be down-regulated during transformation of the parasite from an insect-dwelling promastigote stage to an intracellular amastigote stage that invades mammalian macrophages. In contrast, the major parasite glycolipids, the glycoinositol phospholipids (GIPLs), are shown here to be expressed at near-constant levels in both developmental stages. The structures of the GIPLs from tissue-derived amastigotes have been determined by h.p.l.c. analysis of the deaminated and reduced glycan head groups, and by chemical and enzymic sequencing. The deduced structures appear to form a complete biosynthetic series, ranging from Man alpha 1-4GlcN-phosphatidylinositol (PI) to Gal alpha 1-3Galf beta 1-3Man alpha 1-3Man alpha 1-4GlcN-PI (GIPL-2). A small proportion of GIPL-2 was further extended by addition of a Gal residue in either alpha 1-6 or beta 1-3 linkage. From g.c.-m.s. analysis and mild base treatment, all the GIPLs were shown to contain either alkylacylglycerol or lyso-alkylglycerol lipid moieties, where the alkyl chains were predominantly C18:0, with lower levels of C20:0, C22:0 and C24:0. L. major amastigotes also contained at least two PI-specific phospholipase C-resistant glycolipids which are absent from promastigotes. These neutral glycolipids were resistant to both mild acid and mild base hydrolysis, contained terminal beta-Gal residues and were not lost during extensive purification of amastigotes from host cell membranes. It is likely that these glycolipids are glycosphingolipids acquired from the mammalian host. The GIPL profile of L. major amastigotes is compared with the profiles found in L. major promastigotes and L. donovani amastigotes.

Implication of TNF family members in the immune response to the infection with the intracellular protozoan parasite "Leishmania major"

Suite à une infection avec le protozoaire Leishmania major (L. major), les souris sensibles de souche BALB/c développent des lésions progressives associées à une maturation des cellules CD4+ TH2 sécrétant de l'IL-4. A l'inverse, les souris résistantes de souche C57BL/6 guérissent à terme, sous l'influence de l'expansion des cellules CD4+ TH1 produisant de l'IFNy qui a un effet synergique avec le TNF ("tumor necrosis factor") sur l'activation des macrophages et leur fonction leishmanicide. Lors de notre étude nous avons montré que des souris C57BL/6 doublement déficientes en TNF et FasL ("Fas ligand") infectées par L. major ne guérissaient ni leur lésions ni ne contrôlaient la réplication de parasites malgré une réponse de type TH1. Bien que l'activité de synthétase inductible de l'oxyde nitrique ("iNOs") soit comparable chez les souris doublement ou simplement déficientes, seules celles déficientes en FasL ont démontré une incapacité à contrôler la réplication parasitaire. De surcroît il est apparu que le FasL a un effet synergique avec l'IFNy. L'adjonction de FasL à une culture cellulaire de macrophages stimulés par l'IFNy conduit à une activation de ces cellules. Celle-ci est démontrée par l'augmentation de la production de TNF et de NO par les macrophages ainsi que par l'élimination des parasites intracellulaires par ces mêmes cellules. Alors que le FasL et l'IFNy semblent essentiels au contrôle de la réplication des pathogènes intracellulaires, la contribution de TNF s'oriente davantage vers le contrôle de l'inflammation. L'activation macrophagique via Fas précède la mort cellulaire qui survient quelques jours plus tard. Cette mort cellulaire programmée était indépendante de la cascade enzymatique des caspases, au vu de l'absence d'effet de l'inhibiteur non-spécifique ZVAD-fmk des caspases. Ces résultats suggèrent que l'interaction Fas-FasL agit comme une costimulation nécessaire à une activation efficace des macrophages, la mort cellulaire survenant consécutivement à l'activation des macrophages.¦-¦Upon infection with the protozoan parasite Leishmania major (L. major), susceptible BALB/c mice develop non healing lesions associated with the maturation of CD4+ TH2 cells secreting IL-4. In contrast, resistant C57BL/6 mice are able to heal their lesions, because of CD4+ TH1 cell expansion and production of high levels of IFNy, which synergizes with tumour necrosis factor (TNF) in activating macrophages to their microbicidal state. In our study we showed that C57BL/6 mice lacking both TNF and Fas ligand (FasL) infected with L. major neither resolved their lesions nor controlled L. major replication despite a strong TH1 response. Although comparable inducible nitric oxide synthase (iNOs) was measured in single or double deficient mice, only mice deficient in FasL failed to control the parasite replication. Moreover FasL synergized with IFNy for the induction of leishmanicidal activity within macrophages infected with L. major in vitro. Addition of FasL to IFNy stimulated macrophages led to their activation, as reflected by the secretion of tumour necrosis factor and nitrite oxide, as well as the induction of their microbicidal activity, resulting in the killing of intracellular L. major. While FasL along with IFNy and iNOs appeared to be essential for the complete control of intracellular pathogen replication, the contribution of TNF appeared more important in controlling the inflammation on the site of infection. Macrophage activation via Fas pathway preceded cell death, which occurred a few days after Fas mediated activation. This program cell death was independent of caspase enzymatic activities as revealed by the lack of effect of ZVAD-fmk, a pan-caspase inhibitor. These results suggested that the Fas-FasL pathway, as part of the classical activation pathway of the macrophages, is essential in the stimulation of macrophage leading to a microbicidal state and to AICD, and may thus contribute to the pathogenesis of L. major infection.

