Fernando Yukio Maeda - Academia.edu (original) (raw)

Papers by Fernando Yukio Maeda

Outbreaks of acute Chagas disease by oral infection have been reported frequently over the last 1... more Outbreaks of acute Chagas disease by oral infection have been reported frequently over the last 10 years, with higher incidence in northern South America, where predominates the Trypanosoma cruzi lineage TcI, responsible for the major cause of resurgent human disease, and a small percentage is identified as TcIV. Mechanisms of oral infection and host cell invasion by metacyclic trypomastigotes of these groups of parasites are poorly understood. To address that question, we analyzed T. cruzi strains isolated from patients with Chagas disease from Venezuela, Guatemala and Brazil, by giving metacyclic trypomastigotes forms by oral route in mice. All parasite strains were poorly infective. Parasites were either undetectable or were detected in small numbers in the mouse stomach 4 days post-infection. At day 30, amastigote nests were found in the stomach and/or in the heart. As compared to TcI lineage, the capacity of TcIV parasites to traverse the gastric mucin-coated transwell filter w...

Expression and release of surface gp82 and g90 molecules of T. cruzi metacyclic trypomastigotes. ... more Expression and release of surface gp82 and g90 molecules of T. cruzi metacyclic trypomastigotes. a Ponceau-S staining of the corresponding Western blot shown in Fig. 1b, to demonstrate equal loading of metacyclic trypomastigotes samples, untreated (−) or treated (+) with 2 mg/ml pepsin, at pH 3.5. The molecular size markers are shown on the left. b Shown in the upper panel is the detergent extract of metacyclic forms of the indicated strains, analyzed by Western blot using monoclonal antibodies to gp82 and gp90. Shown in the lower panel is the conditioned medium obtained from strain 515 MT in PBS, pH 7.2, or in citrate buffer, pH 3.5, analyzed by Western blot. c Western blot of detergent extracts of strains 515 and 1434, revealed with mAb 3 F6. Shown in the lower panels is the corresponding Ponceau-S staining d–e Metacyclic forms of the indicated strains were incubated on ice for 1 h, in absence or in the presence of anti-gp82 mAb 3 F6 (d) or anti-gp90 mAb 1G7 (e). After fixation, t...

Journal of Cell Science, 2020

Endocytosis of caveolae was previously implicated in the repair of plasma membrane wounds. Here w... more Endocytosis of caveolae was previously implicated in the repair of plasma membrane wounds. Here we show that caveolin-1-deficient fibroblasts lacking caveolae upregulate a tubular endocytic pathway, and have a reduced capacity to reseal after permeabilization with pore-forming toxins when compared to wild type cells. Silencing endophilin-A2 expression inhibited fission of endocytic tubules and further reduced plasma membrane repair in cells lacking caveolin-1, supporting a role for tubular endocytosis as an alternative pathway for the removal of membrane lesions. Endophilin-A2 was visualized in association with cholera toxin B-containing endosomes and was recruited to recently formed intracellular vacuoles containing Trypanosoma cruzi, a parasite that utilizes the plasma membrane wounding/repair pathway to invade host cells. Endophilin-A2 deficiency inhibited T. cruzi invasion, and fibroblasts deficient in both caveolin-1 and endophilin-A2 did not survive prolonged exposure to the p...

eLife, 2021

B-cell receptor (BCR)-mediated antigen internalization and presentation are essential for humoral... more B-cell receptor (BCR)-mediated antigen internalization and presentation are essential for humoral memory immune responses. Antigen encountered by B-cells is often tightly associated with the surface of pathogens and/or antigen-presenting cells. Internalization of such antigens requires myosin-mediated traction forces and extracellular release of lysosomal enzymes, but the mechanism triggering lysosomal exocytosis is unknown. Here, we show that BCR-mediated recognition of antigen tethered to beads, to planar lipid-bilayers or expressed on cell surfaces causes localized plasma membrane (PM) permeabilization, a process that requires BCR signaling and non-muscle myosin II activity. B-cell permeabilization triggers PM repair responses involving lysosomal exocytosis, and B-cells permeabilized by surface-associated antigen internalize more antigen than cells that remain intact. Higher affinity antigens cause more B-cell permeabilization and lysosomal exocytosis and are more efficiently pre...

