EVIDENCE for the PARTICIPATION of the SSP-3 ANTIGEN in the INVASION of NONPHAGOCYTIC MAMMALIAN-CELLS BY TRYPANOSOMA-CRUZI

Trypomastigotes of Trypanosoma cruzi have to invade mammalian cells in order to multiply. They bear on their plasma membrane a sialic acid-containing epitope (Ssp-3) defined by a series of monoclonal antibodies (mAbs). Previous investigations have shown that Fab fragments of these mAbs inhibit the attachment of trypomastigotes to 3T3 fibroblasts. To further define the role of Ssp-3 in invasion, here we use, as targets for infection, L cells and CHO cells stably transfected with cDNA coding for the mouse Fc receptors genes. When the trypomastigotes are incubated with small, nonagglutinating amounts of antibodies to Ssp-3, their attachment to the transfected cells is greatly enhanced, without a parallel increase in invasion. the enhancement in attachment is Fc mediated, since it is abolished by treatment of the transfected cells with mAbs to Fc receptors. in contrast, both attachment to, and invasion of, the transfected cells are increased if the parasites are incubated with polyclonal or monoclonal antibodies against T. cruzi surface membrane antigens other than Ssp-3. If, however, antibodies to Ssp-3 are added to the incubation mixtures containing any of the other anti-T. cruzi antibodies, the enhancement of invasion (but not of attachment) is reversed. These results suggest that Ssp-3-bearing molecules participate in the process of parasite internalization.NYU MED CTR,DEPT PATHOL,550 1ST AVE,NEW YORK,NY 10016NYU MED CTR,KAPLAN CANC CTR,NEW YORK,NY 10016SLOAN KETTERING MEM CANC CTR,DEWITT WALLACE LAB,NEW YORK,NY 10021ESCOLA PAULISTA MED SCH,DISCIPLINA BIOL CELULAR,BR-04023 São Paulo,BRAZILESCOLA PAULISTA MED SCH,DISCIPLINA BIOL CELULAR,BR-04023 São Paulo,BRAZILWeb of Scienc

Perfil proteômico hipocampal em epilepsia do lobo temporal

In this study we used proteomics approaches to obtain the protein profile of human epileptic hippocampi (N=6) and control hippocampi obtained from autopsy (N=6). We used two-dimensional gel electrophoresis (2-D) coupled to HPLC and Mass spectroscopy (MALDI-TOF) to identify proteins differentially expressed. Nine proteins were differentially expressed comparing the hippocampus of patients with the hippocampus of control. Proteins that were increased in the hippocampus of patients with TLE were: isoform 1 of serum albumin, HSP 70, dihydropyrimidinase-related protein 2, isoform 1 of myelin basic protein, proton ATPase catalytic subunit A, and dihydrolipoyllysine-residue acethyltransferase component of pyruvate dehydrogenase complex. The expression of isoform 3 of spectrin alpha chain (fodrin) was down-regulated while the proteins glutathione S-transferase P and the DJ-1 (PARK7) were detected only in the hippocampus of patients with TLE. Taken together, our results provide evidence supporting a direct link between metabolic disturb and oxidative damage related to pathophysiology of TLE. Besides, indicates biomarkers for further investigations as therapies targeted to epilepsy.No presente estudo empregou-se o método de proteômica para obter a expressão diferencial de proteínas em hipocampo de pacientes com epilepsia do lobo temporal (ELT) (N=6) comparado a hipocampos controle obtidos por meio de autópsia (N=6). O estudo foi feito por meio de eletroforese bidimensional, acoplada a HPLC e espectroscopia de massa. As proteínas que tiveram expressão aumentada no hipocampo de pacientes com ELT foram: isoforma-1 da soroalbumina, HSP70, diidropirimidinase-2, isoforma-1 da proteína básica da mielina, subunidade catalítica A da próton ATPase, glutationa S-transferase P, proteína DJ-1 (PARK7), e resíduo diidropolilisina do componente acetil-transferase do complexo da piruvato desidrogenase. A expressão da isoforma-3 da cadeia alfa da espectrina (fodrina) foi menor no hipocampo de pacientes com epilepsia do lobo temporal e as proteínas glutationa S-transferase P e PARK7 foram detectadas somente no tecido epiléptico. Assim, nossos resultados trazem evidencias sobre a direta relação entre distúrbio metabólico e dano oxidativo na patofisiologia da ELT. Além disto, o estudo indica biomarcadores para futuras investigações como alvos terapêuticos para epilepsia.UNIFESP Departamento de Neurologia e NeurocirurgiaUNIFESP Departamento de Microbiologia, Imunologia e ParasitologiaFaculdade de Medicina da Universidade de São Paulo Instituto do Coração Departamento de Anatomia PatológicaUNIFESP, Depto. de Neurologia e NeurocirurgiaUNIFESP, Depto. de Microbiologia, Imunologia e ParasitologiaSciEL

