Metacaspases are distant orthologues of mammalian caspases and have been proposed to play a role in programmed cell death in yeast and plants, but little is known about their function in parasitic protozoa. In this study, I found that the single MCA gene of Leishmania major (LmajMCA or MCA) is expressed in actively replicating amastigotes and promastigotes, whereas expression level is significantly lower in nondividing stationary phase promastigotes. In order to determine the role of the metacaspase during promastigote division, an analysis of the events of the L. major cell cycle was performed. Based on these findings, immunofluorescence experiments revealed the presence of LmajMCA in punctate structures throughout the cytoplasm of interphase cells and its accumulation in the kinetoplast (mitochondrial DNA) at the time of the organelle's segregation. LmajMCA also translocates to the nucleus during mitOSis, where it associates with the mitotic spindle. LmajMCA null mutants could not be generated using standard gene deletion techniques. When LmajMCA was expressed from an episome, the only mutants that were viable were those expressing LmajMCA at physiological levels. Over-expression of LmajMCA in promastigotes leads to changes in ploidy and a severe growth retardation, associated with defects in kinetoplast segregation and nuclear division and an impairment of cytokinesis. Several LmajMCA variants lacking their N-terminal and/or C-terminal domains or with active site mutation(s) (C201G, C201-C202G, C202G, H147A) were expressed, with or without tags, in L. major promastigotes, in Escherichia coli, in the yeast Pichia pastoris, and using an in vitro translation kit. In all the systems used, the expression of most LmajMCA variants was barely detectable and was apparently toxic for the host cells. Some encouraging results were obtained using P. pastoris, with the expression and secretion in the culture medium of N- and C-terminally truncated LmajMCA. Thus, this system may fadlitate production of suffident enzyme for the characterisation of the biochemical properties of LmajMCA. Insights into the putative function of the different domains of LmajMCA were gained when it was observed, that the first 101 N-terminal amino adds of LmajMCA worked, in L.major, as a mitochondrion targeting signal. A potential role of the LmajMCA proline-, glutamine- and tyrosine-rich C-terminal domain in proteinprotein interactions was also hypothesized. Together these data suggest that LmajMCA is essential for the correct segregation of the nucleus and kinetoplast and cytokinesis and that the tight regulation of the amount of LmajMCA within the cell is crucial for the development of Leishmania promastigotes. Trypanosoma bruce; possesses five metacaspase genes. Two of the encoded proteins, TbMCA 1 and TbMCA4, possess substitutions at their putative active site residues and thus might lack peptidase activity. Therefore functional studies were performed only on the three other metacaspases. Triple RNAi analysis showed TbMCA2, TbMCA3 and TbMCAS to be essential in the bloodstream form, with induced parasites showing a delay in kinetoplast segregation and being blocked in pre-cytokinesis. Triple null mutants (11 Tbmca21311 Tbmca5) were found to be initially affected in their development, but recovered after several weeks of in vitro adaptation. The initial slow growth rate of the I1Tbmca213l1Tbmca5 mutant was associated with a delayed entry in cytokinesis. This phenotype was similar, although less extreme, to that produced upon triple RNAi of TbMCA2, TbMCA3 and TbMCAS. These data suggest that these metacaspases are essential for T. bruce; bloodstream forms, but they have overlapping functions and their progressive loss can be compensated for by activation of alternative biochemical pathways. Analysis of 11 Tbmca21311 Tbmca5 revealed no greater or lesser susceptibility to stresses reported to initiate programmed cell death, such as treatment with prostaglandin Oz. These metacaspases co-localise with RAB11, a marker for recycling endosomes but variant surface glycoprotein (VSG) recycling processes and the degradation of internalised anti-VSG antibody were found to occur similarly in wild type, I1Tbmca213l1Tbmca5 and triple RNAi-induced parasites. Thus the data provide no support for the direct involvement of T. bruce; metacaspases in programmed cell death and suggest that the proteins have a function associated with RAB 11 vesicles that is independent of known recycling processes of RAB11-positive endosomes. As both the L. major and the T. bruce; metacaspases appear to play crueal roles in cell cycle progression and because metacaspases are absent from the mammalian genome, these metacaspases have potential as novel drug targets

