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Renato A Mortara - One of the best experts on this subject based on the ideXlab platform.
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host cell actin remodeling in response to trypanosoma cruzi trypomastigote versus amastigote entry
Sub-cellular biochemistry, 2008Co-Authors: Renato A Mortara, Walter K. Andreoli, Maria Cecilia Fernandes, Claudio Vieira Da Silva, Adriana Barrinha Fernandes, Carolina Labbate, Solange Da SilvaAbstract:Trypanosoma cruzi is the protozoan parasite that causes Chagas’ disease, a highly prevalent vector-borne disease in Latin America. Chagas’ disease is a major public health problem in endemic regions with an estimated 18 million people are infected with T. cruzi and another 100 million at risk (http://www.who.int/ctd/chagas/disease.htm). During its life cycle, T. cruzi alternates between triatomine insect vectors and mammalian hosts. While feeding on host’s blood, infected triatomines release in their feces highly motile and infective metacyclic Trypomastigotes that may initiate infection. Metacyclic Trypomastigotes promptly invade host cells (including gastric mucosa) and once free in the cytoplasm, differentiate into amastigotes that replicate by binary fission. Just before disruption of the parasite-laden cell, amastigotes differentiate back into Trypomastigotes which are then released into the tissue spaces and access the circulation. Circulating Trypomastigotes that disseminate the infection in the mammalian host may be taken up by feeding triatomines and may also transform, extracellu-larly, into amastigote-like forms.1 Unlike their intracellular counterparts, these amastigote-like forms,2 henceforth called amastigotes, are capable of infecting host cells.3, 4, 5, 6, 7 Studies in which the mechanisms of amastigote invasion of host cells have been compared to metacyclic trypomastigote entry have revealed interesting differences regarding the involvement of the target cell actin microfilament system.
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survival of trypanosoma cruzi metacyclic Trypomastigotes within coxiella burnetii vacuoles differentiation and replication within an acidic milieu
Microbes and Infection, 2006Co-Authors: Walter K. Andreoli, Noemi Nosomi Taniwaki, Renato A MortaraAbstract:Coxiella burnetii, the etiological agent of Q fever, is an obligate intracellular bacterium that resides within acidified vacuoles with secondary lysosomal characteristics. Infective stages of Trypanosoma cruzi, the causative agent of Chagas' disease, actively invade a wide variety of cells, a process followed by lysosomal recruitment. Recently, we have investigated and characterized early events that occur in Vero cells persistently colonized with C. burnetii when doubly infected with T. cruzi trypomastigote forms. Kinetic studies of trypomastigote transfer indicated that parasitophorous vacuoles (PV) of metacyclic Trypomastigotes are rapidly and efficiently fused to C. burnetii vacuoles. Based on these observations we have investigated the behavior of metacyclic Trypomastigotes within C. burnetii vacuoles beyond 12 h of co-infection inside Vero cells. Using indirect immunofluorescence with MAb against different developmental stages, it was possible to follow the T. cruzi differentiation process within C. burnetii vacuoles after up to 96 h post-invasion. We observed that metacyclic Trypomastigotes began to differentiate after 12 h of infection, and 24 h later amastigotes were the prevailing forms within C. burnetii vacuoles. T. cruzi amastigote replication within C. burnetii vacuoles was confirmed using video and time-lapse confocal microscopy and around 36 h of co-infection, cytokinesis took about 70 min to occur. After 72 h, we observed that amastigote forms seemed to escape from C. burnetii vacuoles. Labeling of amastigotes within C. burnetii vacuoles using a polyclonal antibody to C9 complement protein suggested that TcTOX (T. cruzi hemolysin) could play a role in parasite escape from C. burnetii. We concluded that T. cruzi has an outstanding adaptation capability and can survive within a hostile milieu such as C. burnetii vacuoles.
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Acidification modulates the traffic of Trypanosoma cruzi Trypomastigotes in Vero cells harbouring Coxiella burnetii vacuoles
International Journal for Parasitology, 2003Co-Authors: Walter K. Andreoli, Renato A MortaraAbstract:Abstract We studied the fate of different Trypanosoma cruzi trypomastigote forms after they invade Vero cells persistently colonised with Coxiella burnetii . When the invasion step was examined we found that persistent C. burnetii infection per se reduced only tissue-culture trypomastigote invasion, whereas raising vacuolar pH with Bafilomycin A1 and related drugs, increased invasion of both metacyclic and tissue-culture Trypomastigotes when compared with control Vero cells. Kinetic studies of trypomastigote transfer indicated that metacyclic Trypomastigotes parasitophorous vacuoles are more efficiently fused to C. burnetii vacuoles. The higher tissue-culture trypomastigote hemolysin and transialidase activities appear to facilitate their faster escape from the parasitophorous vacuole. Sialic acid deficient Lec-2 cells facilitate the escape of both forms. Endosomal–lysosomal sequential labelling with EEA1, LAMP-1, and Rab7 of the parasitophorous vacuoles formed during the entry of each infective form revealed that the phagosome maturation processes are also distinct. Measurements of C. burnetii vacuolar pH disclosed a marked preference for trypomastigote fusion with more acidic rickettsia vacuoles. Our results thus suggest that intravacuolar pH modulates the traffic of trypomastigote parasitophorous vacuoles in these doubly infected cells.