Characterisation of two soluble metalloexopeptidases in the protozoan parasite Leishmania major.

Two soluble exopeptidases were identified in promastigotes of Leishmania major, using an iodinated model tetrapeptide (LIAY) as substrate. Similar activities were also detected in L. major amastigotes and in different species of Leishmania promastigotes. A carboxy- and an aminopeptidase activity were resolved and isolated by anion exchange and gel permeation chromatographies. A single polypeptide of 62 kDa co-purified with the aminopeptidase activity. Optimum pH was neutral for the carboxypeptidase and neutral to alkaline for the aminopeptidase. Both activities were able to hydrolyse a dipeptide substrate (YL), and were inhibited by 20 microM bestatin and 200 microM 1,10-phenanthroline, but not by leupeptin, iodoacetamide and a range of other inhibitors. These results strongly suggest that both enzymes are metalloexopeptidases and thus represent a novel class of soluble peptidases in Leishmania.

Insights into the function of short interspersed degenerated retroposons in the protozoan parasite Leishmania

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

The effect of climate change on the potential distribution of the European Phlebotomus species and the parasite Leishmania infantum in 2011-2070. A climate envelope modeling approach

Aims: In the Mediterranean areas of Europe, leishmanisasis is one of the most emerging vector-borne diseases. Members of genus Phlebotomus are the primary vectors of the genus Leishmania. To track the human health effect of climate change it is a very important interdisciplinary question to study whether the climatic requirements and geographical distribution of the vectors of human pathogen organisms correlate with each other. Our study intended to explore the potential effects of ongoing climate change, in particular through a potential upward altitudinal and latitudinal shift of the distribution of the parasite Leishmania infantum, its vectors Phlebotomus ariasi, P. neglectus, P. perfiliewi, P. perniciosus, and P. tobbi, and some other sandfly species: P. papatasi, P. sergenti, and P. similis. Methods: By using a climate envelope modelling (CEM) method we modelled the current and future (2011-2070) potential distribution of 8 European sandfly species and L. infantum based on the current distribution using the REMO regional climate model. Results: We found that by the end of the 2060’s most parts of Western Europe can be colonized by sandfly species, mostly by P. ariasi and P. pernicosus. P. ariasi showed the greatest potential northward expansion. For all the studied vectors of L. infantum the entire Mediterranean Basin and South-Eastern Europe seemed to be suitable. L. infantum can affect the Eastern Mediterranean, without notable northward expansion. Our model resulted 1 to 2 months prolongation of the potentially active period of P. neglectus P. papatasi and P. perniciosus for the 2060’s in Southern Hungary. Conclusion: Our findings confirm the concerns that leishmanisais can become a real hazard for the major part of the European population to the end of the 21th century and the Carpathian Basin is a particularly vulnerable area.

