Trypanosoma cruzi extracellular amastigotes trigger the protein kinase D1‐cortactin‐actin pathway during cell invasion

Trypanosoma cruzi extracellular amastigotes (EAs) display unique mechanisms for cell invasion that are highly dependent on host actin filaments. Protein kinase D1 (PKD1) phosphorylates and modulates the activity of cortactin, a key regulator of actin dynamics. We evaluated the role of host cortactin and PKD1 in actin filament dynamics during HeLa cell invasion by EAs. Host cortactin, PKD1 and actin are recruited by EAs based on experiments in fixed and live cells by time lapse confocal microscopy. EAs trigger PKD1 and extracellular signal‐regulated kinase 1/2 activation, but not Src family kinases, and selectively phosphorylate cortactin. Heat‐killed EAs and non‐infective epimastigotes both triggered distinct host responses and did not recruit the molecules studied herein. EA invasion was influenced by depletion or overexpression of host cortactin and PKD1, respectively, suggesting the involvement of both proteins in this event. Collectively, these results show new host cell mechanisms subverted during EA internalization into non‐phagocytic cells.     

Trypanosoma cruzi extracellular amastigotes engage Rac1 and Cdc42 to invade RAW macrophages

Cell invasion by Trypanosoma cruzi extracellular amastigotes (EAs) relies significantly upon the host cell actin cytoskeleton. In past decades EAs have been established as a reliable model for phagocytosis inducer in non-phagocytic cells. Our current hypothesis is that EAs engage a phagocytosis-like mechanism in non-professional phagocytic cells; however, the molecular mechanisms in professional phagocytes still remain unexplored. In this work, we evaluated the involvement of Rac1 and Cdc42 in the actin-dependent internalization of EAs in RAW 264.7 macrophages. Kinetic assays showed similar internalization of EAs in unstimulated RAW and non-phagocytic HeLa cells but increased in LPS/IFN-γ stimulated RAW cells. However, depletion of Rac1, Cdc42 or RhoA inhibited EA internalization similarly in both unstimulated and stimulated RAW cells. Overexpression of active, but not the dominant-negative, construct of Rac1 increased EA internalization. Remarkably, for Cdc42, both the active and the inactive mutants decreased EA internalization when compared to wild type groups. Despite that, both Rac1 and Cdc42 activation mutants were similarly recruited to and colocalized with actin at the EA-macrophage contact sites when compared to their native isoforms. Altogether, these results corroborate that EAs engage phagocytic processes to invade both professional and non-professional phagocytic cells providing evidences of converging actin mediated mechanisms induced by intracellular pathogens in both cell types.     

Trypanosoma cruzi: Multiple actin isovariants are observed along different developmental stages

The expression and biological role of actin during the Trypanosoma cruzi life cycle remains largely unknown. Polyclonal antibodies against a recombinant T. cruzi actin protein were used to confirm its expression in epimastigotes, trypomastigotes, and amastigotes. Although the overall levels of expression were similar, clear differences in the subcellular distribution of actin among the developmental stages were identified. The existence of five actin variants in each developmental stage with distinct patterns of expression were uncovered by immunoblotting of protein extracts separated 2D-SDS gels. The isoelectric points of the actin variants in epimastigotes ranged from 4.45 to 4.9, whereas they ranged from 4.9 to 5.24 in trypomastigotes and amastigotes. To determine if the actin variants found could represent previously unidentified actins, we performed a genomic survey of the T. cruzi GeneDB database and found 12 independent loci encoding for a diverse group of actins and actin-like proteins that are conserved among trypanosomatids.     

Trypanosoma cruzi: Role of δ-Amastin on Extracellular Amastigote Cell Invasion and Differentiation

Trypanosoma cruzi is a protozoan parasite that comprises different phylogenetic groups and is the causative agent of Chagas’ disease. Different T. cruzi strains present differences in infectivity in in vitro and in vivo experimental models, which are likely related to the expression of different virulence factors. Amastin is a surface glycoprotein abundantly expressed on the intracellular mammalian amastigote form of the parasite. In this study, we showed that a highly infective strain (G strain) of extracellular amastigote (EA) invasive forms expressed reduced RNA levels of amastin compared to a less infective strain (CL). The treatment of HeLa cells with recombinant δ-amastin reduced infectivity of EA forms. However, the ectopic expression of δ-amastin accelerated amastigote differentiation into trypomastigotes. Corroborating the virulence behavior in association with amastin expression, the EAs overexpressing amastin were precociously and robustly observed in the liver of susceptible mouse strains (A/JUnib), whereas parasitemia was never detected in in vivo assays. This is the first report on the regulatory role of amastin in the course of both in vitro and in vivo T. cruzi infection.     