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Acidification modulates the traffic of Trypanosoma cruzi Trypomastigotes in Vero cells harbouring Coxiella burnetii vacuoles
International journal for parasitology, 2003Co-Authors: Walter K. Andreoli, Renato A MortaraAbstract:We studied the fate of different Trypanosoma cruzi trypomastigote forms after they invade Vero cells persistently colonised with Coxiella burnetii. When the invasion step was examined we found that persistent C. burnetii infection per se reduced only tissue-culture trypomastigote invasion, whereas raising vacuolar pH with Bafilomycin A1 and related drugs, increased invasion of both metacyclic and tissue-culture Trypomastigotes when compared with control Vero cells. Kinetic studies of trypomastigote transfer indicated that metacyclic Trypomastigotes parasitophorous vacuoles are more efficiently fused to C. burnetii vacuoles. The higher tissue-culture trypomastigote hemolysin and transialidase activities appear to facilitate their faster escape from the parasitophorous vacuole. Sialic acid deficient Lec-2 cells facilitate the escape of both forms. Endosomal ‐ lysosomal sequential labelling with EEA1, LAMP-1, and Rab7 of the parasitophorous vacuoles formed during the entry of each infective form revealed that the phagosome maturation processes are also distinct. Measurements of C. burnetii vacuolar pH disclosed a marked preference for trypomastigote fusion with more acidic rickettsia vacuoles. Our results thus suggest that intravacuolar pH modulates the traffic of trypomastigote parasitophorous vacuoles in these doubly infected cells. q 2003 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved
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phosphatidylinositol specific phospholipase c pi plc cleavage of gpi anchored surface molecules of trypanosoma cruzi triggers in vitro morphological reorganization of Trypomastigotes
Journal of Eukaryotic Microbiology, 2001Co-Authors: Renato A Mortara, F Vandekerckhove, Victor Nussenzweig, Lindamar M S Minelli, Juarez F RamalhopintoAbstract:Trypanosoma cruzi Trypomastigotes treated with phosphatidylinositol-specific phospholipase C (PI-PLC) in vitro are rapidly induced to differentiate into round forms. Using confocal microscopy, we were able to show that Trypomastigotes treated with PI-PLC initiate the process of flagellum remodeling by 30 sec after contact with the enzyme and amastigote-like forms are detected as early as 10 min after PI-PLC treatment. Scanning and transmission electron microscopy indicate that Trypomastigotes undergo a previously undescribed process of flagellum circularization and internalization. Analysis of the flagellar complex with monoclonal antibody 4D9 shows heterogeneous labeling among the parasites, suggesting a remodeling of these molecules. After PI-PLC treatment, parasites rapidly lose the surface marker Ssp-3 and 24 h post-treatment they begin to exhibit a circular nucleus and a rod-shaped kinetoplast. By flow cytometry analysis and confocal microscopy, the Ssp-4 amastigote-specific epitope can be detected on the parasite surface. This indicates that the release of trypomastigote GPI-anchored molecules by exogenous PI-PLC in vitro can trigger morphological changes.
Sergio Schenkman - One of the best experts on this subject based on the ideXlab platform.
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comparative transcriptome profiling of virulent and non virulent trypanosoma cruzi underlines the role of surface proteins during infection
PLOS Pathogens, 2017Co-Authors: Trey A Belew, Sergio Schenkman, Caroline Junqueira, Gabriela F Rodriguesluiz, Bruna M Valente, Antonio Edson R Oliveira, Rafael B Polidoro, Luciana W Zuccherato, Daniella Castanheira Bartholomeu, Ricardo T GazzinelliAbstract:Trypanosoma cruzi, the protozoan that causes Chagas disease, has a complex life cycle involving several morphologically and biochemically distinct stages that establish intricate interactions with various insect and mammalian hosts. It has also a heterogeneous population structure comprising strains with distinct properties such as virulence, sensitivity to drugs, antigenic profile and tissue tropism. We present a comparative transcriptome analysis of two cloned T. cruzi strains that display contrasting virulence phenotypes in animal models of infection: CL Brener is a virulent clone and CL-14 is a clone that is neither infective nor pathogenic in in vivo models of infection. Gene expression analysis of Trypomastigotes and intracellular amastigotes harvested at 60 and 96 hours post-infection (hpi) of human fibroblasts revealed large differences that reflect the parasite’s adaptation to distinct environments during the infection of mammalian cells, including changes in energy sources, oxidative stress responses, cell cycle control and cell surface components. While extensive transcriptome remodeling was observed when Trypomastigotes of both strains were compared to 60 hpi amastigotes, differences in gene expression were much less pronounced when 96 hpi amastigotes and Trypomastigotes of CL Brener were compared. In contrast, the differentiation of the avirulent CL-14 from 96 hpi amastigotes to extracellular Trypomastigotes was associated with considerable changes in gene expression, particularly in gene families encoding surface proteins such as trans-sialidases, mucins and the mucin associated surface proteins (MASPs). Thus, our comparative transcriptome analysis indicates that the avirulent phenotype of CL-14 may be due, at least in part, to a reduced or delayed expression of genes encoding surface proteins that are associated with the transition of amastigotes to Trypomastigotes, an essential step in the establishment of the infection in the mammalian host. Confirming the role of members of the trans-sialidase family of surface proteins for parasite differentiation, transfected CL-14 constitutively expressing a trans-sialidase gene displayed faster kinetics of trypomastigote release in the supernatant of infected cells compared to wild type CL-14.