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Genome sequencing of three Leishmania species (L.infantum, L.major and L.brazitiensis) that cause different types of leishmaniasis has identified only a few species-specific genes that may contribute to Leishmania tropism in the host. This thesis describes the preliminary analysis of four Leishmania infantum species-specific genes and the functional analysis of one selected gene from the preliminary analysis, LinJ31.3030, which codes for a putative phosphatase. Transgenic parasites deleted for this sequence have been generated by sequential gene replacement and these analyses have demonstrated the presence of four LinJ31.3030 alleles rather than the two expected for this diploid organism. These data correlate with recent deep sequencing of the L.infantum strain, suggesting that chromosome 31 might be tetrasomic in this and other species. In phenotypic analysis, the LinJ31.3030 deleted mutants demonstrated a reduced flagella length compared to the flagella of wild type promastigotes. In-depth bioinforrnatic analysis suggests that Linj31.3030 codes for a cofilin phosphatase. As reported for chronophin (the human cofilin phosphatase), recombinant Linj31.3030 protein has enzyme activity against a phospho-cofilin peptide. If the identification of Linj31.3030 as a cofilin phosphatase is verified, this enzyme may modulate the flagellar malfunction phenotype observed in Leishmania cofilin deletion mutants, impacting on parasite motility and infectivity.

Leishmaniasis is a worldwide disease prevalent in many tropical and sub tropical countries. Treatment of Leishmaniasis by chemotherapy is not wholly effective and is usually accompanied by unpleasant side effects. The development of an effective and inexpensive vaccine represents a practical way to control the disease, however at present no safe and effective vaccine is available. In the first part of the present study, the immunity induced by four different L. mexicana potential vaccines, including killed leishmania vaccine, Soluble L. mexicana Antigen (SLA), L. mexicana gp63 cDNA and CT26 tumour cells transfected with L. mexicana gp63, were compared. It was shown that DNA immunisation using L. mexicana gp63 generated the highest immunity to the parasite among the four tested vaccines where the killed leishmania vaccine and L. mexicana gp63 transfected CT26 tumour cells did not generate significant immunity. The efficacy of DNA immunisation by intramuscular injection or using gene gun, in generating immunity to leishmania was compared. Gene gun immunisation induced more immunity to the parasite and high levels of Th1 immune response, which were detected, one week after immunisation through determination of the IgG2a levels in blood serum. Gene gun immunisation also induced long-lasting CTL activity, which was detectable before and during the course of infection for up to 6 months. Immunogenicity of MHC class I restricted peptides derived from L. mexicana gp63 have been investigated. Using 'SYFPEITHI' software, four peptides with high affinity to human HLA-A2 and four peptides with high affinity to mouse H2-Ld were predicted, synthesized and tested in HHD II and BALB/c mice respectively. Only three of the peptides predicted with high affinity to HLA-A2 were immunogenic but only two of them were likely to be naturally processed, however, none were protective in HHD II mice against leishmania infection. Purification and application of OX40L, a ligand for T-cell co-stimulatory receptor, was investigated in L. mexicana BALB/c model. In addition to purification by protein A sepharose, the murine OX40L-IgG fusion protein produced by B9B8E2 cells (cells transfected with OX40L and IgG) was successfully purified by two novel resins, MBI & MEP. The biological activity of the OX40L-IgG purified by MBI resin was significantly higher than that of MEP or protein A sepharose resins. Application of OX40L-IgG resulted in healing of leishmania lesions or delaying in development of the lesions in leishmania-infected mice.