B-cell receptor (BCR)-mediated antigen internalization and presentation are essential for humoral... more B-cell receptor (BCR)-mediated antigen internalization and presentation are essential for humoral memory immune responses. Antigen encountered by B-cells is often tightly associated with the surface of pathogens and/or antigen-presenting cells. Internalization of such antigens requires myosin-mediated traction forces and extracellular release of lysosomal enzymes, but the mechanism triggering lysosomal exocytosis is unknown. Here we show that BCR-mediated recognition of antigen tethered to beads, to planar lipid-bilayers or expressed on cell surfaces causes localized plasma membrane (PM) permeabilization, a process that requires BCR signaling and non-muscle myosin II activity. B-cell permeabilization triggers PM repair responses involving lysosomal exocytosis, and B-cells permeabilized by surface-associated antigen internalize more antigen than cells that remain intact. Higher affinity antigens cause more B-cell permeabilization and lysosomal exocytosis and are more efficiently pres...

Infection and Immunity, 2019

The molecular mechanisms underlying biological differences between two Leishmania species that ca... more The molecular mechanisms underlying biological differences between two Leishmania species that cause cutaneous disease, L. major and L. amazonensis , are poorly understood. In L. amazonensis , reactive oxygen species (ROS) signaling drives differentiation of nonvirulent promastigotes into forms capable of infecting host macrophages.

PLoS pathogens, Jun 15, 2018

Iron is essential for many cellular processes, but can generate highly toxic hydroxyl radicals in... more Iron is essential for many cellular processes, but can generate highly toxic hydroxyl radicals in the presence of oxygen. Therefore, intracellular iron accumulation must be tightly regulated, by balancing uptake with storage or export. Iron uptake in Leishmania is mediated by the coordinated action of two plasma membrane proteins, the ferric iron reductase LFR1 and the ferrous iron transporter LIT1. However, how these parasites regulate their cytosolic iron concentration to prevent toxicity remains unknown. Here we characterize Leishmania Iron Regulator 1 (LIR1), an iron responsive protein with similarity to membrane transporters of the major facilitator superfamily (MFS) and plant nodulin-like proteins. LIR1 localizes on the plasma membrane of L. amazonensis promastigotes and intracellular amastigotes. After heterologous expression in Arabidopsis thaliana, LIR1 decreases the iron content of leaves and worsens the chlorotic phenotype of plants lacking the iron importer IRT1. Consist...

Parasites & vectors, 2016

Outbreaks of acute Chagas disease by oral infection have been reported frequently over the last t... more Outbreaks of acute Chagas disease by oral infection have been reported frequently over the last ten years, with higher incidence in northern South America, where Trypanosoma cruzi lineage TcI predominates, being responsible for the major cause of resurgent human disease, and a small percentage is identified as TcIV. Mechanisms of oral infection and host-cell invasion by these parasites are poorly understood. To address that question, we analyzed T. cruzi strains isolated from chagasic patients in Venezuela, Guatemala and Brazil. Trypanosoma cruzi metacyclic trypomastigotes were orally inoculated into mice. The mouse stomach collected four days later, as well as the stomach and the heart collected 30 days post-infection, were processed for histological analysis. Assays to mimic parasite migration through the gastric mucus layer were performed by counting the parasites that traversed gastric mucin-coated transwell filters. For cell invasion assays, human epithelial HeLa cells were inc...

Infection and Immunity, 2014

ABSTRACTTrypanosoma cruzi, the agent of Chagas disease, binds to diverse extracellular matrix pro... more ABSTRACTTrypanosoma cruzi, the agent of Chagas disease, binds to diverse extracellular matrix proteins. Such an ability prevails in the parasite forms that circulate in the bloodstream and contributes to host cell invasion. Whether this also applies to the insect-stage metacyclic trypomastigotes, the developmental forms that initiate infection in the mammalian host, is not clear. UsingT. cruziCL strain metacyclic forms, we investigated whether fibronectin bound to the parasites and affected target cell invasion. Fibronectin present in cell culture medium bound to metacyclic forms and was digested by cruzipain, the majorT. cruzicysteine proteinase. G strain, with negligible cruzipain activity, displayed a minimal fibronectin-degrading effect. Binding to fibronectin was mediated by gp82, the metacyclic stage-specific surface molecule implicated in parasite internalization. When exogenous fibronectin was present at concentrations higher than cruzipain can properly digest, or fibronecti...