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expression of trypomastigote trans sialidase in metacyclic forms of trypanosoma cruzi increases parasite escape from its parasitophorous vacuole
Cellular Microbiology, 2006Co-Authors: Sergio S C Rubindecelis, Haruki Uemura, Nobuko Yoshida, Sergio SchenkmanAbstract:Summary Trypanosoma cruzi actively invades mammalian cells by forming parasitophorous vacuoles (PVs). After entry, the parasite has to escape from these vacuoles in order to replicate inside the host cell cytosol. Trans -sialidase (TS), a parasite enzyme that is used to obtain sialic acid from host glycoconjugates, has been implicated in cell invasion and PV exit, but how the enzyme acts in these processes is still unknown. Here we show that Trypomastigotes derived from infected mammalian cells express and release 20 times more TS activity than axenic metacyclic Trypomastigotes, which correspond to the infective forms derived from the insect vector. Both forms have the same capacity to invade mammalian cells, but cell derived Trypomastigotes exit earlier from the vacuole. To test whether high TS expression is responsible for this increased exit from the PV, trypomastigote TS was expressed on the surface of metacyclic forms. Transfected and non-transfected metacyclics attached to and invaded HeLa or CHO cells equally. In contrast, metacyclics expressing TS on the surface escaped earlier from the vacuole than non-transfected metacyclics, or metacyclics expressing TS in their cytoplasm. Sialic acid may act as a barrier, which is removed by surface and/or secreted TS, because all types of parasites escaped earlier from the vacuoles of sialic acid-deficient Lec 2 cells than wild-type CHO cells. In addition, Trypomastigotes and metacyclic forms expressing TS differentiated earlier into amastigotes. These results indicate that the increased expression of TS in cell-derived Trypomastigotes is responsible for the earlier exit from the PV to the cytoplasm and their subsequent differentiation into amastigotes.
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Expression of trypomastigote trans‐sialidase in metacyclic forms of Trypanosoma cruzi increases parasite escape from its parasitophorous vacuole
Cellular microbiology, 2006Co-Authors: Sergio S. C. Rubin-de-celis, Nobuko Yoshida, Haruki Uemura, Sergio SchenkmanAbstract:Summary Trypanosoma cruzi actively invades mammalian cells by forming parasitophorous vacuoles (PVs). After entry, the parasite has to escape from these vacuoles in order to replicate inside the host cell cytosol. Trans -sialidase (TS), a parasite enzyme that is used to obtain sialic acid from host glycoconjugates, has been implicated in cell invasion and PV exit, but how the enzyme acts in these processes is still unknown. Here we show that Trypomastigotes derived from infected mammalian cells express and release 20 times more TS activity than axenic metacyclic Trypomastigotes, which correspond to the infective forms derived from the insect vector. Both forms have the same capacity to invade mammalian cells, but cell derived Trypomastigotes exit earlier from the vacuole. To test whether high TS expression is responsible for this increased exit from the PV, trypomastigote TS was expressed on the surface of metacyclic forms. Transfected and non-transfected metacyclics attached to and invaded HeLa or CHO cells equally. In contrast, metacyclics expressing TS on the surface escaped earlier from the vacuole than non-transfected metacyclics, or metacyclics expressing TS in their cytoplasm. Sialic acid may act as a barrier, which is removed by surface and/or secreted TS, because all types of parasites escaped earlier from the vacuoles of sialic acid-deficient Lec 2 cells than wild-type CHO cells. In addition, Trypomastigotes and metacyclic forms expressing TS differentiated earlier into amastigotes. These results indicate that the increased expression of TS in cell-derived Trypomastigotes is responsible for the earlier exit from the PV to the cytoplasm and their subsequent differentiation into amastigotes.