Orientadora : Iara Taborda de Messias Reason / Dissertaçao (mestrado) - Universidade Federal do Paraná, Setor de Ciencias da Saúde, Programa de Pós-Graduaçao em Ciencias Farmaceuticas. Defesa: Curitiba, 2005 / Inclui bibliografia / Área de concentraçao: Análises clínicas / Resumo: A Leishmania (Viannia) braziliensis, é o agente causador da leishmaniose cutânea mucosa, doença endêmica em algumas regiões do Brasil, que representa um grande problema de saúde pública para o país. A ativação do complemento na superfície de promastigotas de Leishmania é um importante fator para a infectividade do parasita em hospedeiros mamíferos, permitindo o seu ataque e invasão de macrófagos via receptores do complemento. A Lectina Ligante da Manose (MBL), possui a capacidade de ativar o complemento e também atua como uma eficiente opsonina. Neste estudo investigou-se a interação de MBL purificada com a superfície de promastigotas da L. braziliensis e a deposição de MBL, C1q, C4 e C3 na superfície do parasita após interação com soro humano normal (SHN), não imune. Foi também avaliada a influência das vias clássica, alternativa e das lectinas no efeito lítico do complemento sobre os parasitas. Observou-se que tanto a MBL do soro como o complexo MBL-MASP purificado ligam-se à superfície da L. braziliensis, através de ensaios de imunofluorescência e de inibição utilizando-se anticorpo monoclonal anti-MBL humana e microscopia confocal. A ligação da MBL demonstrou ser específica para manose presente na superfície do parasita. Foi também observado que a deposição da MBL na superfície da L. braziliensis não alterou o efeito lítico do complemento sobre os parasitas. Deposição de C1q, C4 e C3 foi observada após incubação do parasita com SHN. Ácido etilenodiamino tetraacetico (EDTA), mas não ácido etileno glicol-bis ?-aminoetileter N,N,N,N-tetraacético (EGTA) inibiram a deposição de C3 na superfície do parasita, indicando o envolvimento da via alternativa neste processo. Esses resultados indicam que a MBL pode ligar-se a L. braziliensis através de carboidrato específico na superfície do parasita e fornece evidências para um mecanismo de ativação do complemento independente de anticorpo. Palavras Chave: Leishmania (Viannia) braziliensis, MBL, complemento, lectina-ligante da manose, via clássica, via alternativa. / Abstract: Leishmania (Viannia) braziliensis (L.braziliensis) is the causative agent of mucocutaneous leishmaniasis, a disease that presents high endemicity in some regions of Brazil that represents a major public health problem for the country. Activation of complement on the surface of Leishmania promastigotes appears to be an important factor for parasite infectivity in the mammalian host, allowing their attachment and invasion of macrophages via complement receptors. Mannan-binding lectin (MBL) is a well-known complement activator and an efficient opsonine. In this study, we have investigated whether serum and purified MBL bind to live promastigotes of L. braziliensis and evaluated the deposition of MBL, C1q, C4 and C3 on the parasite surface after interaction with non-immune human serum (NHS). Using immunofluorescence assays we have observed that both serum MBL and purified MBL-MASP complex bind to the surface of L. braziliensis using a mouse monoclonal anti-human MBL antibody and confocal microscopy. MBL binding to parasite surface was shown to be specific for mannose present on the surface of parasites, by inhibition assays. It was observed that the presence of MBL doesn't modify the lytic effect of complement on the parasites. Deposition of C1q, C4 and C3 was observed after parasite incubation with NHS. Ethylenediamine tetra acetic acid (EDTA) but not ethylene glicol-bis ?-aminoethylether N, N, N, N-tetra acetic acid (EGTA) abolished C3 deposition on parasite surface, indicating the involvement of the alternative pathway in this process. Our results indicate that L. braziliensis binds MBL and that this is mediated by specific carbohydrate on the surface of parasites and provide evidences for antibody-independent mechanisms complement activation on parasite surface. Keywords: Leishmania (Viannia) braziliensis; mannose-binding lectin; complement; MBL; alternative pathway; classic pathway.