Differential apoptosis-like cell death in amastigote and trypomastigote forms from Trypanosoma cruzi-infected heart cells in vitro

Apoptosis, type-I of programmed cell death (PCD-I), is not restricted to multicellular organisms since many apoptotic features have been described in different trypanosomatids, including Trypanosoma cruzi. Our present aim was to monitor, by different morphological markers, the occurrence of apoptosis-like death in amastigotes and trypomastigotes of T.cruzi (Y strain) during the infection of heart culture cells. We documented the differential occurrence of PCD-I in amastigotes and trypomastigotes, with distinct death rates noticed between these two parasite-distinct forms. Fluorescence microscopy and flow cytometry analysis using different hall markers of apoptosis (phosphatidylserine exposure, collapse of mitochondrial membrane potential and DNA fragmentation) showed that amastigotes present higher levels of apoptosis-like cell death as compared to trypomastigotes. It is possible that the higher levels of PCD-I in these highly multiplicative forms may contribute to the control of the parasite burden within the host cells. On the other hand, the apoptosis-like occurrence in the infective but non-proliferative stage of the parasite (trypomastigotes) may play a role in parasite evasion mechanisms as suggested for other parasites.     

Trypanosoma cruzi: A Specific Surface Marker for the Amastigote Form1

ABSTRACT. Among the known life cycle stages of Trypanosoma cruzi only the amastigote form bound lactoferrin (LF), a glycoprotein produced by neutrophils. This capacity was readily demonstrable by indirect immunofluorescence in amastigotes derived from mice, a mammalian cell culture, or grown in an axenic medium. No LF binding was detectable on trypomastigotes from blood or mammalian cells, insect-derived metacyclics or epimastigotes, or on epimastigotes grown in Warren's medium. Serum levels of LF were increased in mice acutely infected with T. cruzi, and amastigotes from the spleens of these animals were found to have the glycoprotein on their surface. The amastigote LF receptor may have biological significance in parasite-host interaction since mononuclear phagocytes also express a LF receptor, and treatment of these cells with LF has been shown to increase their capacities to take up and kill T. cruzi amastigotes in vitro. The LF receptor is the first marker for T. cruzi amastigotes for which a naturally occurring ligand has been described.     

Trypanosoma cruzi: single cell live imaging inside infected tissues

Although imaging the live Trypanosoma cruzi parasite is a routine technique in most laboratories, identification of the parasite in infected tissues and organs has been hindered by their intrinsic opaque nature. We describe a simple method for in vivo observation of live single‐cell Trypanosoma cruzi parasites inside mammalian host tissues. BALB/c or C57BL/6 mice infected with DsRed‐CL or GFP‐G trypomastigotes had their organs removed and sectioned with surgical blades. Ex vivo organ sections were observed under confocal microscopy. For the first time, this procedure enabled imaging of individual amastigotes, intermediate forms and motile trypomastigotes within infected tissues of mammalian hosts.     

Evaluation of thiosemicarbazones and semicarbazones as potential agents anti-Trypanosoma cruzi

Synthetic thiosemicarbazones and semicarbazones were evaluated for their Trypanosoma cruzi trypomastigotes obtained from LLC-MK2 cell cultures. In general, thiosemicarbazone derivatives were most effective and among them the 4-N-(2′-methoxy styryl)-thiosemicarbazone was chosen, to compare the in vitro effect against amastigotes of T. cruzi lodged in mouse peritoneal and human macrophages. A potent trypanocidal effect was observed that was more pronounced against parasites internalized in human macrophages. A potential target for this compound was also evaluated by measuring the nitric oxide synthase activity through NADPH consumption. A significant decrease in enzyme activity was observed. In contrast to the cytotoxic effect observed with benznidazole, no macrophage toxicity was observed for any of the compounds, indicating that their activity was specific for the parasite forms investigated.     

Trypanosoma cruzi extracellular amastigotes selectively trigger the PI3K/Akt and Erk pathways during HeLa cell invasion

Cell invasion by Trypanosoma cruzi extracellular amastigotes involves different signaling pathways to induce phagocytosis-like mechanisms. Previous works indicated that PI3K/Akt, Src and Erk might be involved in EA invasion; however, participation of these molecules in this process remains elusive. Here, we observed that EA activated Akt, Erk but not Src. Interference of EA invasion with specific inhibitors corroborated this observation. Our results show that EA is capable of selectively triggering complex signaling pathways. Activation of PI3K/Akt and Erk, kinases related to actin cytoskeleton rearrangement and phagocytosis, reinforces the idea that T. cruzi EA subverts the phagocytic machinery during invasion.     