Description of Leishmania equatorensis sp. n. (Kinetoplastida: Trypanosomatidae), a new parasite infecting arboreal mammals in Ecuador

Characterization is given of a new parasite, Leishmania equatoriensis sp.n. wich was isolated from the viscera of a sloth (Choloepus hoffmanni) and a squirrel (Sciurus granatensis), captured in humid tropical forest onthe Pacific Coast of Ecuador. Data based on biological and molecular criteria, as well as numerical zymotaxonomical analysis, indicate that this parasite is a new species of the L. brasiliensis complex. L. equatoriensis is cleary distinguishable form all other known species within this complex, using the following molecular criteria: reactivity patterns with specific monoclonal antibodies, isoenzyme electrophoresis, and restriction-endonuclease fragment patterns of kinetoplast DNA (k-DNA).

Both the Fas ligand and inducible nitric oxide synthase are needed for control of parasite replication within lesions in mice infected with Leishmania major whereas the contribution of tumor necrosis factor is minimal.

Following infection with the protozoan parasite Leishmania major, C57BL/6 mice develop a small lesion that heals spontaneously. Resistance to infection is associated with the development of CD4(+) Th1 cells producing gamma interferon (IFN-gamma) and tumor necrosis factor (TNF), which synergize in activating macrophages to their microbicidal state. We show here that C57BL/6 mice lacking both TNF and Fas ligand (FasL) (gld TNF(-/-) mice) infected with L. major neither resolved their lesions nor controlled Leishmania replication despite the development of a strong Th1 response. Comparable inducible nitric oxide synthase (iNOS) activities were detected in lesions of TNF(-/-), gld TNF(-/-), and gld mice, but only gld and gld TNF(-/-) mice failed to control parasite replication. Parasite numbers were high in gld mice and even more elevated in gld TNF(-/-) mice, suggesting that, in addition to iNOS, the Fas/FasL pathway is required for successful control of parasite replication and that TNF contributes only a small part to this process. Furthermore, FasL was shown to synergize with IFN-gamma for the induction of leishmanicidal activity within macrophages infected with L. major in vitro. Interestingly, TNF(-/-) mice maintained large lesion size throughout infection, despite being able to largely control parasite numbers. Thus, IFN-gamma, FasL, and iNOS appear to be essential for the complete control of parasite replication, while the contribution of TNF is more important in controlling inflammation at the site of parasite inoculation.

Stage-specific proteophosphoglycan from Leishmania mexicana amastigotes - Structural characterization of novel mono-, di-, and triphosphorylated phosphodiester-linked oligosaccharides