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mucin like molecules form a negatively charged coat that protects trypanosoma cruzi Trypomastigotes from killing by human anti alpha galactosyl antibodies
Journal of Cell Science, 2000Co-Authors: Vera Lucia Pereirachioccola, Luiz R. Travassos, Michael A. J. Ferguson, Mauricio M Rodrigues, Alvaro Acostaserrano, Correia I De Almeida, T Soutopadron, Sergio SchenkmanAbstract:In the presence of sialic acid donors Trypanosoma cruzi acquires up to 10(7) sialic acid residues on its surface, in a reaction catalyzed by its unique trans-sialidase. Most of these sialic acid residues are incorporated into mucin-like glycoproteins. To further understand the biological role of parasite sialylation, we have measured the amount of mucin in this parasite. We found that both epimastigote and trypomastigote forms have the same number of mucin molecules per surface area, although Trypomastigotes have less than 10% of the amount of glycoinositol phospholipids, the other major surface glycoconjugate of T. cruzi. Based on the estimated surface area of each mucin, we calculated that these molecules form a coat covering the entire trypomastigote cell. The presence of the surface coat is shown by transmission electron microscopy of Ruthenium Red-stained parasites. The coat was revealed by binding of antibodies isolated from Chagasic patients that react with high affinity to alpha-galactosyl epitopes present in the mucin molecule. When added to the trypomastigote, these antibodies cause an extensive structural perturbation of the parasite coat with formation of large blebs, ultimately leading to parasite lysis. Interestingly, lysis is decreased if the mucin coat is heavily sialylated. Furthermore, addition of MgCl2 reverses the protective effect of sialylation, suggesting that the sialic acid negative charges stabilize the surface coat. Inhibition of sialylation by anti-trans-sialidase antibodies, found in immunized animals, or human Chagasic sera, also increase killing by anti-alpha-galactosyl antibodies. Therefore, the large amounts of sialylated mucins, forming a surface coat on infective trypomastigote forms, have an important structural and protective role.
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passive transfer of a monoclonal antibody specific for a sialic acid dependent epitope on the surface of trypanosoma cruzi Trypomastigotes reduces infection in mice
Infection and Immunity, 1997Co-Authors: G Franchin, Sergio Schenkman, Vera Lucia Pereirachioccola, Mauricio M RodriguesAbstract:Trypanosoma cruzi, the parasite that causes Chagas' disease, proliferates in the cytosol of mammalian cells. When the trypomastigote forms exit the infected cell, they become extensively sialylated because the parasite contains an enzyme called trans-sialidase. This enzyme efficiently catalyzes the transfer of bound sialic acid residues from host glycoconjugates to acceptors containing terminal beta-galactosyl residues on the parasite surface. The sialic acid acceptors are developmentally regulated mucin-like glycoproteins that are extremely abundant on the trypomastigote surface. In the present study, we determined whether passive transfer of monoclonal antibodies specific for sialic acid acceptors could reduce the acute infection induced by T. cruzi in a highly susceptible mouse strain. We found that passive transfer to naive mice of an immunoglobulin G1 monoclonal antibody directed to a sialylated epitope of these mucin-like glycoproteins significantly decreased parasitemia and the number of tissue parasites as measured by a DNA probe specific for T. cruzi. Upon challenge with Trypomastigotes, mice which received this antibody also had a significant increase in survival. A statistically significant reduction in parasitemia could be accomplished with relatively small doses of immunoglobulin, and Fab fragments alone could not mediate protective immunity. The precise mechanism of parasite elimination is unknown; however, this monoclonal antibody does not lyse Trypomastigotes in vitro in the presence of human complement or mouse spleen cells.
Walter K. Andreoli - One of the best experts on this subject based on the ideXlab platform.
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host cell actin remodeling in response to trypanosoma cruzi trypomastigote versus amastigote entry
Sub-cellular biochemistry, 2008Co-Authors: Renato A Mortara, Walter K. Andreoli, Maria Cecilia Fernandes, Claudio Vieira Da Silva, Adriana Barrinha Fernandes, Carolina Labbate, Solange Da SilvaAbstract:Trypanosoma cruzi is the protozoan parasite that causes Chagas’ disease, a highly prevalent vector-borne disease in Latin America. Chagas’ disease is a major public health problem in endemic regions with an estimated 18 million people are infected with T. cruzi and another 100 million at risk (http://www.who.int/ctd/chagas/disease.htm). During its life cycle, T. cruzi alternates between triatomine insect vectors and mammalian hosts. While feeding on host’s blood, infected triatomines release in their feces highly motile and infective metacyclic Trypomastigotes that may initiate infection. Metacyclic Trypomastigotes promptly invade host cells (including gastric mucosa) and once free in the cytoplasm, differentiate into amastigotes that replicate by binary fission. Just before disruption of the parasite-laden cell, amastigotes differentiate back into Trypomastigotes which are then released into the tissue spaces and access the circulation. Circulating Trypomastigotes that disseminate the infection in the mammalian host may be taken up by feeding triatomines and may also transform, extracellu-larly, into amastigote-like forms.1 Unlike their intracellular counterparts, these amastigote-like forms,2 henceforth called amastigotes, are capable of infecting host cells.3, 4, 5, 6, 7 Studies in which the mechanisms of amastigote invasion of host cells have been compared to metacyclic trypomastigote entry have revealed interesting differences regarding the involvement of the target cell actin microfilament system.
