Differentiation Of Epimastigotes Research Articles

Disruption of the inositol phosphorylceramide synthase gene affects Trypanosoma cruzi differentiation and infection capacity.

Sphingolipids (SLs) are essential components of all eukaryotic cellular membranes. In fungi, plants and many protozoa, the primary SL is inositol-phosphorylceramide (IPC). Trypanosoma cruzi is a protozoan parasite that causes Chagas disease (CD), a chronic illness for which no vaccines or effective treatments are available. IPC synthase (IPCS) has been considered an ideal target enzyme for drug development because phosphoinositol-containing SL is absent in mammalian cells and the enzyme activity has been described in all parasite forms of T. cruzi. Furthermore, IPCS is an integral membrane protein conserved amongst other kinetoplastids, including Leishmania major, for which specific inhibitors have been identified. Using a CRISPR-Cas9 protocol, we generated T. cruzi knockout (KO) mutants in which both alleles of the IPCS gene were disrupted. We demonstrated that the lack of IPCS activity does not affect epimastigote proliferation or its susceptibility to compounds that have been identified as inhibitors of the L. major IPCS. However, disruption of the T. cruzi IPCS gene negatively affected epimastigote differentiation into metacyclic trypomastigotes as well as proliferation of intracellular amastigotes and differentiation of amastigotes into tissue culture-derived trypomastigotes. In accordance with previous studies suggesting that IPC is a membrane component essential for parasite survival in the mammalian host, we showed that T. cruzi IPCS null mutants are unable to establish an infection in vivo, even in immune deficient mice.

The Kinetoplastid-Specific Protein TcCAL1 Plays Different Roles During In Vitro Differentiation and Host-Cell Invasion in Trypanosoma cruzi.

In the pathogen Typanosoma cruzi, the calcium ion (Ca2+) regulates key processes for parasite survival. However, the mechanisms decoding Ca2+ signals are not fully identified or understood. Here, we investigate the role of a hypothetical Ca2+-binding protein named TcCAL1 in the in vitro life cycle of T. cruzi. Results showed that the overexpression of TcCAL1 fused to a 6X histidine tag (TcCAL1-6xHis) impaired the differentiation of epimastigotes into metacyclic trypomastigotes, significantly decreasing metacyclogenesis rates. When the virulence of transgenic metacyclic trypomastigotes was explored in mammalian cell invasion assays, we found that the percentage of infection was significantly higher in Vero cells incubated with TcCAL1-6xHis-overexpressing parasites than in controls, as well as the number of intracellular amastigotes. Additionally, the percentage of Vero cells with adhered metacyclic trypomastigotes significantly increased in samples incubated with TcCAL1-6xHis-overexpressing parasites compared with controls. In contrast, the differentiation rates from metacyclic trypomastigotes to axenic amastigotes or the epimastigote proliferation in the exponential phase of growth have not been affected by TcCAL1-6xHis overexpression. Based on our findings, we speculate that TcCAL1 exerts its function by sequestering intracellular Ca2+ by its EF-hand motifs (impairing metacyclogenesis) and/or due to an unknown activity which could be amplified by the ion binding (promoting cell invasion). This work underpins the importance of studying the kinetoplastid-specific proteins with unknown functions in pathogen parasites.

Involvement of STI1 protein in the differentiation process of Trypanosoma cruzi

The protozoan Trypanosoma cruzi is a parasite exposed to several environmental stressors inside its invertebrate and vertebrate hosts. Although stress conditions are involved in its differentiation processes, little information is available about the stress response proteins engaged in these activities. This work reports the first known association of the stress-inducible protein 1 (STI1) with the cellular differentiation process in a unicellular eukaryote. Albeit STI1 expression is constitutive in epimastigotes and metacyclic trypomastigotes, higher protein levels were observed in late growth phase epimastigotes subjected to nutritional stress. Analysis by indirect immunofluorescence revealed that T. cruzi STI1 (TcSTI1) is located throughout the cell cytoplasm, with some cytoplasmic granules appearing in greater numbers in late growing epimastigotes and late growing epimastigotes subjected to nutritional stress. We observed that part of the fluorescence signal from both TcSTI1 and TcHSP70 colocalized around the nucleus. Gene silencing of sti1 in Trypanosoma brucei did not affect cell growth. Similarly, the growth of T. cruzi mutant parasites with a single allele sti1 gene knockout was not affected. However, the differentiation of epimastigotes in metacyclic trypomastigotes (metacyclogenesis) was compromised. Lower production rates and numbers of metacyclic trypomastigotes were obtained from the mutant parasites compared with the wild-type parasites. These data indicate that reduced levels of TcSTI1 decrease the rate of in vitro metacyclogenesis, suggesting that this protein may participate in the differentiation process of T. cruzi.