Chromatin modifications in trypanosomes due to stress

Trypanosomatids are parasites of worldwide distribution with relevant importance in human and veterinary health, which inhabit invertebrate and vertebrate hosts, such that they are exposed to large environmental variations during their life cycle. the signalling mechanisms and molecular basis that lead these parasites to adjust to such distinct conditions are beginning to be understood, and are somehow related to modifications in gene expression. Although the control of gene expression in this group of organisms happens predominantly at the post-transcriptional level, they present modifications in chromatin that has been implicated in transcription initiation, replication and DNA repair. Here, we explore the current scenario of chromatin alterations in these protozoans and how these changes affect transcription, replication and DNA repair in response to environmental modifications.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, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilWeb of Scienc

Mechanism of resistance to lysis by the alternative complement pathway in Trypanosoma cruzi trypomastigotes: effect of specific monoclonal antibody

ESCOLA PAULISTA MED,DEPT MICROBIOL IMMUNOL & PARASITOL,RUA BOTUCATU 862 80 ANDAR,BR-04023 SAO PAULO,SP,BRAZILESCOLA PAULISTA MED,DEPT MICROBIOL IMMUNOL & PARASITOL,RUA BOTUCATU 862 80 ANDAR,BR-04023 SAO PAULO,SP,BRAZILWeb of Scienc

Stress Induces Changes in the Phosphorylation of Trypanosoma cruzi RNA Polymerase II, Affecting Its Association with Chromatin and RNA Processing

The phosphorylation of the carboxy-terminal heptapeptide repeats of the largest subunit of RNA polymerase II (Pol II) controls several transcription-related events in eukaryotes. Trypanosomatids lack these typical repeats and display an unusual transcription control. RNA Pol II associates with the transcription site of the spliced leader (SL) RNA, which is used in the trans-splicing of all mRNAs transcribed on long polycistronic units. We found that Trypanosoma cruzi RNA Pol II associated with chromatin is highly phosphorylated. When transcription is inhibited by actinomycin D, the enzyme runs off from SL genes, remaining hyperphosphorylated and associated with polycistronic transcription units. Upon heat shock, the enzyme is dephosphorylated and remains associated with the chromatin. Transcription is partially inhibited with the accumulation of housekeeping precursor mRNAs, except for heat shock genes. DNA damage caused dephosphorylation and transcription arrest, with RNA Pol II dissociating from chromatin although staying at the SL. in the presence of calyculin A, the hyperphosphorylated form detached from chromatin, including the SL loci. These results indicate that in trypanosomes, the unusual RNA Pol II is phosphorylated during the transcription of SL and polycistronic operons. Different types of stresses modify its phosphorylation state, affecting pre-RNA processing