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survival of trypanosoma cruzi metacyclic Trypomastigotes within coxiella burnetii vacuoles differentiation and replication within an acidic milieu
Microbes and Infection, 2006Co-Authors: Walter K. Andreoli, Noemi Nosomi Taniwaki, Renato A MortaraAbstract:Coxiella burnetii, the etiological agent of Q fever, is an obligate intracellular bacterium that resides within acidified vacuoles with secondary lysosomal characteristics. Infective stages of Trypanosoma cruzi, the causative agent of Chagas' disease, actively invade a wide variety of cells, a process followed by lysosomal recruitment. Recently, we have investigated and characterized early events that occur in Vero cells persistently colonized with C. burnetii when doubly infected with T. cruzi trypomastigote forms. Kinetic studies of trypomastigote transfer indicated that parasitophorous vacuoles (PV) of metacyclic Trypomastigotes are rapidly and efficiently fused to C. burnetii vacuoles. Based on these observations we have investigated the behavior of metacyclic Trypomastigotes within C. burnetii vacuoles beyond 12 h of co-infection inside Vero cells. Using indirect immunofluorescence with MAb against different developmental stages, it was possible to follow the T. cruzi differentiation process within C. burnetii vacuoles after up to 96 h post-invasion. We observed that metacyclic Trypomastigotes began to differentiate after 12 h of infection, and 24 h later amastigotes were the prevailing forms within C. burnetii vacuoles. T. cruzi amastigote replication within C. burnetii vacuoles was confirmed using video and time-lapse confocal microscopy and around 36 h of co-infection, cytokinesis took about 70 min to occur. After 72 h, we observed that amastigote forms seemed to escape from C. burnetii vacuoles. Labeling of amastigotes within C. burnetii vacuoles using a polyclonal antibody to C9 complement protein suggested that TcTOX (T. cruzi hemolysin) could play a role in parasite escape from C. burnetii. We concluded that T. cruzi has an outstanding adaptation capability and can survive within a hostile milieu such as C. burnetii vacuoles.
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Acidification modulates the traffic of Trypanosoma cruzi Trypomastigotes in Vero cells harbouring Coxiella burnetii vacuoles
International Journal for Parasitology, 2003Co-Authors: Walter K. Andreoli, Renato A MortaraAbstract:Abstract We studied the fate of different Trypanosoma cruzi trypomastigote forms after they invade Vero cells persistently colonised with Coxiella burnetii . When the invasion step was examined we found that persistent C. burnetii infection per se reduced only tissue-culture trypomastigote invasion, whereas raising vacuolar pH with Bafilomycin A1 and related drugs, increased invasion of both metacyclic and tissue-culture Trypomastigotes when compared with control Vero cells. Kinetic studies of trypomastigote transfer indicated that metacyclic Trypomastigotes parasitophorous vacuoles are more efficiently fused to C. burnetii vacuoles. The higher tissue-culture trypomastigote hemolysin and transialidase activities appear to facilitate their faster escape from the parasitophorous vacuole. Sialic acid deficient Lec-2 cells facilitate the escape of both forms. Endosomal–lysosomal sequential labelling with EEA1, LAMP-1, and Rab7 of the parasitophorous vacuoles formed during the entry of each infective form revealed that the phagosome maturation processes are also distinct. Measurements of C. burnetii vacuolar pH disclosed a marked preference for trypomastigote fusion with more acidic rickettsia vacuoles. Our results thus suggest that intravacuolar pH modulates the traffic of trypomastigote parasitophorous vacuoles in these doubly infected cells.
