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- ItemAcesso aberto (Open Access)Analysis and chromosomal mapping of Leishmania (Leishmania) amazonensis amastigote expressed sequence tags(Instituto Oswaldo Cruz, Ministério da Saúde, 2007-09-01) Gentil, Luciana Girotto [UNIFESP]; Lasakosvitsch, Fernanda [UNIFESP]; Franco da Silveira, José [UNIFESP]; Santos, Márcia Regina Machado dos [UNIFESP]; Barbiéri, Clara Lúcia [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)The characterization of expressed sequence tags (ESTs) generated from a cDNA library of Leishmania (Leishmania) amazonensis amastigotes is described. The sequencing of 93 clones generated new L. (L.) amazonensis ESTs from which 32% are not related to any other sequences in database and 68% presented significant similarities to known genes. The chromosome localization of some L. (L.) amazonensis ESTs was also determined in L. (L.) amazonensis and L. (L.) major. The characterization of these ESTs is suitable for the genome physical mapping, as well as for the identification of genes encoding cysteine proteinases implicated with protective immune responses in leishmaniasis.
- ItemAcesso aberto (Open Access)A century of research: what have we learned about the interaction of Trypanosoma cruzi with host cells?(Instituto Oswaldo Cruz, Ministério da Saúde, 2009-07-01) Alves, Maria Julia Manso; Mortara, Renato Arruda [UNIFESP]; Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP)Since the discovery of Trypanosoma cruzi and the brilliant description of the then-referred to new tripanosomiasis by Carlos Chagas 100 years ago, a great deal of scientific effort and curiosity has been devoted to understanding how this parasite invades and colonises mammalian host cells. This is a key step in the survival of the parasite within the vertebrate host, and although much has been learned over this century, differences in strains or isolates used by different laboratories may have led to conclusions that are not as universal as originally interpreted. Molecular genotyping of the CL-Brener clone confirmed a genetic heterogeneity in the parasite that had been detected previously by other techniques, including zymodeme or schizodeme (kDNA) analysis. T. cruzi can be grouped into at least two major phylogenetic lineages: T. cruzi I, mostly associated with the sylvatic cycle and T. cruzi II, linked to human disease; however, a third lineage, T. cruziIII, has also been proposed. Hybrid isolates, such as the CL-Brener clone, which was chosen for sequencing the genome of the parasite (Elias et al. 2005, El Sayed et al. 2005a), have also been identified. The parasite must be able to invade cells in the mammalian host, and many studies have implicated the flagellated trypomastigotes as the main actor in this process. Several surface components of parasites and some of the host cell receptors with which they interact have been described. Herein, we have attempted to identify milestones in the history of understanding T. cruzi- host cell interactions. Different infective forms of T. cruzi have displayed unexpected requirements for the parasite to attach to the host cell, enter it, and translocate between the parasitophorous vacuole to its final cytoplasmic destination. It is noteworthy that some of the mechanisms originally proposed to be broad in function turned out not to be universal, and multiple interactions involving different repertoires of molecules seem to act in concert to give rise to a rather complex interplay of signalling cascades involving both parasite and cellular components.
- ItemSomente MetadadadosCloning and characterisation of a cysteine proteinase gene expressed in amastigotes of Leishmania (L.) amazonensis(Elsevier B.V., 2003-04-01) Lasakosvitsch, F.; Gentil, L. G.; Santos, MRM dos; Silveira, J. F. da; Barbieri, C. L.; Universidade Federal de São Paulo (UNIFESP)The present study describes the cloning and characterisation of a gene encoding a cysteine proteinase isoform, Llacys1, expressed in amastigote forms of Leishmania (L.) amazonensis. Recombinant clones containing the Llacys1 gene were isolated from genomic DNA by PCR amplification and screening of an amastigote cDNA library. Sequence analysis of the Llacys1 gene showed a high identity to sequence of Leishmania (L.) pifanoi Lpcys1, Leishmania (L.) major cpa, Leishmania (L.) mexicana LCPa, and Leishmania (L.) chagasi Ldccys2. the Llacys1 gene is present in a single copy per L. (L.) amazonensis haploid genome and was mapped on a chromosome of approximately 700 kb. Two transcripts of the Llacys1 gene were identified, one of 2.4 kb transcribed in both forms of L (L.) amazonensis, and another of 1.6 kb weakly expressed in amastigotes. Related forms of Llacys1 gene exist in other species of Leishmania genus, including L (L.) major, L. (L.) mexicana, L. (L.) chagasi and Leishmania (V.) braziliensis. the Llacys1 expression in Escherichia coli was obtained when the nucleotide sequence corresponding to the signal sequence was deleted, suggesting that this signal sequence was recognised by Escherichia coli and cleaved, generating a truncated protein. (C) 2003 Australian Society for Parasitology Inc. Published by Elsevier B.V. All rights reserved