Identification of a Golgi-localized UDP-N-acetylglucosamine transporter in Trypanosoma cruzi.

BackgroundNucleotide sugar transporters (NSTs) play an essential role in translocating nucleotide sugars into the lumen of the endoplasmic reticulum and Golgi apparatus to be used as substrates in glycosylation reactions. This intracellular transport is an essential step in the biosynthesis of glycoconjugates.ResultsWe have identified a family of 11 putative NSTs in Trypanosoma cruzi, the etiological agent of Chagas’ disease. A UDP-N-acetylglucosamine transporter, TcNST1, was identified by a yeast complementation approach. Based on a phylogenetic analysis four candidate genes were selected and used for complementation assays in a Kluyveromyces lactis mutant strain. The transporter is likely expressed in all stages of the parasite life cycle and during differentiation of epimastigotes to infective metacyclics. Immunofluorescence analyses of a GFP-TcNST1 fusion protein indicate that the transporter is localized to the Golgi apparatus. As many NSTs are multisubstrate transporters, we also tested the capacity of TcNST1 to transport GDP-Man.ConclusionsWe have identified a UDP-N-acetylglucosamine transporter in T. cruzi, which is specifically localized to the Golgi apparatus and seems to be expressed, at the mRNA level, throughout the parasite life cycle. Functional studies of TcNST1 will be important to unravel the role of NSTs and specific glycoconjugates in T. cruzi survival and infectivity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0601-7) contains supplementary material, which is available to authorized users.

The GTPase TcRjl of the human pathogen Trypanosoma cruzi is involved in the cell growth and differentiation

The protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas Disease, undergoes through a complex life cycle where rounds of cell division and differentiation occur initially in the gut of triatominae vectors and, after transmission, inside of infected cells in vertebrate hosts. Members of the Ras superfamily of GTPases are molecular switches which play pivotal regulatory functions in cell growth and differentiation. We have previously described a novel GTPase in T. cruzi, TcRjl, which belongs to the RJL family of Ras-related GTP binding proteins. Here we show that most of TcRjl protein is found bound to GTP nucleotides and may be locked in this stage. In addition, we show that TcRjl is located close to the kinetoplast, in a region corresponding possibly to flagellar pocket of the parasite and the expression of a dominant-negative TcRjl construct (TcRjlS37N) displays a significative growth phenotype in reduced serum medium. Remarkably, overexpression of TcRjl inhibits differentiation of epimastigotes to trypomastigote forms and promotes the accumulation of intermediate differentiation stages. Our data suggest that TcRjl might play a role in the control of the parasite growth and differentiation.

Biochemical characterization of a protein tyrosine phosphatase from Trypanosoma cruzi involved in metacyclogenesis and cell invasion

Protein tyrosine phosphatases (PTPs) form a large family of enzymes involved in the regulation of numerous cellular functions in eukaryotes. Several protein tyrosine phosphatases have been recently identified in trypanosomatides. Here we report the purification and biochemical characterization of TcPTP1, a protein tyrosine phosphatase from Trypanosoma cruzi, the causing agent of Chagas’ disease. The enzyme was cloned and expressed recombinantly in Escherichia coli and purified by Ni-affinity chromatography. Biochemical characterization of recombinant TcPTP1 with the PTP pseudo-substrate pNPP allowed the estimation of a Michaelis–Menten constant Km of 4.5mM and a kcat of 2.8s−1. We were able to demonstrate inhibition of the enzyme by the PTP1b inhibitor BZ3, which on its turn was able to accelerate the differentiation of epimastigotes into metacyclic forms of T. cruzi induced by nutritional stress. Additionally, this compound was able to inhibit by 50% the infectivity of T. cruzi trypomastigotes in a separate cellular assay. In conclusion our results indicate that TcPTP1 is of importance for cellular differentiation and invasivity of this parasite and thus is a valid target for the rational drug design of potential antibiotics directed against T. cruzi.