Subcellular Biochemistry, 2013

Gp82 is a surface glycoprotein expressed in Trypanosoma cruzi metacyclic trypomastigotes, the par... more Gp82 is a surface glycoprotein expressed in Trypanosoma cruzi metacyclic trypomastigotes, the parasite forms from the insect vector that initiate infection in the mammalian host. Studies with metacyclic forms generated in vitro, as counterparts of insect-borne parasites, have shown that gp82 plays an essential role in host cell invasion and in the establishment of infection by the oral route. Among the gp82 properties relevant for infection are the gastric mucin-binding capacity and the ability to induce the target cell signaling cascades that result in actin cytoskeleton disruption and lysosome exocytosis, events that facilitate parasite internalization. The gp82 sequences from genetically divergent T. cruzi strains are highly conserved, displaying >90 % identity. Both the host cell-binding sites, as well as the gastric mucin-binding sequence of gp82, are localized in the C-terminal domain of the molecule. In the gp82 structure model, the main cell-binding site consists of an α-helix, which connects the N-terminal β-propeller domain to the C-terminal β-sandwich domain, where the second cell binding site is nested. The two cell binding sites are fully exposed on gp82 surface. Downstream and close to the α-helix is the gp82 gastric mucin-binding site, which is partially exposed. All available data support the notion that gp82 is structurally suited for metacyclic trypomastigote invasion of host cells and for initiating infection by the oral route.

Frontiers in Immunology, 2012

Cell signaling is an essential requirement for mammalian cell invasion by Trypanosoma cruzi. Depe... more Cell signaling is an essential requirement for mammalian cell invasion by Trypanosoma cruzi. Depending on the parasite strain and the parasite developmental form, distinct signaling pathways may be induced. In this short review, we focus on the data coming from studies with metacyclic trypomastigotes (MT) generated in vitro and tissue culturederived trypomastigotes (TCT), used as counterparts of insect-borne and bloodstream parasites, respectively. During invasion of host cells by MT or TCT, intracellular Ca 2+ mobilization and host cell lysosomal exocytosis are triggered. Invasion mediated by MT surface molecule gp82 requires the activation of mammalian target of rapamycin (mTOR), phosphatidylinositol 3-kinase (PI3K), and protein kinase C (PKC) in the host cell, associated with Ca 2+-dependent disruption of the actin cytoskeleton. In MT, protein tyrosine kinase, PI3K, phospholipase C, and PKC appear to be activated. TCT invasion, on the other hand, does not rely on mTOR activation, rather on target cell PI3K, and may involve the host cell autophagy for parasite internalization. Enzymes, such as oligopeptidase B and the major T. cruzi cysteine proteinase cruzipain, have been shown to generate molecules that induce target cell Ca 2+ signal. In addition, TCT may trigger host cell responses mediated by transforming growth factor β receptor or integrin family member. Further investigations are needed for a more complete and detailed picture of T. cruzi invasion.

Malaria Journal, 2007

Objective The objective of this study was to investigate whether the infection of C57BL/6 mice by... more Objective The objective of this study was to investigate whether the infection of C57BL/6 mice by P. berghei ANKA, which causes severe malaria, was modulated by co-infection with Trypanosoma cruzi. Methods Groups of C57BL/6 mice were infected either with P. berghei ANKA, T. cruzi strain G, or with both parasites. The presence of parasites was checked by microscopic examination of blood samples. Symptoms of neurological or respiratory disorders, as well as mortality, were registered. Breakdown of the blood brain barrier was determined by injecting the dye Evans blue. Histological sections of the lung were prepared and stained with hematoxilin-eosin. Results All mice infected only with P. berghei ANKA died within 7–11 days post-infection, either with symptoms of cerebral malaria or with respiratory abnormalities. The animals co-infected with T. cruzi strain G survived longer, without any of the referred to symptoms. Protection against the early death by severe malaria was effective wh...