La leishmaniose est causée par un parasite protozoaire du genre Leishmania. Cette maladie infecte environ 12 millions de personnes dans le monde et en menace 350 millions dans 98 pays. Il existe trois formes majeures de leishmaniose : cutanée, mucocutanée et viscérale. L'infection se produit par le dépôt des parasites sous forme de promastigotes dans la peau de l'hôte mammifère via la piqûre d’un phlébotome. Les parasites peuvent migrer au sein de la matrice extracellulaire avant d’infecter les macrophages. Bien que la plupart des études réalisées jusqu’ici aient été consacrées aux interactions des parasites Leishmania avec leurs cellules cibles, quelques interactants extracellulaires ont déjà été identifiés. Dans cette étude, nous avons étudié pour la première fois le répertoire d’interactions de 24 souches de promastigotes intacts, vivants (6 espèces aux différents tropismes) avec environ ~70 biomolécules de la matrice extracellulaire de l’hôte à l’échelle moléculaire en utilisant des puces à protéines et à glycosaminoglycanes et la résonance plasmonique de surface en mode imagerie. Nous avons identifié 27 nouveaux partenaires (23 protéines et 4 glycosaminoglycanes) des promastigotes de Leishmania. Les souches partagent des partenaires communs tels que le plasminogène, TEM-8 et la tropoélastine, qui est dégradée in vitro par la majorité des souches. Les Leishmania se lient à plusieurs régulateurs de l’angiogenèse et à des glycosaminoglycanes. Dans une seconde partie, nous avons cloné deux protéines de L. major, l’énolase et la superoxyde dismutase, toutes deux identifiées dans le sécrétome de Leishmania, afin d’étudier leur répertoire d’interactions. L’énolase possède un répertoire d’interactions (13 partenaires) supérieur à celui de la superoxyde dismutase (6 partenaires) mais toutes deux interagissent également avec le plasminogène, l’ectodomaine de TEM-8, l’endostatine et l’héparine. Enfin, dans une troisième partie, nous avons créé une base de données, LeishMatrixDB, qui recense toutes les interactions des parasites Leishmania, ou leurs molécules, avec les composants de la matrice extracellulaire de l’hôte décrites dans la littérature / Leishmaniasis is a vector-borne disease caused by parasitic protozoa of the genus Leishmania. 12 million people are presently infected worldwide and the disease threatens 350 million people in 98 countries around the world. There are three main types of the disease: cutaneous, mucocutaneous and visceral. Infection occurs by the deposition of promastigote form into the mammalian skin via the bite of phlebotomine sandflies within the extracellular matrix proteins prior infecting macrophages. Most studies have focused on the interaction of Leishmania promastigotes with their cellular targets, some extracellular partners have been identified. In this study, we investigated for the first time the interplay between 24 strains of intact, live, parasites (6 species of different tropisms) and ~70 biomolecules of the host extracellular matrix at the molecular level using protein and glycosaminoglycan arrays probed by surface plasmon resonance imaging. We have identified 27 new partners (23 proteins and 4 glycosaminoglycans) of Leishmania promastigotes. All strains tested shared 3 common partners such as plasminogen, TEM-8 and tropoelastin, which is degraded in vitro by most Leishmania tested. Leishmania bound to several regulators of angiogenesis and to glycosaminoglycans. In a second part, we cloned two L. major proteins, enolase and superoxyde dismutase, both identified in Leishmania secretome in order to study their interaction repertoire. Enolase had a larger interaction repertoire (13 partners) than superoxide dismutase (6 partners) but both bound to plasminogen, ectodomain of TEM-8, endostatin and heparin. In a third part, we have created a database, LeishMatrixDB, which lists all the interactions of Leishmania, or their molecules, with host extracellular components from the literature