- ItemSomente MetadadadosCross-priming of long lived protective CD8(+) T cells against Trypanosoma cruzi infection: Importance of a TLR9 agonist and CD4(+) T cells(Elsevier B.V., 2007-08-10) Alencar, Bruna C. G. de; Araujo, Adriano F. S.; Penido, Marcus L. O.; Gazzinelli, Ricardo T.; Rodrigues, Mauricio M.; Universidade Federal de São Paulo (UNIFESP); Fiocruz MS; Universidade Federal de Minas Gerais (UFMG)We recently described that vaccination of mice with a gluthatione S transferase fusion protein representing amino acids 261-500 of the Amastigote Surface Protein-2 efficiently cross-primed protective CD8(+) T cells against a lethal challenge with the human protozoan parasite Trypanosoma cruzi. in this study, we initially established that this protective immunity was long lived. Subsequently, we studied the importance of TLR9 agonist CpG ODN 1826, TLR4 and CD4(+) T cells for the generation of these protective CD8(+) T cells. We found that: (i) the TLR9 agonist CpG ODN 1826 improved the efficiency of protective immunity; (ii) TLR4 is not relevant for priming of specific CD8(+) T cells; (iii) CD4(+) T cells are critical for priming of memory/protective CD8(+) T cells. (c) 2007 Elsevier B.V. All rights reserved.
- ItemSomente MetadadadosIdentification of a 30 kDa antigen from Leishmania (L.) chagasi amastigotes implicated in protective cellular responses in a murine model(Elsevier B.V., 2000-04-24) Pinto, A. R.; Beyrodt, CGP; Antonio, R.; Lopes, M.; Barbieri, C. L.; Universidade Federal de São Paulo (UNIFESP); Inst Adolfo LutzAn antigen of apparent molecular mass of 30 kDa, termed p30, was purified from Leishmania (L.) chagasi amastigotes after separation of parasite extracts by sodium dodecyl sulfate-polyacrylamide eel eletroctrophoresis followed by electroelution. the use of the purified antigen in lymphocyte cultures from BALB/c mice previously immunised with L. (L.) chagasi amastigotes led to high levels of proliferation. Animal immunisation with p30 plus complete Freund's adjuvant either by subcutaneous or intraperitoneal route led to comparable antigenic stimulation. Similar stimulation indices induced by p30 were also obtained when animals were immunised with Corynebacterium parvum as adjuvant by the intraperitoneal route. Detection of IL-2 and IFN-gamma in the supernatants from lymphocytes stimulated by p30 and inhibition of the production of these lymphokines in the presence of anti-CD4, strongly indicated the involvement of the Th1 subset in the responses elicited by p30 antigen. Immunisation of BALB/c mice with p30 provided partial protection against challenge with L. (L.) chagasi amastigotes, indicating a protective role for p30 and that Th1 can be related to accquired resistance to visceral leishmaniasis in a murine model. Further characterisation studies were performed by the use of a monoclonal antibody directed to a cysteine proteinase of 30 kDa from L. (L.) amazonensis amastigotes. Despite the cross-reactivity presented by p30 from both Leishmania species, the p30 from L. (L.) chagasi amastigotes lacks proteolytic activity. (C) 2000 Published by Elsevier Science Ltd on behalf of the Australian Society for Parasitology Inc. All rights reserved.
- ItemSomente MetadadadosImmunologically relevant strain polymorphism in the Amastigote Surface Protein 2 of Trypanosoma cruzi(Elsevier B.V., 2007-07-01) Claser, Carla; Espindola, Noeli Maria; Sasso, Gisela; Vaz, Adelaide Jose; Boscardin, Silvia B.; Rodrigues, Mauricio M.; Universidade Federal de São Paulo (UNIFESP); Universidade de São Paulo (USP)Several evidences suggest that the Amastigote Surface Protein-2 (ASP-2) of Trypanosonia cruzi is an important target for immunity during infection. Based on this, we considered it important to evaluate its strain polymorphism. Initially, we observed the presence of conserved cross-reactive epitopes in amastigotes of all parasite strains tested. in addition, the predicted amino acid sequences of the genes isolated from the cDNA of amastigotes of CL-Brener, Tulahuen, Colombian and G strains displayed a high degree of identity (> 80%) to the previously described C Genes of ASP-2. Unexpectedly, Sylvio X10/4 and G strains expressed a new isoform of ASP-2 with limited identity to the previously described genes, but with a high degree of identity when compared to each other. Immunological studies confirmed the presence of cross-reactive epitopes between recombinant proteins representing the different isoforms of ASP-2. However, the genetic vaccination of mice with the new isoform of asp-2 gene expressed by the G strain failed to provide the same degree of protective immunity to a challenge by parasites of the Y strain as did asp-2 genes of Y or CL-Brener strains. in summary, we found that few strains can express different isoforms of ASP-2 which may not share cross-protective epitopes. (c) 2007 Elsevier Masson SAS. All rights reserved.