Acta Tropica, 2012

Protozoan parasites of the genus Trypanosoma can infect virtually all mammalian species. Within t... more Protozoan parasites of the genus Trypanosoma can infect virtually all mammalian species. Within this genus, Trypanosoma dionisii from bats and Trypanosoma cruzi that causes Chagas' disease, belonging to the subgenus Schizotrypanum, can invade mammalian cells. The mechanisms of cell invasion by T. dionisii are poorly understood. To address that question, metacyclic trypomastigotes (MT) and human epithelial HeLa cells were used. Similarly to genetically divergent T. cruzi strains G (TcI) and CL (TcVI), associated, respectively with marsupial and human infections, T. dionisii infectivity increased under nutritional stress, a condition that induces host cell lysosome exocytosis required for parasite internalization. For efficient internalization, T. dionisii depended on MT protein tyrosine kinase (PTK) and Ca 2+ mobilization from acidocalcisomes, whereas T. cruzi strains also relied on phosphatidylinositol 3-kinase (PI3K), protein kinase C (PKC) and Ca 2+ released from thapsigargin-sensitive compartments. T. dionisii-induced signaling in host cells implicated PKC and Ca 2+ mobilized from thapsigargin-sensitive stores, like T. cruzi, but without PI3K involvement. Unlike T. cruzi, T. dionisii metacyclic forms did not use l-proline as source of energy required for internalization. Molecules related to T. cruzi surface glycoproteins involved in MT-host cell interaction were undetectable in T. dionisii. The difference in the surface profile of the two species was also inferred from the susceptibility of T. dionisii metacyclic forms to complement-mediated lysis, as opposed to complete resistance of T. cruzi. In summary, the two Trypanosoma species display distinct surface profiles but invade host cells through a common mechanism involving lysosome mobilization to the site of parasite entry.

Acta Tropica, 2011

A new genotype of Trypanosoma cruzi, associated with bats from anthropic areas, was recently desc... more A new genotype of Trypanosoma cruzi, associated with bats from anthropic areas, was recently described. Here we characterized a T. cruzi strain from this new genetic group, which could be a potential source of infection to humans. Metacyclic trypomastigotes (MT) of this strain, herein designated BAT, were compared to MT of well characterized CL and G strains, as regards the surface profile and infectivity toward human epithelial HeLa cells. BAT strain MT expressed gp82, the surface molecule recognized by monoclonal antibody 3F6 and known to promote CL strain invasion by inducing lysosomal exocytosis, as well as mucin-like molecules, but lacked gp90, which functions as a negative regulator of invasion in G strain. A set of experiments indicated that BAT strain internalization is gp82-mediated, and requires the activation of host cell phosphatidylinositol 3-kinase, protein kinase C and the mammalian target of rapamycin. MT of BAT strain were able to migrate through a gastric mucin layer, a property associated with p82 and relevant for oral infection. Gp82 was found to be a highly conserved molecule. Analysis of the BAT strain gp82 domain, containing the cell binding-and gastric mucin-binding sites, showed 91 and 93% sequence identity with G and CL strains, respectively. Hela cell invasion by BAT strain MT was inhibited by purified mucin-like molecules, which were shown to affect lysosome exocytosis required for MT internalization. Although MT of BAT strain infected host cells in vitro, they were less effective than G or CL strains in infecting mice either orally or intraperitoneally.

Acta Tropica, 2009

The phylogenetic proximity between Trypanosoma cruzi and Trypanosoma (Schizotrypanum) dionisii su... more The phylogenetic proximity between Trypanosoma cruzi and Trypanosoma (Schizotrypanum) dionisii suggests that these parasites might explore similar strategies to complete their life cycles. T. cruzi is the etiological agent of the life threatening Chagas' disease, whereas T. dionisii is a bat trypanosome and probably not capable of infecting humans. Here we sought to compare mammalian cell invasion and intracellular traffic of both trypanosomes and determine the differences and similarities in this process. The results presented demonstrate that T. dionisii is highly infective in vitro, particularly when the infection process occurs without serum and that the invasion is similarly affected by agents known to interfere with T. cruzi invasion process. Our results indicate that the formation of lysosomal-enriched compartments is part of a cell-invasion mechanism retained by related trypanosomatids, and that residence and further escape from a lysosomal compartment may be a common requisite for successful infection. During intracellular growth, parasites share a few epitopes with T. cruzi amastigotes and trypomastigotes. Unexpectedly, in heavily infected cells, amastigotes and trypomastigotes were found inside the host cell nucleus. These findings suggest that T. dionisii although sharing some features in host cell invasion with T. cruzi, has unique behaviors that deserve to be further explored.