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Acidification modulates the traffic of Trypanosoma cruzi Trypomastigotes in Vero cells harbouring Coxiella burnetii vacuoles
International journal for parasitology, 2003Co-Authors: Walter K. Andreoli, Renato A MortaraAbstract:We studied the fate of different Trypanosoma cruzi trypomastigote forms after they invade Vero cells persistently colonised with Coxiella burnetii. When the invasion step was examined we found that persistent C. burnetii infection per se reduced only tissue-culture trypomastigote invasion, whereas raising vacuolar pH with Bafilomycin A1 and related drugs, increased invasion of both metacyclic and tissue-culture Trypomastigotes when compared with control Vero cells. Kinetic studies of trypomastigote transfer indicated that metacyclic Trypomastigotes parasitophorous vacuoles are more efficiently fused to C. burnetii vacuoles. The higher tissue-culture trypomastigote hemolysin and transialidase activities appear to facilitate their faster escape from the parasitophorous vacuole. Sialic acid deficient Lec-2 cells facilitate the escape of both forms. Endosomal ‐ lysosomal sequential labelling with EEA1, LAMP-1, and Rab7 of the parasitophorous vacuoles formed during the entry of each infective form revealed that the phagosome maturation processes are also distinct. Measurements of C. burnetii vacuolar pH disclosed a marked preference for trypomastigote fusion with more acidic rickettsia vacuoles. Our results thus suggest that intravacuolar pH modulates the traffic of trypomastigote parasitophorous vacuoles in these doubly infected cells. q 2003 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved
Luiz R. Travassos - One of the best experts on this subject based on the ideXlab platform.
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mucin like molecules form a negatively charged coat that protects trypanosoma cruzi Trypomastigotes from killing by human anti alpha galactosyl antibodies
Journal of Cell Science, 2000Co-Authors: Vera Lucia Pereirachioccola, Luiz R. Travassos, Michael A. J. Ferguson, Mauricio M Rodrigues, Alvaro Acostaserrano, Correia I De Almeida, T Soutopadron, Sergio SchenkmanAbstract:In the presence of sialic acid donors Trypanosoma cruzi acquires up to 10(7) sialic acid residues on its surface, in a reaction catalyzed by its unique trans-sialidase. Most of these sialic acid residues are incorporated into mucin-like glycoproteins. To further understand the biological role of parasite sialylation, we have measured the amount of mucin in this parasite. We found that both epimastigote and trypomastigote forms have the same number of mucin molecules per surface area, although Trypomastigotes have less than 10% of the amount of glycoinositol phospholipids, the other major surface glycoconjugate of T. cruzi. Based on the estimated surface area of each mucin, we calculated that these molecules form a coat covering the entire trypomastigote cell. The presence of the surface coat is shown by transmission electron microscopy of Ruthenium Red-stained parasites. The coat was revealed by binding of antibodies isolated from Chagasic patients that react with high affinity to alpha-galactosyl epitopes present in the mucin molecule. When added to the trypomastigote, these antibodies cause an extensive structural perturbation of the parasite coat with formation of large blebs, ultimately leading to parasite lysis. Interestingly, lysis is decreased if the mucin coat is heavily sialylated. Furthermore, addition of MgCl2 reverses the protective effect of sialylation, suggesting that the sialic acid negative charges stabilize the surface coat. Inhibition of sialylation by anti-trans-sialidase antibodies, found in immunized animals, or human Chagasic sera, also increase killing by anti-alpha-galactosyl antibodies. Therefore, the large amounts of sialylated mucins, forming a surface coat on infective trypomastigote forms, have an important structural and protective role.
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Glycosylphosphatidylinositol-anchored mucin-like glycoproteins isolated from Trypanosoma cruzi Trypomastigotes initiate the synthesis of proinflammatory cytokines by macrophages.
Journal of immunology (Baltimore Md. : 1950), 1997Co-Authors: Maristela M. De Camargo, Igor C Almeida, Luiz R. Travassos, Michael A. J. Ferguson, Maria E. S. Pereira, Ricardo T GazzinelliAbstract:Components of Trypanosoma cruzi able to induce the production of IL-12 and other proinflammatory cytokines by macrophages were identified. Murine inflammatory macrophages were cultured with live parasites or with cellular components from different developmental forms of T. cruzi (i.e., Trypomastigotes, amastigotes, metacyclic Trypomastigotes, and epimastigotes), and the cytokine levels were measured after 24 and 48 h. Our results indicate that live Trypomastigotes or live amastigotes (but not live epimastigotes or live metacyclic Trypomastigotes) as well as trypomastigote extracts (but not extracts derived from epimastigotes) induce IL-12 and TNF-alpha synthesis by macrophages. Such biological activity is enhanced in membrane preparations from Trypomastigotes. Further enrichment of the trypomastigote-derived monokine-inducing factor was obtained by solvent extraction and hydrophobic-interaction chromatography. The resultant purified molecules are a family of closely related glycoconjugates with predominant species at 70 to 80 and 120 to 200 kDa. These molecules are composed of carbohydrate chains O-linked to a polypeptide backbone that is anchored to the trypomastigote membrane via a glycosylphosphatidylinositol structure. The trypomastigote-derived glycoconjugates are active in inducing cytokine synthesis by macrophages at concentrations of 100 ng/ml. These effects are highly potentiated by IFN-gamma. Mapping of the glycoconjugate molecules to characterize the structural requirements for macrophage activation suggested that nonsaturated acyl fatty acid chains and periodate-sensitive units from the glycosylphosphatidylinositol anchor are important elements for the infective trypomastigote form to initiate cytokine synthesis by macrophages.