Intracellular amastigotes of the protozoan parasite Leishmania mexicana secrete a macromolecular proteophosphoglycan (aPPG) into the phagolysosome of their host cell, the mammalian macrophage. The structures of aPPG glycans were analyzed by a combination of high pH anion exchange high pressure liquid chromatography, gas chromatography-mass spectrometry, enzymatic digestions, electrospray-mass spectrometry as well as H-1 and P-31 NMR spectroscopy. Some glycans are identical to oligosaccharides known from Leishmania mexicana promastigote lipophosphoglycan and secreted acid phosphatase, However, the majority of the aPPG glycans represent amastigote stage-specific and novel structures. These include neutral glycans ([Glc beta(1-3)](1-2)Gal beta 1-4Man, Gal beta 1-3Gal beta 1-4Man, Gal beta 1-3Glc beta 1-3Gal beta 1-4Man), several monophosphorylated glycans containing the conserved phosphodisaccharide backbone (R-3-[PO4-6-Gal]beta 1-4Man) but carrying stage-specific modifications (R = Gal beta 1-, [Glc beta 1-3](1-2)Glc beta 1-), and monophosphorylated aPPG tri- and tetrasaccharides that are uniquely phosphorylated on the terminal hexose (PO4-6-Glc beta 1-3Gal beta 1-4Man, PO4-6-Glc beta 1-3Glc beta 1-3Gal beta 1-4Man, PO4-6-Gal beta 1-3Glc beta 1-3Gal beta 1-4Man), In addition aPPG contains highly unusual di- and triphosphorylated glycans whose major species are PO4-6-Glc beta 1-3Glc beta 1-3[PO4-6-Gal]beta 1-4Man, PO4-6-Gal beta 1-3Glc beta 1-3 [PO4-6-Gal]beta 1-4Man, PO4-6-GaL beta 1-3Glc beta 1-3Glc beta 1-3[PO4-6-Gal]beta 1-4Man, PO4-6-Glc beta 1-3[PO4-6-Glc]beta 1-3[PO4-6-Gal]beta 1-4Man, PO4-6Gal beta 1-3[PO4-6-Glc]beta 1-3Glc beta 1-3[PO4-6-Gal]beta 1-4Man, and PO4-6-Glc beta 1-3[PO4-6-Glc]beta 1-3Glc beta 1-3[PO4-6-Gal]beta 1-4Man. These glycans are linked together by the conserved phosphodiester R-Man alpha 1-PO4-6-Gal-R or the novel phosphodiester R-Man alpha 1-PO4-6-Glc-R and are connected to Ser(P) of the protein backbone most likely via the linkage R-Man alpha 1-PO4-Ser. The variety of stage-specific glycan structures in Leishmania mexicana aPPG suggests the presence of developmentally regulated amastigote glycosyltransferases which may be potential anti-parasite drug targets.

Identification and chromosomal localization of one locus of Leishmania (L.) major related with resistance to itraconazole

Ergosterol is an important compound responsible to maintain integrity and fluidity of Leishmania spp. membranes. Starting from an overexpression/selection method, our group has isolated and mapped nine different loci of Leishmania (L.) major related to resistance against two inhibitors of the ergosterol biosynthesis pathway, terbinafine (TBF) and itraconazole (ITZ). Individual functional analysis after overexpression induction of these loci in the presence of TBF and/or ITZ [or the ITZ analog ketoconazole (CTZ)] have shown low but significant levels of resistance after transfection into L. major wild-type parasites. In this work, we have shown the insert mapping and chromosomal identification of one of these loci (cosItz2). Functional analysis experiments associated with chromosomal localization by comparison at genomic database allowed us to identify two prospective gene-protein systems not related to the ergosterol biosynthesis and capable to confer wild-type cells resistance to ITZ-CTZ after transfection. We expected that this approach can open new insights for a better understanding of mechanisms of ITZ-CTZ action and resistance in Leishmania resulting in new strategies for the leishmaniasis treatment.

Cell homeostasis in a Leishmania major mutant overexpressing the spliced leader RNA is maintained by an increased proteolytic activity

Although several stage-specific genes have been identified in Leishmania, the molecular mechanisms governing developmental gene regulation in this organism are still not well understood. We have previously reported an attenuation of virulence in Leishmania major and L braziliensis carrying extra-copies of the spliced leader RNA gene. Here, we surveyed the major differences in proteome and transcript expression profiles between the spliced leader RNA overexpressor and control lines using two-dimensional gel electrophoresis and differential display reverse transcription PCR, respectively. Thirty-nine genes related to stress response, cytoskeleton, proteolysis, cell cycle control and proliferation, energy generation, gene transcription, RNA processing and post-transcriptional regulation have abnormal patterns of expression in the spliced leader RNA overexpressor line. The evaluation of proteolytic pathways in the mutant revealed a selective increase of cysteine protease activity and an exacerbated ubiquitin-labeled protein population. Polysome profile analysis and measurement of cellular protein aggregates showed that protein translation in the spliced leader RNA overexpressor line is increased when compared to the control line. We found that L major promastigotes maintain homeostasis in culture when challenged with a metabolic imbalance generated by spliced leader RNA surplus through modulation of intracellular proteolysis. However, this might interfere with a fine-tuned gene expression control necessary for the amastigote multiplication in the mammalian host. (c) 2010 Elsevier Ltd. All rights reserved.