Outbreaks of acute Chagas disease by oral infection have been reported frequently over the last 1... more Outbreaks of acute Chagas disease by oral infection have been reported frequently over the last 10 years, with higher incidence in northern South America, where predominates the Trypanosoma cruzi lineage TcI, responsible for the major cause of resurgent human disease, and a small percentage is identified as TcIV. Mechanisms of oral infection and host cell invasion by metacyclic trypomastigotes of these groups of parasites are poorly understood. To address that question, we analyzed T. cruzi strains isolated from patients with Chagas disease from Venezuela, Guatemala and Brazil, by giving metacyclic trypomastigotes forms by oral route in mice. All parasite strains were poorly infective. Parasites were either undetectable or were detected in small numbers in the mouse stomach 4 days post-infection. At day 30, amastigote nests were found in the stomach and/or in the heart. As compared to TcI lineage, the capacity of TcIV parasites to traverse the gastric mucin-coated transwell filter w...

Expression and release of surface gp82 and g90 molecules of T. cruzi metacyclic trypomastigotes. ... more Expression and release of surface gp82 and g90 molecules of T. cruzi metacyclic trypomastigotes. a Ponceau-S staining of the corresponding Western blot shown in Fig. 1b, to demonstrate equal loading of metacyclic trypomastigotes samples, untreated (−) or treated (+) with 2 mg/ml pepsin, at pH 3.5. The molecular size markers are shown on the left. b Shown in the upper panel is the detergent extract of metacyclic forms of the indicated strains, analyzed by Western blot using monoclonal antibodies to gp82 and gp90. Shown in the lower panel is the conditioned medium obtained from strain 515 MT in PBS, pH 7.2, or in citrate buffer, pH 3.5, analyzed by Western blot. c Western blot of detergent extracts of strains 515 and 1434, revealed with mAb 3 F6. Shown in the lower panels is the corresponding Ponceau-S staining d–e Metacyclic forms of the indicated strains were incubated on ice for 1 h, in absence or in the presence of anti-gp82 mAb 3 F6 (d) or anti-gp90 mAb 1G7 (e). After fixation, t...

Journal of Cell Science, 2020

Endocytosis of caveolae was previously implicated in the repair of plasma membrane wounds. Here w... more Endocytosis of caveolae was previously implicated in the repair of plasma membrane wounds. Here we show that caveolin-1-deficient fibroblasts lacking caveolae upregulate a tubular endocytic pathway, and have a reduced capacity to reseal after permeabilization with pore-forming toxins when compared to wild type cells. Silencing endophilin-A2 expression inhibited fission of endocytic tubules and further reduced plasma membrane repair in cells lacking caveolin-1, supporting a role for tubular endocytosis as an alternative pathway for the removal of membrane lesions. Endophilin-A2 was visualized in association with cholera toxin B-containing endosomes and was recruited to recently formed intracellular vacuoles containing Trypanosoma cruzi, a parasite that utilizes the plasma membrane wounding/repair pathway to invade host cells. Endophilin-A2 deficiency inhibited T. cruzi invasion, and fibroblasts deficient in both caveolin-1 and endophilin-A2 did not survive prolonged exposure to the p...

eLife, 2021

B-cell receptor (BCR)-mediated antigen internalization and presentation are essential for humoral... more B-cell receptor (BCR)-mediated antigen internalization and presentation are essential for humoral memory immune responses. Antigen encountered by B-cells is often tightly associated with the surface of pathogens and/or antigen-presenting cells. Internalization of such antigens requires myosin-mediated traction forces and extracellular release of lysosomal enzymes, but the mechanism triggering lysosomal exocytosis is unknown. Here, we show that BCR-mediated recognition of antigen tethered to beads, to planar lipid-bilayers or expressed on cell surfaces causes localized plasma membrane (PM) permeabilization, a process that requires BCR signaling and non-muscle myosin II activity. B-cell permeabilization triggers PM repair responses involving lysosomal exocytosis, and B-cells permeabilized by surface-associated antigen internalize more antigen than cells that remain intact. Higher affinity antigens cause more B-cell permeabilization and lysosomal exocytosis and are more efficiently pre...

