Expression and cellular trafficking of GP82 and GP90 glycoproteins during Trypanosoma cruzi metacyclogenesis

Background: the transformation of noninfective epimastigotes into infective metacyclic trypomastigotes (metacyclogenesis) is a fundamental step in the life cycle of Trypanosoma cruzi, comprising several morphological and biochemical changes. GP82 and GP90 are glycoproteins expressed at the surface of metacyclic trypomastigote, with opposite roles in mammalian cell invasion. GP82 is an adhesin that promotes cell invasion, while GP90 acts as a negative regulator of parasite internalization. Our understanding of the synthesis and intracellular trafficking of GP82 and GP90 during metacyclogenesis is still limited. Therefore, we decided to determine whether GP82 and GP90 are expressed only in fully differentiated metacyclic forms or they start to be expressed in intermediate forms undergoing differentiation.Methods: Parasite populations enriched in intermediate forms undergoing differentiation were analyzed by quantitative real-time PCR, Western blot, flow cytometry and immunofluorescence to assess GP82 and GP90 expression.Results: We found that GP82 and GP90 mRNAs and proteins are expressed in intermediate forms and reach higher levels in fully differentiated metacyclic forms. Surprisingly, GP82 and GP90 presented distinct cellular localizations in intermediate forms compared to metacyclic trypomastigotes. in intermediate forms, GP82 is localized in organelles at the posterior region and colocalizes with cruzipain, while GP90 is localized at the flagellar pocket region.Conclusions: This study discloses new aspects of protein expression and trafficking during T. cruzi differentiation by showing that the machinery involved in GP82 and GP90 gene expression starts to operate early in the differentiation process and that different secretion pathways are responsible for delivering these glycoproteins toward the cell surface.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, BR-04023062 São Paulo, BrazilUniv Fed Rio de Janeiro, Inst Biofis Carlos Chagas Filho, BR-21949900 Rio de Janeiro, RJ, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, BR-04023062 São Paulo, BrazilWeb of Scienc

Composições microbicidas, processos para sua obtenção, construções gênicas, processos para o controle de pestes

Em 17/03/2016: Anuidade de pedido de patente de invenção no prazo ordinárioDepositadaA presente invenção é relacionada a fatores antimicrobianos extraídos de envoltórios protetores (cascas) de ovos de insetos. A maioria destes fatores compreende peptídeos e/ou proteínas obtidas de tais involuntários e/ou de seus respectivos genes codificantes, em construções gênicas artificiais. São descritas composições microbicidas e processos para sua obtenção, bem como construções gênicas e processos para o controle de pestes

Cell fractionation of parasitic protozoa: a review

Cell fractionation, a methodological strategy for obtaining purified organelle preparations, has been applied successfully to parasitic protozoa by a number of investigators. Here we present and discuss the work of several groups that have obtained highly purified subcellular fractions from trypanosomatids, Apicomplexa and trichomonads, and whose work have added substantially to our knowledge of the cell biology of these parasites

Electron microscopy and cytochemistry analysis of the endocytic pathway of pathogenic protozoa

58 p. : il.Endocytosis is essential for eukaryotic cell survival and has been well characterized in mammal and yeast cells. Among protozoait is also important for evading from host immune defenses and to support in tense proliferation characteristic of some life cycle stages.Here we focused on the contribution of morphological and cytochemical studies to the understanding of endocytosisin Trichomonas, Giardia, Entamoeba, Plasmodium, andtrypanosomatids, mainly Trypanosoma cruzi, and also Trypanosoma brucei and Leishmania

Endocytosis in Trypanosoma cruzi

4 p. : il.Endocytic activity is particularly intense in Trypanosoma cruzi epimastigotes, while in amastigotes and trypomastigotes it is untraceable. Cargo molecules enters through the cytostome or flagellar pocket at the parasite anterior region, goes along a branched early endosomal network of tubules and vesicles spread from nuclear periphery to the posterior pole, until delivery to reservosomes, the final compartment. Reservosomes are acid compartments that store protein and lipid cargo and also accumulate lysosomal hydrolases, modulating digestive activity. Although T. cruzi infective forms are unable to uptake molecules, its lysosome related organelles represent a potential targets for anti-parasitic chemotherapy