The Hus1 homologue of Leishmania major encodes a nuclear protein that participates in DNA damage response

The protozoan parasite Leishmania presents a dynamic and plastic genome in which gene amplification and chromosome translocations are common phenomena. Such plasticity hints at the necessity of dependable genome maintenance pathways. Eukaryotic cells have evolved checkpoint control systems that recognize altered DNA structures and halt cell cycle progression allowing DNA repair to take place. In these cells, the PCNA-related heterotrimeric complex formed by the proteins Hus1, Rad9, and Rad1 is known to participate in the early steps of replicative stress sensing and signaling. Here we show that the Hus1 homolog of Leishmania major is a nuclear protein that improves the cell capability to cope with replicative stress. Overexpression of LmHus1 confers resistance to the genotoxic drugs hydroxyurea (HU) and methyl methanesulfonate (MMS) and resistance to HU correlates to reduced net DNA damage upon LmHus1 expression. (C) 2011 Elsevier B.V. All rights reserved.

Macrophages induce neutrophil apoptosis through membrane TNF, a process amplified by Leishmania major.

Neutrophils are recruited to the site of parasite inoculation within a few hours of infection with the protozoan parasite Leishmania major. In C57BL/6 mice, which are resistant to infection, neutrophils are cleared from the site of s.c. infection within 3 days, whereas they persist for at least 10 days in susceptible BALB/c mice. In the present study, we investigated the role of macrophages (MPhi) in regulating neutrophil number. Inflammatory cells were recruited by i.p. injection of either 2% starch or L. major promastigotes. Neutrophils were isolated and cultured in the presence of increasing numbers of MPhi. Extent of neutrophil apoptosis positively correlated with the number of MPhi added. This process was strictly dependent on TNF because MPhi from TNF-deficient mice failed to induce neutrophil apoptosis. Assays using MPhi derived from membrane TNF knock-in mice or cultures in Transwell chambers revealed that contact with MPhi was necessary to induce neutrophil apoptosis, a process requiring expression of membrane TNF. L. major was shown to exacerbate MPhi-induced apoptosis of neutrophils, but BALB/c MPhi were not as potent as C57BL/6 MPhi in this induction. Our results emphasize the importance of MPhi-induced neutrophil apoptosis, and membrane TNF in the early control of inflammation.

Cathepsin B-like and cell death in the unicellular human pathogen Leishmania.

In several studies reporting cell death (CD) in lower eukaryotes and in the human protozoan parasite Leishmania, proteolytic activity was revealed using pan-caspase substrates or inhibitors such as carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK). However, most of the lower eukaryotes do not encode caspase(s) but MCA, which differs from caspase(s) in its substrate specificity and cannot be accountable for the recognition of Z-VAD-FMK. In the present study, we were interested in identifying which enzyme was capturing the Z-VAD substrate. We show that heat shock (HS) induces Leishmania CD and leads to the intracellular binding of Z-VAD-FMK. We excluded binding and inhibition of Z-VAD-FMK to Leishmania major metacaspase (LmjMCA), and identified cysteine proteinase C (LmjCPC), a cathepsin B-like (CPC) enzyme, as the Z-VAD-FMK binding enzyme. We confirmed the specific interaction of Z-VAD-FMK with CPC by showing that Z-VAD binding is absent in a Leishmania mexicana strain in which the cpc gene was deleted. We also show that parasites exposed to various stress conditions release CPC into a soluble fraction. Finally, we confirmed the role of CPC in Leishmania CD by showing that, when exposed to the oxidizing agent hydrogen peroxide (H(2)O(2)), cpc knockout parasites survived better than wild-type parasites (WT). In conclusion, this study identified CPC as the substrate of Z-VAD-FMK in Leishmania and as a potential additional executioner protease in the CD cascade of Leishmania and possibly in other lower eukaryotes.