Involvement of protein kinase C isoenzymes in Trypanosoma cruzi metacyclogenesis induced by oleic acid

Previously, we showed that oleic acid (OA) induces Trypanosoma cruzi metacyclogenesis through a signaling pathway involving de novo diacylglycerol biosynthesis and simultaneous protein kinase C (PKC) activation. Herein, we demonstrated that OA also triggers a transient Ca(2+) signal in epimastigotes, necessary for parasite differentiation, that could account for PKC activation. In addition, we found that this free fatty acid (FFA) directly stimulated in vitro the activity of T. cruzi PKC in a dose-response way. We determined the presence of classical and novel PKC isoenzymes that were differentially expressed in the infective amastigotes (alpha and delta) and tripomastigotes (alpha, beta, and gamma) and in the non-infective epimastigotes (alpha, beta, gamma, and delta). We also demonstrated that OA induced in epimastigotes the translocation of PKC alpha, beta, gamma, and delta to the membrane, indicating a selective effect of this FFA. To establish a correlation between T. cruzi metacyclogenesis induced by OA and the activation of a particular PKC isoenzyme, the specific PKC inhibitors Ro 32-0432 and Rottlerin (9-30 nM and 5-35 microM, respectively) were employed. These compounds, even at the lowest concentrations assayed, abrogated both epimastigote differentiation and membrane translocation of PKC beta, gamma, and delta. These findings strongly support a key role for classical and novel PKC isoenzymes in the signaling pathways involved in T. cruzi metacyclogenesis induced by OA.

Activity of 8.O.4′-neolignans against Trypanosoma cruzi

The in vitro evaluation of a series of 8.O.4'-neolignans (in their ketone, alcohol, and acetylated forms) on the proliferation of Trypanosoma cruzi led to the detection of three compounds with interesting activities against T. cruzi epimastigotes. These compounds also inhibited the in vitro differentiation of epimastigotes to trypomastigotes. When tested against HeLa cells, the same compounds did not affect the cell vitality and showed a low influence on cell viability as measured by dimethylthiazol diphenyltetrazolium bromide reduction. The three phenylpropanoid moieties tested did not show any activity against T. cruzi. One of the tested compounds, compound 4 (3,4-methylenedioxi7-oxo-1'-allyl-3',5'-dimethoxy-8.O.4'-neolignan) showed the best performance in both trypanocide and cytotoxic assays, suggesting that it should be considered as a leading structure for further research.

Small-Subunit rRNA Processome Proteins Are Translationally Regulated during Differentiation of Trypanosoma cruzi

We used differential display to select genes differentially expressed during differentiation of epimastigotes into metacyclic trypomastigotes in the protozoan parasite Trypanosoma cruzi. One of the selected clones had a sequence similar to that of the small-subunit (SSU) processome protein Sof1p, which is involved in rRNA processing. The corresponding T. cruzi protein, TcSof1, displayed a nuclear localization and is downregulated during metacyclogenesis. Heterologous RNA interference assays showed that depletion of this protein impaired growth but did not affect progression through the cell cycle, suggesting that ribosome synthesis regulation and the cell cycle are uncoupled in this parasite. Quantitative PCR (qPCR) assays of several SSU processome-specific genes in T. cruzi also showed that most of them were regulated posttranscriptionally. This process involves the accumulation of mRNA in the polysome fraction of metacyclic trypomastigotes, where TcSof1 cannot be detected. Metacyclic trypomastigote polysomes were purified and separated by sucrose gradient sedimentation. Northern blot analysis of the sucrose gradient fractions showed the association of TcSof1 mRNA with polysomes, confirming the qPCR data. The results suggest that the mechanism of regulation involves the blocking of translation elongation and/or termination.

Platelet-activating factor-like activity isolated from Trypanosoma cruzi

Platelet-activating factor is a phospholipid mediator that exhibits a wide variety of physiological and pathophysiological effects, including induction of inflammatory response, chemotaxis and cellular differentiation. Trypanosoma cruzi, the etiological agent of Chagas' disease, is transmitted by triatomine insects and while in the triatomine midgut the parasite differentiates from a non-infective epimastigote stage into the pathogenic trypomastigote metacyclic form. We have previously demonstrated that platelet activating factor triggers in vitro cell differentiation of T. cruzi. Here we show a platelet activating factor-like activity isolated from lipid extract of T. cruzi epimastigotes incubated in the presence of [ 14C]acetate. Trypanosoma cruzi-platelet activating factor-like lipid induced the aggregation of rabbit platelets, which was prevented by platelet activating factor-acetylhydrolase. Mouse macrophage infection by T. cruzi was stimulated when epimastigotes were kept for 5 days in the presence of T. cruzi-platelet activating factor, before interacting with the macrophages. The differentiation of epimastigotes into metacyclic trypomastigotes was also triggered by T. cruzi-platelet activating factor. These effects were abrogated by a platelet activating factor antagonist, WEB 2086. Polyclonal antibody raised against mouse platelet activating factor receptor showed labelling for T. cruzi epimastigotes using immunoblotting and immunofluorescence assays. These data suggest that T. cruzi contain the components of an autocrine platelet activating factor-like ligand–receptor system that modulates cell differentiation towards the infectious stage.