- ItemSomente MetadadadosInhibition of Leishmania (Leishmania) amazonensis growth and infectivity by aureobasidin A(Oxford Univ Press, 2007-03-01) Tanaka, Ameria K. [UNIFESP]; Valero, Valderez B [UNIFESP]; Takahashi, Helio K. [UNIFESP]; Straus, Anita H. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Objectives: To study the effect of aureobasidin A, an inhibitor of inositol phosphorylceramide (IPC) synthase, on Leishmania growth and infectivity.Methods: Effects of aureobasidin A were determined for: (i) promastigote growth in axenic culture; (ii) promastigote infectivity in macrophage monolayers; (iii) development of footpad lesions in BALB/c mice; (iv) differentiation of amastigotes into promastigotes.Results: Aureobasidin A (20 mu M) inhibited 90% of Leishmania (Leishmania) amazonensis promastigote growth in axenic culture, but the parasites remained viable, i.e. growth curves returned to normal after aureobasidin A was removed from culture medium. the aureobasidin A IC50 was determined by MTT assay as 4.1 mu M for L. (L.) amazonensis promastigotes, 12.6 mu M for Leishmania (Leishmania) major and 13.7 mu M for Leishmania (Viannia) braziliensis. There was a significant delay in infection when L. (L.) amazonensis promastigotes pre-treated with aureobasidin A were inoculated into BALB/c mouse footpads. When aureobasidin A was added to cultured macrophages infected with amastigotes, the number of infected macrophages was reduced by >90%.Conclusions: Aureobasidin A is an interesting pharmacological tool to investigate the effect of lipid metabolism inhibition in Leishmania spp.
- ItemSomente MetadadadosInvasion of MDCK epithelial cells with altered expression of Rho GTPases by Trypanosoma cruzi amastigotes and metacyclic trypomastigotes of strains from the two major phylogenetic lineages(Elsevier B.V., 2004-04-01) Fernandes, A. B.; Mortara, R. A.; Universidade Federal de São Paulo (UNIFESP)In order to invade mammalian cells, Trypanosoma cruzi infective forms cause distinct rearrangements of membrane and host cell cytoskeletal components. Rho GTPases have been shown to regulate three separate signal transduction pathways, linking plasma membrane receptors to the assembly of distinct actin filament structures. Here, we examined the role of Rho GTPases on the interaction between different T cruzi infective forms of strains from the two major phylogenetic lineages with nonpolarized MDCK cells transfected with different Rho GTPase constructs. We compared the infectivity of amastigotes isolated from infected cells (intracellular amastigotes) with forms generated from the axenic differentiation of trypomastigotes (extracellular amastigotes), and also with metacyclic trypomastigotes.No detectable effect of GTPase expression was observed on metacyclic trypomastigote invasion and parasites of Y and CL (T cruzi 11) strains invaded to similar degrees all MDCK transfectants, and were more infective than either G or Tulahuen (T cruzi I) strains. Intracellular amastigotes were complement sensitive and showed very low infectivity towards the different transfectants regardless of the parasite strain. Complement-resistant T cruzi I extracellular amastigotes, especially of the G strain, were more infective than T cruzi 11 parasites, particularly for the Rac1V12 constitutively active GTPase transfectant. the fact that in Rac1N17 dominant-negative cells, the invasion of G strain extracellular amastigotes was specifically inhibited suggested an important role for Rac1 in this process. (C) 2004 Elsevier SAS. All rights reserved.