A human astrocytoma cell line is highly susceptible to infection with Trypanosoma cruzi

Astrocytes play a vital role in neuronal protection, homeostasis, vascular interchange and the local immune response. Some viruses and parasites can cross the blood-brain barrier and infect glia. Trypanosoma cruzi, the aetiological agent of Chagas disease, can seriously compromise the central nervous system, mainly in immune-suppressed individuals, but also during the acute phase of the infection. In this report, the infective capacity of T. cruzi in a human astrocyte tumour-derived cell line was studied. Astrocytes exposed to trypomastigotes (1:10 ratio) produced intracellular amastigotes and new trypomastigotes emerged by day 4 post-infection (p.i.). At day 6 p.i., 93% of the cells were infected. Using flow cytometry, changes were observed in both the expression of major histocompatibility complex class I and II molecules and the chemokine secretion pattern of astrocytes exposed to the parasite. Blocking the low-density lipoprotein receptor on astrocytes did not reduce parasite intracellular infection. Thus, T. cruzi can infect astrocytes and modulate the immune response during central nervous system infection.     

All <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> developmental forms present lysosome-related organelles

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

Lysosomal exocytosis: An important event during invasion of lamp deficient cells by extracellular amastigotes of Trypanosoma cruzi

Trypanosoma cruzi is an obligate intracellular organism in vertebrate hosts. Lysosomes are involved in parasite invasion. LAMP-1 and LAMP-2 are the most abundant glycoproteins of the lysosomal membrane. This study is the first report on the invasion of T. cruzi extracellular amastigotes (EA) in single LAMP-1 or LAMP-2 knockouts, respectively, or in two independent LAMP-1/2 double-knockout cell lines. When compared to their respective wild type clones, the EA show higher infectivity in LAMP-2 knockouts, but no difference was seen in LAMP-1 knockout cells. Similarly, EA invasion rate was higher for one of the double knockout clones but not for the other. Higher lysosomal exocytosis correlated with a higher invasion rate and early lysosomal marker acquisition. These findings suggest that lysosomal exocytosis is important to EA cell invasion. Also, phagolysosome maturation in knockout cell lines differed from previous results revealing that EA enter cells by a mechanism other than receptor-mediated phagocytosis.     

3D reconstruction of Trypanosoma cruzi-macrophage interaction shows the recruitment of host cell organelles towards parasitophorous vacuoles during its biogenesis

Trypanosoma cruzi has a complex life cycle where two infective developmental stages, known as trypomastigote and amastigote, can be found in the vertebrate host. Both forms can invade a large variety of cellular types and induce the formation of a parasitophorous vacuole (PV), that, posteriorly, disassembles and releases the parasites into the host cell cytoplasm. The biogenesis of T. cruzi PVs has not been analyzed in professional phagocytic cells. We investigated the biogenesis of PVs containing trypomastigotes or amastigotes in peritoneal macrophages. We observed the presence of profiles of the endoplasmic reticulum and lysosomes from the host cell near PVs at early stages of interaction in both developmental stages, suggesting that both organelles may participate as possible membrane donors for the formation of the PVs. The Golgi complex, however, was observed only near already formed PVs. Electron microscopy tomography and FIB-SEM microscopy followed by 3D reconstruction of entire PVs containing amastigotes or trypomastigotes confirmed the presence of both endoplasmic reticulum and lysosomes in the initial stages of PV formation. In addition, Golgi complex and mitochondria localize around PVs during their biogenesis. Taken together these observations provide a whole view of the invasion process in a professional phagocytic cell.     

Lactoferrin effects on the interaction of blood forms of Trypanosoma cruzi with mononuclear phagocytes

Mouse macrophages and human monocytes displayed increased capacities to take up blood trypomastigotes of Trypanosoma cruzi after a 24-h and 2-h lactoferrin (LF) pretreatment, respectively. Lactoferrin binding to trypomastigotes was not detectable by indirect immunofluorescence and pretreatment of the parasite with LF did not affect its capacity to interact with macrophages. Macrophages treated with LF also displayed a greater capacity to kill T. cruzi, whether the treatment was applied before or after parasite internalization. Since serum levels of LF increase during T. cruzi infection, the noted effects might play a role in host defense.     