B-cell receptor (BCR)-mediated antigen internalization and presentation are essential for humoral... more B-cell receptor (BCR)-mediated antigen internalization and presentation are essential for humoral memory immune responses. Antigen encountered by B-cells is often tightly associated with the surface of pathogens and/or antigen-presenting cells. Internalization of such antigens requires myosin-mediated traction forces and extracellular release of lysosomal enzymes, but the mechanism triggering lysosomal exocytosis is unknown. Here we show that BCR-mediated recognition of antigen tethered to beads, to planar lipid-bilayers or expressed on cell surfaces causes localized plasma membrane (PM) permeabilization, a process that requires BCR signaling and non-muscle myosin II activity. B-cell permeabilization triggers PM repair responses involving lysosomal exocytosis, and B-cells permeabilized by surface-associated antigen internalize more antigen than cells that remain intact. Higher affinity antigens cause more B-cell permeabilization and lysosomal exocytosis and are more efficiently pres...

Infection and Immunity, 2019

The molecular mechanisms underlying biological differences between two Leishmania species that ca... more The molecular mechanisms underlying biological differences between two Leishmania species that cause cutaneous disease, L. major and L. amazonensis , are poorly understood. In L. amazonensis , reactive oxygen species (ROS) signaling drives differentiation of nonvirulent promastigotes into forms capable of infecting host macrophages.

PLoS pathogens, Jun 15, 2018

Iron is essential for many cellular processes, but can generate highly toxic hydroxyl radicals in... more Iron is essential for many cellular processes, but can generate highly toxic hydroxyl radicals in the presence of oxygen. Therefore, intracellular iron accumulation must be tightly regulated, by balancing uptake with storage or export. Iron uptake in Leishmania is mediated by the coordinated action of two plasma membrane proteins, the ferric iron reductase LFR1 and the ferrous iron transporter LIT1. However, how these parasites regulate their cytosolic iron concentration to prevent toxicity remains unknown. Here we characterize Leishmania Iron Regulator 1 (LIR1), an iron responsive protein with similarity to membrane transporters of the major facilitator superfamily (MFS) and plant nodulin-like proteins. LIR1 localizes on the plasma membrane of L. amazonensis promastigotes and intracellular amastigotes. After heterologous expression in Arabidopsis thaliana, LIR1 decreases the iron content of leaves and worsens the chlorotic phenotype of plants lacking the iron importer IRT1. Consist...

Parasites & vectors, 2016

Outbreaks of acute Chagas disease by oral infection have been reported frequently over the last t... more Outbreaks of acute Chagas disease by oral infection have been reported frequently over the last ten years, with higher incidence in northern South America, where Trypanosoma cruzi lineage TcI predominates, being responsible for the major cause of resurgent human disease, and a small percentage is identified as TcIV. Mechanisms of oral infection and host-cell invasion by these parasites are poorly understood. To address that question, we analyzed T. cruzi strains isolated from chagasic patients in Venezuela, Guatemala and Brazil. Trypanosoma cruzi metacyclic trypomastigotes were orally inoculated into mice. The mouse stomach collected four days later, as well as the stomach and the heart collected 30 days post-infection, were processed for histological analysis. Assays to mimic parasite migration through the gastric mucus layer were performed by counting the parasites that traversed gastric mucin-coated transwell filters. For cell invasion assays, human epithelial HeLa cells were inc...

Infection and Immunity, 2014

ABSTRACTTrypanosoma cruzi, the agent of Chagas disease, binds to diverse extracellular matrix pro... more ABSTRACTTrypanosoma cruzi, the agent of Chagas disease, binds to diverse extracellular matrix proteins. Such an ability prevails in the parasite forms that circulate in the bloodstream and contributes to host cell invasion. Whether this also applies to the insect-stage metacyclic trypomastigotes, the developmental forms that initiate infection in the mammalian host, is not clear. UsingT. cruziCL strain metacyclic forms, we investigated whether fibronectin bound to the parasites and affected target cell invasion. Fibronectin present in cell culture medium bound to metacyclic forms and was digested by cruzipain, the majorT. cruzicysteine proteinase. G strain, with negligible cruzipain activity, displayed a minimal fibronectin-degrading effect. Binding to fibronectin was mediated by gp82, the metacyclic stage-specific surface molecule implicated in parasite internalization. When exogenous fibronectin was present at concentrations higher than cruzipain can properly digest, or fibronecti...