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glycoconjugates of trypanosoma cruzi a 74 kd antigen of Trypomastigotes specifically reacts with lytic anti α galactosyl antibodies from patients with chronic chagas disease
Journal of Clinical Laboratory Analysis, 1993Co-Authors: Igor C Almeida, Greice M Krautz, Antoniana U Krettli, Luiz R. TravassosAbstract:Protective, lytic antibodies are believed to be correlated with active Typanosoma cruzi infection. In patients with chronic infection, antibodies lysing trypomastigote forms recognize chiefly α-galactosyl structures at the parasite surface. The target molecules on cell-derived Trypomastigotes that react with anti-α-galactosyl antibodies (anti-Gal) from patients with chronic Chagas disease were investigated. Glycoconjugates were isolated from Trypomastigotes and shown to absorb purified Chagasic (Ch) anti-Gal effectively as well as lytic antibodies from Ch sera. Active fractions were F2 (74 kD and 95.6 kD) and F3 (120–200 kD). A differential reactivity with antibodies from untreated Ch patients (trypanolytic) and from treated, presumably cured, individuals (not trypanolytic) was evident using F2 and F3 antigenic fractions. No cross-reactivity with heterologous sera (other infections) was observed. The F2 glycoconjugate (mostly 74 kD) can be used in the diagnosis of active Chagas infection, replacing the quantitative determination of complementmediated lysis. With the present sample of patients' sera and normal human sera, it showed 100% sensitivity and specificity. © 1993 Wiley-Liss, Inc.
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Complement-mediated lysis of Trypanosoma cruzi Trypomastigotes by human anti-alpha-galactosyl antibodies.
Journal of Immunology, 1991Co-Authors: Igor C Almeida, Sandra R. Milani, Philip A J Gorin, Luiz R. TravassosAbstract:Antibodies that lyse Trypomastigotes in a complement-mediated reaction are believed to be the main participants in the protection against virulent Trypanosoma cruzi. Antibodies with a specificity for alpha-galactosyl-containing determinants--generally called antiGal--were studied to determine their role in the lysis of trypomastigote forms. The titers of antiGal markedly increase in Chagas's disease. In the present study we demonstrate binding of this antibody to T. cruzi and the complement-mediated lysis of Trypomastigotes by antiGal. Lysis of metacyclic Trypomastigotes by whole Chagasic (Ch) serum or isolated antiGal fractions was equally inhibited by alpha- but not by beta-galactosides. Most of the lytic power of the Ch antiGal as well as of the whole Ch serum was removed by absorption on Synsorb-linked Gal alpha 1, 3Gal beta 1, 4GlcNAc followed by rabbit erythrocyte absorption. The Ch antiGal had a lower affinity for melibiose bound to agarose than for the trisaccharide linked to Synsorb, and was several times more effective in the immunolysis of Trypomastigotes than the corresponding antiGal from normal human serum. Lytic antibodies were partly absorbed by Serratia marcescens but not by Escherichia coli O111. A human volunteer immunized with an S. marcescens vaccine elicited a specific antiGal response that was lytic to Trypomastigotes (70% lysis). We suggest that in vivo high-affinity antiGal antibody clones, as occur in Ch patients, may significantly contribute to the destruction of the parasite, whereas low-affinity antiGal clones are much less effective in the protection against T. cruzi infection.
Victor Nussenzweig - One of the best experts on this subject based on the ideXlab platform.
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phosphatidylinositol specific phospholipase c pi plc cleavage of gpi anchored surface molecules of trypanosoma cruzi triggers in vitro morphological reorganization of Trypomastigotes
Journal of Eukaryotic Microbiology, 2001Co-Authors: Renato A Mortara, F Vandekerckhove, Victor Nussenzweig, Lindamar M S Minelli, Juarez F RamalhopintoAbstract:Trypanosoma cruzi Trypomastigotes treated with phosphatidylinositol-specific phospholipase C (PI-PLC) in vitro are rapidly induced to differentiate into round forms. Using confocal microscopy, we were able to show that Trypomastigotes treated with PI-PLC initiate the process of flagellum remodeling by 30 sec after contact with the enzyme and amastigote-like forms are detected as early as 10 min after PI-PLC treatment. Scanning and transmission electron microscopy indicate that Trypomastigotes undergo a previously undescribed process of flagellum circularization and internalization. Analysis of the flagellar complex with monoclonal antibody 4D9 shows heterogeneous labeling among the parasites, suggesting a remodeling of these molecules. After PI-PLC treatment, parasites rapidly lose the surface marker Ssp-3 and 24 h post-treatment they begin to exhibit a circular nucleus and a rod-shaped kinetoplast. By flow cytometry analysis and confocal microscopy, the Ssp-4 amastigote-specific epitope can be detected on the parasite surface. This indicates that the release of trypomastigote GPI-anchored molecules by exogenous PI-PLC in vitro can trigger morphological changes.