Trypanosoma cruzi

Trypanosoma cruzi is the etiological agent of American trypanosomiasis, or Chagas disease, and is transmitted mainly by blood-sucking reduviid insects in endemic countries. Metacyclic trypomastigotes released in the feces during the insect blood meal enter a mammalian host through skin wounds or mucosal membranes and invade sur- rounding cells. After cell invasion, metacyclic trypomastigotes are restrained within a parasitophorous vacuole (PV), from where they escape, transform into amastigotes, and multiply in the cytosol. Later, following binary division, amastigotes differentiate back into highly motile trypomastigotes that are released upon cell lysis. They can infect neighboring cells, migrate to different tissues, or be ingested by an insect vector. The parasites in the tissues, as- sociated with an immune response, contribute to the chronic symptoms of the disease. Reactive oxygen species (ROS), among other factors, play an important role during parasite multiplication and interstage transformation.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)CNPq: 424729/2018FAPESP: 2018/09948-

TRANS-SIALIDASE and SIALIC-ACID ACCEPTERS FROM INSECT TO MAMMALIAN STAGES of TRYPANOSOMA-CRUZI

ESCOLA PAULISTA MED,DEPT MICROBIOL IMMUNOL & PARASITOL,R BOTUCATU 862,BR-04023062 São Paulo,SP,BRAZILESCOLA PAULISTA MED,DEPT MICROBIOL IMMUNOL & PARASITOL,R BOTUCATU 862,BR-04023062 São Paulo,SP,BRAZILWeb of Scienc

Chromatin and nuclear organization in Trypanosoma cruzi

A total of 100 years have passed since the discovery of the protozoan Trypanosoma cruzi, the etiologic agent of Chagas' disease. Since its discovery, the molecular and cellular biology of this early divergent eukaryote, as well as its interactions with the mammalian and insect hosts, has progressed substantially. It is now clear that this parasite presents unique mechanisms controlling gene expression, DNA replication, cell cycle and differentiation, generating several morphological forms that are adopted to survive in different hosts, in recent years, the relationship between the chromatin structure and nuclear organization with the unusual transcription, splicing, DNA replication and DNA repair mechanisms have been investigated in T. cruzi. This article reviews the relevant aspects of these mechanisms in relation to chromatin and nuclear organization.Universidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, BrazilInst Butantan, Parasitol Lab, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, BrazilWeb of Scienc

A Novel Monoclonal Antibody Against the C-terminus of beta-Tubulin Recognizes Endocytic Organelles in Trypanosoma cruzi

Microtubule cytoskeleton is a dynamic structure involved in the maintenance of eukaryote cell shape, motion of cilia and flagellum, and intracellular movement of vesicles and organelles. Many antibodies against tubulins have been described, most of them against the C-terminal portion, which is exposed at the outside of the microtubules. By generating a novel set of monoclonal antibodies against the cytoskeleton of Trypanosoma cruzi, a flagellate protozoan that causes Chagas' disease, we selected a clone (mAb 3G4) that recognizes beta-tubulin. The epitope for mAb 3G4 was mapped by pepscan to a highly conserved sequence motif found between alpha-helices 11 and 12 of the C-terminus of beta-tubulin in eukaryotes. It labels vesicular structures in both T. cruzi and mammalian cells, colocalizing respectively with a major cysteine protease (Cruzipain) and lysosome associated protein (LAMP2) respectively, but it does not label regular microtubules on these cellular models. We propose that the epitope recognized by mAb 3G4 is exposed only in a form of tubulin associated with endosomes.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ Fed Sao Paulo, Dept Microbiol Imunol & Parasitol, BR-04023062 Sao Paulo, BrazilUniv Fed Sao Paulo, Inst Ciencia & Tecnol, BR-12231280 Sao Jose Dos Campos, BrazilForschungszentrum Karlsruhe, KIT, Inst Biol Interfaces, D-76021 Karlsruhe, GermanyUniv Fed Sao Paulo, Dept Microbiol Imunol & Parasitol, BR-04023062 Sao Paulo, BrazilUniv Fed Sao Paulo, Inst Ciencia & Tecnol, BR-12231280 Sao Jose Dos Campos, BrazilWeb of Scienc