- ItemSomente MetadadadosInvolvement of host cell heparan sulfate proteoglycan in Trypanosoma cruzi amastigote attachment and invasion(Cambridge Univ Press, 2011-04-01) Bambino-Medeiros, R.; Oliveira, F. O. R.; Calvet, C. M.; Vicente, D.; Toma, L. [UNIFESP]; Krieger, M. A.; Meirelles, M. N.; Pereira, M. C. S.; Fiocruz MS; Universidade Federal de São Paulo (UNIFESP); Inst Biol Mol Parana FIOCRUZCell surface glycosaminoglycans (GAGs) play an important role in the attachment and invasion process of a variety of intracellular pathogens. We have previously demonstrated that heparan sulfate proteoglycans (HSPG) mediate the invasion of trypomastigote forms of Trypanosoma cruzi in cardiomyocytes. Herein, we analysed whether GAGs are also implicated in amastigote invasion. Competition assays with soluble GAGs revealed that treatment of T. cruzi amastigotes with heparin and heparan sulfate leads to a reduction in the infection ratio, achieving 82% and 65% inhibition of invasion, respectively. Other sulfated GAGs, such as chondroitin sulfate, dermatan sulfate and keratan sulfate, had no effect on the invasion process. in addition, a significant decrease in infection occurred after interaction of amastigotes with GAG-deficient Chinese Hamster Ovary (CHO) cells, decreasing from 20% and 28% in wild-type CHO cells to 5% and 9% in the mutant cells after 2 h and 4 h of infection, respectively. These findings suggest that amastigote invasion also involves host cell surface heparan sulfate proteoglycans. the knowledge of the mechanism triggered by heparan sulfate-binding T. cruzi proteins may provide new potential candidates for Chagas disease therapy.
- ItemAcesso aberto (Open Access)Mammalian cell invasion and intracellular trafficking by Trypanosoma cruzi infective forms(Academia Brasileira de Ciências, 2005-03-01) Mortara, Renato Arruda [UNIFESP]; Andreoli, Walter Kindro [UNIFESP]; Taniwaki, Noemi Nosomi [UNIFESP]; Fernandes, Adriana Barrinha [UNIFESP]; Silva, Claudio Vieira da [UNIFESP]; Fernandes, Maria Cecília Di Ciero [UNIFESP]; L'Abbate, Carolina [UNIFESP]; Silva, Solange da [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Trypanosoma cruzi, the etiological agent of Chagas disease, occurs as different strains or isolates that may be grouped in two major phylogenetic lineages: T. cruzi I, associated with the sylvatic cycle and T. cruzi II, linked to the human disease. In the mammalian host the parasite has to invade cells and many studies implicated the flagellated trypomastigotes in this process. Several parasite surface components and some of host cell receptors with which they interact have been identified. Our work focused on how amastigotes, usually found growing in the cytoplasm, can invade mammalian cells with infectivities comparable to that of trypomastigotes. We found differences in cellular responses induced by amastigotes and trypomastigotes regarding cytoskeletal components and actin-rich projections. Extracellularly generated amastigotes of T. cruzi I strains may display greater infectivity than metacyclic trypomastigotes towards cultured cell lines as well as target cells that have modified expression of different classes of cellular components. Cultured host cells harboring the bacterium Coxiella burnetii allowed us to gain new insights into the trafficking properties of the different infective forms of T. cruzi, disclosing unexpected requirements for the parasite to transit between the parasitophorous vacuole to its final destination in the host cell cytoplasm.
- ItemSomente MetadadadosTrypanosoma cruzi: Effect of protein kinase inhibitors and cytoskeletal protein organization and expression on host cell invasion by amastigotes and metacyclic trypomastigotes(Academic Press Inc, 1998-09-01) Procopio, D. O.; Silva, S. da; Cunningham, C. C.; Mortara, R. A.; Universidade Federal de São Paulo (UNIFESP)Although trypomastigotes are regarded as the classic infective forms of T. cruzi, amastigotes generated extracellularly or released from infected cells during lysis may circulate and infect other cells. We have compared the infectivity of metacyclic trypomastigotes and extracellular amastigotes toward HeLa and Vero cells and observed that amastigotes were capable of invading both HeLa and Vero cells to a much higher degree than the corresponding metacyclic forms. Second, cell microfilament or microtubule disruption inhibited amastigote but not trypomastigote entry. Third, cells with altered expression in cytoskeletal components (ABP or gelsolin) internalize amastigotes and trypomastigotes with highly contrasting fashion. Fourth, protein kinase inhibitors such as genistein and staurosporine affect the internalization of amastigotes and trypomastigotes in a host-cell-dependent manner. Our results suggest that extracellular amastigotes and metacyclic trypomastigotes utilize mechanisms to invade host cells with particular features for each I: cruzi form and for each host cell. When internalized, both forms associate to lysosomes of HeLa cells. (C) 1998 Academic Press.