Subcellular Biochemistry, 2013

Gp82 is a surface glycoprotein expressed in Trypanosoma cruzi metacyclic trypomastigotes, the par... more Gp82 is a surface glycoprotein expressed in Trypanosoma cruzi metacyclic trypomastigotes, the parasite forms from the insect vector that initiate infection in the mammalian host. Studies with metacyclic forms generated in vitro, as counterparts of insect-borne parasites, have shown that gp82 plays an essential role in host cell invasion and in the establishment of infection by the oral route. Among the gp82 properties relevant for infection are the gastric mucin-binding capacity and the ability to induce the target cell signaling cascades that result in actin cytoskeleton disruption and lysosome exocytosis, events that facilitate parasite internalization. The gp82 sequences from genetically divergent T. cruzi strains are highly conserved, displaying >90 % identity. Both the host cell-binding sites, as well as the gastric mucin-binding sequence of gp82, are localized in the C-terminal domain of the molecule. In the gp82 structure model, the main cell-binding site consists of an α-helix, which connects the N-terminal β-propeller domain to the C-terminal β-sandwich domain, where the second cell binding site is nested. The two cell binding sites are fully exposed on gp82 surface. Downstream and close to the α-helix is the gp82 gastric mucin-binding site, which is partially exposed. All available data support the notion that gp82 is structurally suited for metacyclic trypomastigote invasion of host cells and for initiating infection by the oral route.

Frontiers in Immunology, 2012

Cell signaling is an essential requirement for mammalian cell invasion by Trypanosoma cruzi. Depe... more Cell signaling is an essential requirement for mammalian cell invasion by Trypanosoma cruzi. Depending on the parasite strain and the parasite developmental form, distinct signaling pathways may be induced. In this short review, we focus on the data coming from studies with metacyclic trypomastigotes (MT) generated in vitro and tissue culturederived trypomastigotes (TCT), used as counterparts of insect-borne and bloodstream parasites, respectively. During invasion of host cells by MT or TCT, intracellular Ca 2+ mobilization and host cell lysosomal exocytosis are triggered. Invasion mediated by MT surface molecule gp82 requires the activation of mammalian target of rapamycin (mTOR), phosphatidylinositol 3-kinase (PI3K), and protein kinase C (PKC) in the host cell, associated with Ca 2+-dependent disruption of the actin cytoskeleton. In MT, protein tyrosine kinase, PI3K, phospholipase C, and PKC appear to be activated. TCT invasion, on the other hand, does not rely on mTOR activation, rather on target cell PI3K, and may involve the host cell autophagy for parasite internalization. Enzymes, such as oligopeptidase B and the major T. cruzi cysteine proteinase cruzipain, have been shown to generate molecules that induce target cell Ca 2+ signal. In addition, TCT may trigger host cell responses mediated by transforming growth factor β receptor or integrin family member. Further investigations are needed for a more complete and detailed picture of T. cruzi invasion.

Malaria Journal, 2007

Objective The objective of this study was to investigate whether the infection of C57BL/6 mice by... more Objective The objective of this study was to investigate whether the infection of C57BL/6 mice by P. berghei ANKA, which causes severe malaria, was modulated by co-infection with Trypanosoma cruzi. Methods Groups of C57BL/6 mice were infected either with P. berghei ANKA, T. cruzi strain G, or with both parasites. The presence of parasites was checked by microscopic examination of blood samples. Symptoms of neurological or respiratory disorders, as well as mortality, were registered. Breakdown of the blood brain barrier was determined by injecting the dye Evans blue. Histological sections of the lung were prepared and stained with hematoxilin-eosin. Results All mice infected only with P. berghei ANKA died within 7–11 days post-infection, either with symptoms of cerebral malaria or with respiratory abnormalities. The animals co-infected with T. cruzi strain G survived longer, without any of the referred to symptoms. Protection against the early death by severe malaria was effective wh...