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the induction of trypanosoma cruzi trypomastigote to amastigote transformation by low ph
Parasitology, 1995Co-Authors: Stephen Tomlinson, F Vandekerckhove, Ute Frevert, Victor NussenzweigAbstract:Following cell invasion, Trypanosoma cruzi Trypomastigotes transform into amastigotes, which are the mammalian replicative forms of the parasite. Although amastigotes represent a critical stage in the life-cycle of T. cruzi , little is known of the factors controlling trypomastigote to amastigote transformation. Kanbera et al. (1990) observed that exposure of Trypomastigotes to acidic pH induced their transformation into rounded forms resembling amastigotes. We confirm their observation and, using two strains of T. cruzi , establish that these transformants are ultrastructurally and biochemically indistinguishable from natural amastigotes. Incubation of Trypomastigotes in medium at pH 5·0 for 2 h was sufficient to trigger their transformation into forms resembling amastigotes. Electron microscopical analysis confirmed that the kinetoplast structure, and general morphological features of the acid-induced, extracellular amastigotes were indistinguishable from those of intracellular-derived amastigotes. The extracellular transformation was accompanied by the acquisition of the stage-specific surface antigen of the naturally transformed amastigotes (Ssp-4), and loss of a stagespecific trypomastigote antigen (Ssp-3). Trypomastigotes incubated at neutral pH did not transform into amastigotes, and did not acquire the Ssp-4 epitope or lose the Ssp-3 epitope. Finally, acid-induced amastigotes subsequently incorporated [ 3 H]thymidine into their DNA, indicating that the important replicative property of intracellular amastigotes is also exhibited by these in vitro transformants. This effect of low pH appears to be of physiological relevance, and acid-induced extracellular transformation appears to represent a valid experimental technique for studies of the molecular mechanisms involved in the differentiation process.
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Role of sialic acid in the resistance of Trypanosoma cruzi Trypomastigotes to complement.
Journal of Immunology, 1994Co-Authors: Stephen Tomlinson, F Vandekerckhove, L C Pontes De Carvalho, Victor NussenzweigAbstract:Trypomastigotes of Trypanosoma cruzi, mammalian infective forms of the parasite, express an unusual cell surface trans-sialidase. This enzyme enables the parasite to rapidly sialylate its surface when supplied with alpha(2,3)-linked sialic acid from glycoconjugates in serum or on cell surfaces. Here we used a novel fluorescence-based, trypomastigote lysis assay to evaluate the role of sialic acid on the parasite's plasma membrane in providing protection against the complement cascade. Trypomastigotes were desialylated, and sialic acid removal was confirmed by a chemical assay and also by flow cytometry with the use of a mAb that recognizes a T. cruzi-sialylated epitope. Compared with sialylated Trypomastigotes, which were completely refractory to lysis by human serum, only about 5% of the desialylated Trypomastigotes were lysed by complement. However, further analysis revealed that the desialylated parasites had been resialylated during exposure to serum complement. Next we incubated desialylated Trypomastigotes with samples of desialylated human serum. Although the sialidase-treated serum retained its full hemolytic activity, lysis of Trypomastigotes increased only from 5 to 24%. This increase correlated with an enhanced deposition of complement protein C3 on the parasite surface. The ratio of C3b to lytically inactive iC3b was increased for desialylated, compared with sialylated, parasites. We conclude that although parasite sialic acid promotes C3b cleavage into iC3b, this mechanism alone does not account for the robust resistance of these parasites to complement lysis.
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a novel cell surface trans sialidase of trypanosoma cruzi generates a stage specific epitope required for invasion of mammalian cells
Cell, 1991Co-Authors: Sergio Schenkman, Man Shiow Jiang, Gerald W Hart, Victor NussenzweigAbstract:Abstract When Trypomastigotes of T. cruzi emerge from cells of the mammalian host, they contain little or no sialic acids on their surfaces. However, rapidly upon entering the circulation, they express a unique cell surface trans-sialidase activity. This enzyme specifically transfers α(2–3)-linked sialic acid from extrinsic host-derived macromolecules to parasite surface molecules, leading to the assembly of Ssp-3, a trypomastigote-specific epitope. The T. cruzi trans-sialidase does not utilize cytidine 5′ monophospho-N-acetylneuraminic acid as a donor substrate, but readily transfers sialic acid from exogenously supplied α(2–3)-sialyllactose. Monoclonal antibodies that recognize sialic acid residues of Ssp-3 inhibit attachment of Trypomastigotes to host cells, suggesting that the unusual trans-sialidase provides Ssp-3 with structural features required for target cell recognition.