New insights into the morphology of Trypanosoma cruzi reservosome

7 p. : il.Reservosomes are late endosomes present only in members of the Schizotrypanum subgenus of the Trypanosoma genus and are defined as the site of storage of endocytosed macromolecules and lysosomal enzymes. They have been extensively described in Trypanosoma cruzi epimastigote: are bounded by a membrane unit, present an electron-dense protein matrix with electron-lucent lipid inclusions, being devoid of inner membranes. Here we performed a detailed ultrastructural analysis of these organelles using a variety of electron microscopy techniques, including ultrathin sectioning, uranyl acetate stained preparations, and freeze fracture, either in intact epimastigotes or in isolated reservosomes. New informations were obtained. First, both isolated and in situ reservosomes presented small profiles of inner membranes that are morphologically similar to the membrane surrounding the organelle. In uranyl acetate stained preparations, internal membrane profiles turned out to be longer than they appeared in ultrathin section images and traversed the organelle diameter. Internal vesicles were also found. Second, endocytosed cargo are not associated with internal vesicles and reach reservosomes on board of vesicles that fuse with the boundary membrane, delivering cargo directly into reservosome lumen. Third, electron-lucent bodies with saturated lipid core surrounded by a membrane monolayer and with unusual rectangular shape were also observed. Fourth, it was possible to demonstrate the presence of intramembranous particles on the E face of both internal vesicles and the surrounding membrane. Collectively, these results indicate that reservosomes have a complex internal structure, which may correlate with their multiple functions

All Trypanosoma cruzi developmental forms present lysosome-related organelles

13 p. : il.Trypanosoma cruzi epimastigote forms concentrate their major protease, cruzipain, in the same compartment where these parasites store macromolecules obtained from medium and for this ability these organelles were named as reservosomes. Intracellular digestion occurs mainly inside reservosomes and seems to be modulated by cruzipain and its natural inhibitor chagasin that also concentrates in reservosomes. T. cruzi mammalian forms, trypomastigotes and amastigotes, are unable to capture macromolecules by endocytosis, but also express cruzipain and chagasin, whose role in infectivity has been described. In this paper, we demonstrate that trypomastigotes and amastigotes also concentrate cruzipain, chagasin as well as serine carboxypeptidase in hydrolase-rich compartments of acidic nature. The presence of P-type proton ATPase indicates that this compartment is acidiWed by the same enzyme as epimastigote endocytic compartments. Electron microscopy analyzes showed that these organelles are placed at the posterior region of the parasite body, are single membrane bound and possess an electron-dense matrix with electronlucent inclusions. Three-dimensional reconstruction showed that these compartments have diVerent size and shape in trypomastigotes and amastigotes. Based on these evidences, we suggest that all T. cruzi developmental stages present lysosome-related organelles that in epimastigotes have the additional and unique ability of storing cargo

Evolutionary conservation of actin-binding proteins in Trypanosoma cruzi and unusual subcellular localization of the actin homologue

11 p. : il.The actin cytoskeleton controls pivotal cellular processes such as motility and cytokinesis, as well as cell-cell and cellsubstrate interactions. Assembly and spatial organization of actin filaments are dynamic events regulated by a large repertoire of actin-binding proteins. This report presents the first detailed characterization of the Trypanosoma cruzi actin (TcActin). Protein sequence analysis and homology modelling revealed that the overall structure of T. cruzi actin is conserved and that the majority of amino-acid changes are concentrated on the monomer surface. Immunofluorescence assays using specific polyclonal antibody against TcActin revealed numerous rounded and punctated structures spread all over the parasitic body. No pattern differences could be found between epimastigotes and trypomastigotes or amastigotes. Moreover, in detergent extracts, TcActin was localized only in the soluble fraction, indicating its presence in the G-actin form or in short filaments dissociated from the microtubule cytoskeleton. The trypanosomatid genome was prospected to identify actin-binding and actin-related conserved proteins. The main proteins responsible for actin nucleation and treadmilling in higher eukaryotes are conserved in T. cruzi

Subcellular proteomics of Trypanosoma cruzi reservosomes

13 p. : il.Reservosomes are the endpoint of the endocytic pathway in Trypanosoma cruzi epimastigotes. These organelles have the particular ability to concentrate proteins and lipids obtained from medium together with the main proteolytic enzymes originated from the secretory pathway, being at the same time a storage organelle and the main site of protein degradation. Subcellular proteomics have been extensively used for profiling organelles in different cell types. Here, we combine cell fractionation and LC-MS/MS analysis to identify reservosome-resident proteins. Starting from a purified reservosome fraction, we established a protocol to isolate reservosome membranes. Transmission electron microscopy was applied to confirm the purity of the fractions. To achieve a better coverage of identified proteins we analyzed the fractions separately and combined the results. LC-MS/MS analysis identified in total 709 T. cruzi-specific proteins; of these, 456 had predicted function and 253 were classified as hypothetical proteins. We could confirm the presence of most of the proteins validated by previous work and identify new proteins from different classes such as enzymes, proton pumps, transport proteins, and others. The definition of the reservosome protein profile is a good tool to assess their molecular signature, identify molecular markers, and understand their relationship with different organelles