Acta Tropica, 2012

Protozoan parasites of the genus Trypanosoma can infect virtually all mammalian species. Within t... more Protozoan parasites of the genus Trypanosoma can infect virtually all mammalian species. Within this genus, Trypanosoma dionisii from bats and Trypanosoma cruzi that causes Chagas' disease, belonging to the subgenus Schizotrypanum, can invade mammalian cells. The mechanisms of cell invasion by T. dionisii are poorly understood. To address that question, metacyclic trypomastigotes (MT) and human epithelial HeLa cells were used. Similarly to genetically divergent T. cruzi strains G (TcI) and CL (TcVI), associated, respectively with marsupial and human infections, T. dionisii infectivity increased under nutritional stress, a condition that induces host cell lysosome exocytosis required for parasite internalization. For efficient internalization, T. dionisii depended on MT protein tyrosine kinase (PTK) and Ca 2+ mobilization from acidocalcisomes, whereas T. cruzi strains also relied on phosphatidylinositol 3-kinase (PI3K), protein kinase C (PKC) and Ca 2+ released from thapsigargin-sensitive compartments. T. dionisii-induced signaling in host cells implicated PKC and Ca 2+ mobilized from thapsigargin-sensitive stores, like T. cruzi, but without PI3K involvement. Unlike T. cruzi, T. dionisii metacyclic forms did not use l-proline as source of energy required for internalization. Molecules related to T. cruzi surface glycoproteins involved in MT-host cell interaction were undetectable in T. dionisii. The difference in the surface profile of the two species was also inferred from the susceptibility of T. dionisii metacyclic forms to complement-mediated lysis, as opposed to complete resistance of T. cruzi. In summary, the two Trypanosoma species display distinct surface profiles but invade host cells through a common mechanism involving lysosome mobilization to the site of parasite entry.

Acta Tropica, 2011

A new genotype of Trypanosoma cruzi, associated with bats from anthropic areas, was recently desc... more A new genotype of Trypanosoma cruzi, associated with bats from anthropic areas, was recently described. Here we characterized a T. cruzi strain from this new genetic group, which could be a potential source of infection to humans. Metacyclic trypomastigotes (MT) of this strain, herein designated BAT, were compared to MT of well characterized CL and G strains, as regards the surface profile and infectivity toward human epithelial HeLa cells. BAT strain MT expressed gp82, the surface molecule recognized by monoclonal antibody 3F6 and known to promote CL strain invasion by inducing lysosomal exocytosis, as well as mucin-like molecules, but lacked gp90, which functions as a negative regulator of invasion in G strain. A set of experiments indicated that BAT strain internalization is gp82-mediated, and requires the activation of host cell phosphatidylinositol 3-kinase, protein kinase C and the mammalian target of rapamycin. MT of BAT strain were able to migrate through a gastric mucin layer, a property associated with p82 and relevant for oral infection. Gp82 was found to be a highly conserved molecule. Analysis of the BAT strain gp82 domain, containing the cell binding-and gastric mucin-binding sites, showed 91 and 93% sequence identity with G and CL strains, respectively. Hela cell invasion by BAT strain MT was inhibited by purified mucin-like molecules, which were shown to affect lysosome exocytosis required for MT internalization. Although MT of BAT strain infected host cells in vitro, they were less effective than G or CL strains in infecting mice either orally or intraperitoneally.

Acta Tropica, 2009

The phylogenetic proximity between Trypanosoma cruzi and Trypanosoma (Schizotrypanum) dionisii su... more The phylogenetic proximity between Trypanosoma cruzi and Trypanosoma (Schizotrypanum) dionisii suggests that these parasites might explore similar strategies to complete their life cycles. T. cruzi is the etiological agent of the life threatening Chagas' disease, whereas T. dionisii is a bat trypanosome and probably not capable of infecting humans. Here we sought to compare mammalian cell invasion and intracellular traffic of both trypanosomes and determine the differences and similarities in this process. The results presented demonstrate that T. dionisii is highly infective in vitro, particularly when the infection process occurs without serum and that the invasion is similarly affected by agents known to interfere with T. cruzi invasion process. Our results indicate that the formation of lysosomal-enriched compartments is part of a cell-invasion mechanism retained by related trypanosomatids, and that residence and further escape from a lysosomal compartment may be a common requisite for successful infection. During intracellular growth, parasites share a few epitopes with T. cruzi amastigotes and trypomastigotes. Unexpectedly, in heavily infected cells, amastigotes and trypomastigotes were found inside the host cell nucleus. These findings suggest that T. dionisii although sharing some features in host cell invasion with T. cruzi, has unique behaviors that deserve to be further explored.