Reservosome: an endocytic compartment in epimastigote forms of the protozoan Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae). Correlation between endocytosis of nutrients and cell differentiation

Reservosomes are large membrane-bound organelles found at the posterior end of epimastigote forms of Trypanosoma cruzi, but absent in amastigotes and trypomastigotes. We have transferred bloodstream trypomastigotes to LIT medium supplemented with gold-labelled transferrin in order to analyse, at the ultrastructural level, the occurrence of reservosomes and endocytosis during the trypomastigote to epimastigote differentiation. After 24 h, the trypomastigotes differentiated into amastigotes, which adhered to each other forming large clusters. Electron-dense vesicles were detected close to the Golgi complex in cells with intermediary characteristics between amastigotes and epimastigotes, but typical reservosomes at the posterior cell tip were still absent. Transferrin-gold complexes were observed only bound to the surface of clustered cells. After 72 h, epimastigotes were observed being released from the clusters and free-swimming epimastigotes appeared, containing electron-dense vesicles at their posterior region. Typical reservosomes, labelled with transferrin-gold, were observed only in free-swimming epimastigotes. When fully differentiated epimastigotes were incubated with transferrin-gold complexes and then processed for the immunocytochemical detection of cysteine proteinase, all reservosomes were positive for the enzyme, but co-localization of both markers did not occur in all organelles. Our data demonstrate that in T. cruzi epimastigotes endocytosis is strongly related to reservosome biogenesis during the trypomastigote to epimastigote differentiation process.

Trypanosoma cruzi: cloning and characterization of two genes whose expression is up-regulated in metacyclic trypomastigotes

The differentiation of epimastigotes into metacyclic trypomastigotes (metacyclogenesis) involves the transformation of a replicative non-infective form of Trypanosoma cruzi into a non-replicative infective stage. The study of genes with stage-specific expression may provide insight into the mechanisms involved in the regulation of gene expression in this parasite. We cloned and characterized two genes whose expression is up-regulated in metacyclic trypomastigote, those encoding metacyclin-II (Met-II) and metacyclin-III (Met-III). Nucleotide sequence analysis identified no sequence similarity with sequences available from genetic databases. The deduced amino acid sequences of the genes indicated that Met-III encodes a basic polypeptide whereas Met-II encodes an acidic polypeptide. Northern and Western blot analyses showed that Met-II and Met-III were expressed by metacyclic trypomastigotes, but not by epimastigotes. Antisera directed against the recombinant Met-II and Met-III proteins recognized two polypeptides on Western blots: a 16-kDa and a 24-kDa polypeptide. Immunocytochemistry analysis using electron microscopy showed that metacyclin-II is localized mainly at the kinetoplast whereas metacyclin-III is localized at the nucleus of the parasite. Southern blot analysis, using genomic DNA and T. cruzi chromosomes separated by pulsed-field gel electrophoresis, indicated that these genes were present as single copies on different chromosomes of T. cruzi Dm28c.

Effect of the alkyl-lysophospholipids on the proliferation and differentiation of Trypanosoma cruzi