Biochemical Requirements for Intracellular Invasion by Trypanosoma cruzi: Protein Synthesis1

The effects of irreversible inhibition of protein synthesis by pactamycin in either infective forms of Trypanosoma cruzi or mammalian host cells on cellular invasion by this human pathogen were investigated. Treatment of bloodstream forms of T. cruzi with pactamycin markedly reduced their ability to bind either fibroblast-like cells of monkey origin or myoblasts of rat origin. The number of amastigote forms that could be established intracellularly was also significantly decreased with respect to control values obtained when mock-treated (medium alone) trypomastigotes were incubated with the cells. Pactamycin treatment also reduced the infectivity of T. cruzi trypomastigotes for mice as evidenced by both significantly reduced parasitemia levels and mortality rates when compared with those of control mice infected with mock-treated parasites. Inhibition of protein synthesis in the host cells neither prevented cell infection by untreated trypomastigotes nor altered the percentages of infected cells or the magnitude of the infection in vitro. These results indicate that protein synthesis is a requirement for cell invasion by T. cruzi and that the parasite can establish itself and replicate within cells relying on its own protein synthesis ability.     

Separation of Individual Stages of Trypanosoma cruzi Grown in Cell Culture by Continuous Free-Flow Electrophoresis

The separation of extracellular protozoan parasites from host cells based on a difference in surface charge has been described. However, with Trypanosoma cruzi no method exists for the isolation of pure parasite stages from heterogeneous mixtures. Studies on the electrophoresis of mixed stage populations confirm significant surface charge density differences exist among epimastigotes, trypomastigotes, and amastigotes. In ascending order of electronegativity, amastigotes have the lowest charge density, try-pomastigotes next, followed by epimastigotes. A technique has been developed for the separation of purified populations of parasites based on these charge differences using a continuous free-flow electrophoresis apparatus. The separated populations are morphologically intact and maintain their infectivity to mice. This separation method is applicable for preparative and analytical isolation of pure stages of T. cruzi for biochemical and immunological studies.     

Cellular signaling during the macrophage invasion by Trypanosoma cruzi

We have reported that protein tyrosine kinases play an important role in the invasion of Trypanosoma cruzi into primary resident macrophages. In the present study we carry out immunofluorescence assays, using monoclonal anti-phosphotyrosine antibodies, to reveal an accumulation of tyrosine-phosphorylated residues at the site of parasite association with the macrophage surface, colocalizing with host cell F-actin-rich domains. SDS-PAGE analysis of macrophage cell line IC-21 tyrosine phosphoproteins, labeled with [(35)S] L-methionine, revealed several peptides with increased levels of phosphorylation upon interaction with the parasite. Among them, were detected bands of 140, 120, 112, 94, 73, 67, and 56 kDa that match the molecular weights of proteins described as being tyrosine phosphorylated during events that lead to actin assembly in mononuclear phagocytes. The pretreatment of IC-21 macrophages with the tyrosine kinase inhibitor tyrphostin 23 inhibited trypomastigote uptake showing that tyrosine phosphorylation is important for the parasite penetration in this particular cell line. Immunofluorescence microscopy, using antibodies against p85, the regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase), placed this enzyme also in the same sites, in accordance to what is reported for phagocytosis. We suggest that once the components of T. cruzi trypomastigotes surface are recognized by macrophage receptors, they trigger the activation of a tyrosine phosphorylation cascade, PI 3-kinase recruitment, and assembly of actin filaments at the site of initial cell-to-cell contact, resembling the events described during phagocytosis. These achievements support the model for a phagocytic-like actin-dependent invasion mechanism for T. cruzi trypomastigotes into macrophages.     

Trans-sialidase and mucins of Trypanosoma cruzi: an important interplay for the parasite

A dense glycocalix covers the surface of Trypanosoma cruzi, the agent of Chagas disease. Sialic acid in the surface of the parasite plays an important role in the infectious process, however, T. cruzi is unable to synthesize sialic acid or the usual donor CMP-sialic acid. Instead, T. cruzi expresses a unique enzyme, the trans-sialidase (TcTS) involved in the transfer of sialic acid from host glycoconjugates to mucins of the parasite. The mucins are the major glycoproteins in the insect stage epimastigotes and in the infective trypomastigotes. Both, the mucins and the TcTS are anchored to the plasma membrane by a glycosylphosphatidylinositol anchor. Thus, TcTS may be shed into the bloodstream of the mammal host by the action of a parasite phosphatidylinositol-phospholipase C, affecting the immune system. The composition and structure of the sugars in the parasite mucins is characteristic of each differentiation stage, also, interstrain variations were described for epimastigote mucins. This review focus on the characteristics of the interplay between the trans-sialidase and the mucins of T. cruzi and summarizes the known carbohydrate structures of the mucins.