Alkyl-lysophospholipids (ALPs), designed as potential immunomodulators, have been shown to be cytotoxic for a variety of tumour cells and are under clinical studies for cancer chemotherapy. ET-18-OCH 3, hexadecylphosphocholine and ilmofosine were assayed against the three forms of Trypanosoma cruzi. Incubation with bloodstream trypomastigotes resulted, under different experimental conditions, in higher activity of the compounds in comparison with crystal violet. The ED 50/24 h values were 13.4±2.8 μM and 11.7±0.6 μM for amastigotes and epimastigotes, respectively. ET-18-OCH 3 (0.3 and 0.6 μM) inhibited the differentiation of epimastigotes to trypomastigotes (Dm28C clone) in the range 40–57%. This drug (3.75–15 μM) also caused a time- and dose-dependent inhibition of the intracellular proliferation of amastigotes in heart muscle cells with ED 50 values of 14.3±4.2, 8.9±1.9 and 6.8±0.4 μM, after 1, 2 and 3 days of treatment. Pre-treatment of the parasite with this drug inhibited its interiorization into the host cell. Interestingly, the intracellular differentiation of amastigotes to trypomastigotes was not hampered by the drug. The present results demonstrate the lytic effect of ALPs on the three forms of T. cruzi, as well as the inhibition of both the differentiation to the infective form and the proliferation of parasites interiorized in heart cells. Ultrastructural analysis of epimastigotes treated with the three ALPs showed extensive blebing of the flagellar membrane. As described in tumour cells, the membrane seems to be a primary target of the drugs.

Biological parameters and molecular markers of clone CL Brener--the reference organism of the Trypanosoma cruzi genome project.

Clone CL Brener is the reference organism used in the Trypanosoma cruzi Genome Project. Some biological parameters of CL Brener were determined: (a) the doubling time of epimastigote forms cultured in liver infusion-tryptose (LIT) medium at 28 degrees C is 58 +/- 13 hr; (b) differentiation of epimastigotes to metacyclic trypomastigotes is obtained by incubation in LIT-20% Grace's medium; (c) trypomastigotes infect mammalian cultured cells and perform the complete intracellular cycle at 33 and 37 degrees C; (d) blood forms are highly infective to mice; (e) blood forms are susceptible to nifurtimox and benznidazole. The molecular typing of CL Brener has been determined: (a) isoenzymatic profiles are characteristic of zymodeme ZB; (b) PCR amplification of a 24S alpha ribosomal RNA sequence indicates it belongs to T. cruzi lineage 1; (c) schizodeme, randomly amplified polymorphic DNA (RAPD) and DNA fingerprinting analyses were performed.

Induction of Trypanosoma cruzi Metacyclogenesis in the Gut of the Hematophagous Insect Vector, Rhodnius prolixus, by Hemoglobin and Peptides Carrying αD-Globin Sequences

Trypanosoma cruzi, a protozoan responsible for the American trypanosomiasis (Chagas disease), multiplies and differentiates in the gut of triatomine insect vectors. The effects of hemoglobin and synthetic peptides carrying αD-globin fragments on both the growth and the transformation of T. cruzi epimastigotes (noninfective) into metacyclic trypomastigotes (infective forms) were studied. This differentiation in the insect′s gut is expressed when hemoglobin and synthetic peptides corresponding to residues 30-49 and 35-73 of the αD-globin were added to the plasma diet. However, synthetic peptide 41-73 does not induce differentiation of epimastigotes even in the presence of the two former synthetic peptides. Thus, these data delineate an unusual molecular mechanism which modulates the dynamics of transformation of epimastigotes into metacyclic trypomastigotes in the triatomine vector′s gut.

Stimulation of Trypanosoma cruzi adenylyl cyclase by an alpha D-globin fragment from Triatoma hindgut: effect on differentiation of epimastigote to trypomastigote forms.

A peptide from hindguts of the Triatoma hematophagous Chagas insect vector activates adenylyl cyclase activity in Trypanosoma cruzi epimastigote membranes and stimulates the in vitro differentiation of epimastigotes to metacyclic trypomastigotes. Hindguts were obtained from insects fed 2 days earlier with chicken blood. Purification was performed by gel filtration and HPLC on C18 and C4 columns. SDS/PAGE of the purified peptide showed a single band of about 10 kDa. The following sequence was determined for the 20 amino-terminal residues of this peptide: H2N-Met-Leu-Thr-Ala-Glu-Asp-Lys-Lys-Leu-Ile-Gln- Gln-Ala-Trp-Glu-Lys-Ala-Ala-Ser-His. This sequence is identical to the amino terminus of chicken alpha D-globin. On a Western blot, the peptide immunoreacted with a polyclonal antibody against chicken globin D. A synthetic peptide corresponding to residues 1-40 of the alpha D-globin amino terminus also stimulated adenylyl cyclase activity and promoted differentiation. This 125I-labeled synthetic peptide bound specifically to T. cruzi epimastigote cells. Activation of epimastigote adenylyl cyclase by the hemoglobin-derived peptide may play an important role in T. cruzi differentiation and consequently in the transmission of Chagas disease.

Further characterization ofTrypanosoma cruzi GP57/51 as the major antigen expressed by differentiating epimastigotes

Study of Trypanosoma cruzi metacyclogenesis under chemically defined conditions showed that the expression of a group of acidic polypeptides with a molecular weight of 45-50 kDa is markedly increased on adhesion of epimastigotes to the culture vessels. Immunochemical analysis revealed that these developmentally regulated polypeptides are structurally related to, and possibly homologous with, a major T. cruzi antigen, namely, GP57/51, a glycoprotein that has recently been characterized as a cysteine proteinase. The differentiation of epimastigotes into metacyclic trypomastigotes is accompanied by a 2- to 5-fold reduction in the concentration of GP57/51 antigen as determined by radioimmunoassays using monoclonal antibodies. Two-dimensional polyacrylamide gel electrophoretic analysis of metabolically labelled parasites indicated that this antigen is synthesized as a precursor with a molecular weight of 60 kDa, which is then processed to a level of 45-50 kDa via the formation of at least one intermediate processing product. The observation that expression of GP57/51-related products increases during epimastigote differentiation suggests an important role for this proteinase during the life cycle of T. cruzi.

Trypanosoma cruzi proliferation and differentiation are blocked by topoisomerase II inhibitors

Bacterial topoisomerase II inhibitors (ofloxacin and its commercial derivative Tarivid, nalidixic acid, and novobiocin) were tested as blockers of Trypanosoma cruzi differentiation and proliferation. The transformation of either epimastigotes into metacyclic trypomastigotes or amastigotes into trypomastigotes was inhibited by the drugs in a dose-dependent manner. The inhibition of epimastigote differentiation was also dependent on the time of drug addition to the medium. Proliferation of T. cruzi was also blocked in a dose-dependent manner by the drugs, with the exception of novobiocin, which did not inhibit epimastigote replication and resulted in cell lysis when it was used at high concentrations. On the other hand, the transformation of amastigotes into epimastigotes in axenic culture was not inhibited; this process did not require either kinetoplast (mitochondrial) DNA replication or changes in the DNA network organization. Electron microscopy of cells treated with Tarivid (ofloxacin) showed damage to the kinetoplast, suggesting that this organelle might be the target of the drug. These results indicate that a bacterial-like topoisomerase II plays an important role in T. cruzi proliferation and differentiation.

Biological characterization of Trypanosoma cruzi zymodemes: In vitro differentiation of epimastigotes and infectivity of culture metacyclic trypomastigotes to mice

Thirty-one Trypanosoma cruzi isolates from Chile, Peru, and Bolivia were studied in their capacity to differentiate in vitro from epimastigotes to metacyclic trypomastigotes on TAU-3AAG medium. Zymodeme 1 parasites displayed the best level of differentiation, which ranges from 60 to 90% depending on the isolate. Zymodeme 2 parasites exhibited highly heterogeneous differentiation rates. This differentiation method permits the obtention of large amounts of metacyclic trypomastigotes from zymodeme 1 parasites. Metacyclic trypomastigotes obtained in vitro were infective to nude Balb/c hybrid mice. Zymodeme 1 parasites produced high parasitemias in this murine model; in contrast, zymodeme 2 parasites displayed lower parasitemias. Of a total of 27 T. cruzi isolates, 20 proved to be infective to mice, 12 gave enough parasites for further studies, and 8 of these were used for biological characterization. Results are compared with the infective clone Dm28 and Tulahuén strains maintained since (1954) in mice.

Partial inhibition of trypomastigote entry into cultured mammalian cells by monoclonal antibodies against a surface glycoprotein of Trypanosoma cruzi

Monoclonal antibodies were raised against the surface of trypomastigote forms of Trypanosoma cruzi. Although some of these antibodies reacted against antigens shared by trypomastigote and epimastigote or amastigote forms, the majority were trypomastigote-specific. Trypomastigote-specific monoclonal antibodies recognized all infective stages, including trypomastigotes from the bloodstream of infected mice, insect feces, tissue culture and those resulting from differentiation of epimastigotes in axenic culture media. The monoclonal antibodies H1A10 and 6A2, as well as Fab fragments from H1A10, partially prevented T. cruzi invasion of LLC-MK2 cell monolayers (inhibition of 50–70%) when present throughout the entire experiment. Both antibodies recognized an 85 kDa glycoprotein (Tc-85) of the trypomastigote surface which contains N-acetyl-d-glucosamine and /or sialic acid.