Amastigote and epimastigote stage-specific components of Trypanosoma cruzi characterized by using monoclonal antibodies. Purification and molecular characterization of an 83-kilodalton amastigote protein

Stage-specific mAb have been produced to amastigotes and epimastigotes of Trypanosoma cruzi (Brazil strain). mAb C-1 through C-6 reacted specifically with T. cruzi strains; no cross-reactions were found with membranes of promastigotes or amastigotes of Leishmania species. One mAb produced against the epimastigote membranes (C-5) was found to be specific against this stage by radioimmune binding assay, immunofluorescence, and radioimmunoprecipitation. mAb C-5 recognized a novel epimastigote protein at Mr (greater than 200,000) on immunoprecipitation with radiolabeled epimastigotes. Three amastigote stage-specific monoclonal antibodies were produced against membrane-enriched preparations of T. cruzi (Brazil strain) amastigotes grown in axenic culture (C-1 through C-3). By indirect immunofluorescence assay, monoclonal antibody C-2 bound only to T. cruzi amastigotes; no reaction with either tissue culture-derived trypomastigotes or epimastigotes was observed. mAb C-1 and C-2 each specifically immunoprecipitated a single protein molecule with Mr 83,000 from [35S]-methionine-labeled amastigotes. mAb C-2 was also used to affinity purify an 83-kDa Ag that was recognized by human Chagasic sera from patients of endemic countries of Latin America in an enzyme immunoassay. Amino acid composition and preliminary sequence data of the 83-kDa protein are presented. These mAb and/or purified Ag may be useful in studying stage differentiation, monitoring transformation, and for further taxonomic, epidemiologic, and immunologic studies of Chagas' disease.     

Interleukin-10 expression after intramuscular DNA electrotransfer: kinetic studies

A set of monoclonal antibodies that recognizes a Trypanosoma cruzi 45-kDa protein was produced and used to characterize this molecule and study its role in trypanosome adhesion to heart myoblasts. We found that the 45-kDa protein is a surface mucin, is expressed only in invasive trypomastigotes, but not in noninvasive epimastigotes or amastigotes, and is released by the trypanosome in culture medium. One of the monoclonal antibodies (Mab B5) from this set inhibits the attachment of trypomastigotes to heart myoblasts preventing trypanosome entry, whereas the others (Mabs B4 and F1) do not. This inhibition was seen with the B5 hybridoma culture supernatant, with the purified Mab B5 IgG or with Mab B5 Fab fragments. These novel findings identify the 45-kDa mucin as a new T. cruzi ligand that is used by invasive forms of this organism to adhere to heart myoblasts.     

Extracellular killing of Trypanosoma cruzi amastigotes by human eosinophils

Granules released from human eosinophils upon interaction with Trypanosoma cruzi amastigotes in vitro were seen attached to the surface of non-internalized parasites by electron microscopy. Amastigote damage was preceded by the binding of eosinophil granule material to its membrane, and eosinophil granule major basic protein (MBP) bound to the parasite surface was readily detectable. Additional evidence of eosinophil cytotoxicity for extracellular amastigotes was the observation that amastigotes trapped between two eosinophils, without being ingested by either one, were destroyed at the interface. Amastigotes isolated from the spleens of infected mice or grown in culture were similarly sensitive to the lytic effects of purified MBP. These results demonstrate the ability of human eosinophils to lyse T. cruzi amastigotes extracellularly in the absence of antibody and suggest that MBP may be involved in the effect. Thus, eosinophils, known to be capable of destroying phagocytosed amastigotes, could also contribute to the clearance of these parasites through extracellular killing.     

Human chagasic serum contains antibodies capable of inhibiting Trypanosoma cruzi egress from tissue culture cells

Previous studies at our laboratory have shown that an antibody (antiegressin) present in the serum of chronically infected mice is capable of inhibiting the egress of Trypanosoma cruzi from infected BALB/c fibroblasts. We have used this in vitro system to evaluate whether human chagasic serum is also capable of inhibiting T. cruzi egress. BALB/c fibroblasts were infected with tissue culture-derived parasites. Five-percent solutions of the individual human serum samples in culture medium were added to the wells, and the number of parasites released was determined at day 5 after infection. The cells cultured with serum from infected individuals released between 37% and 72% fewer parasites than those cultured with control serum. A similar reduction in parasite egress resulted from incubation with the protein-A purified IgG fraction from 3 of these human samples. Immunocytochemical staining employing antineuraminidase antibodies supported the notion that the reduction in parasite levels is due to inhibition at the point of parasite egress. These results indicate that human serum of individuals infected with T. cruzi is capable of inhibiting release of the parasite from infected tissue culture cells and that the phenomenon of egress-inhibition may be relevant during infection of human subjects.     

Production of amastigotes from metacyclic trypomastigotes of Trypanosoma cruzi

Attempts to recreate all the developmental stages of Trypanosoma cruzi in vitro have thus far been met with partial success. It is possible, for instance, to produce trypomastigotes in tissue culture and to obtain metacyclic trypomastigotes in axenic conditions. Even though T. cruzi amastigotes are known to differentiate from trypomastigotes and metacyclic trypomastigotes, it has only been possible to generate amastigotes in vitro from the tissue-culture-derived trypomastigotes. The factors and culture conditions required to trigger the transformation of metacyclic trypomastigotes into amastigotes are as yet undetermined. We show here that pre-incubation of metacyclic trypomastigotes in culture (MEMTAU) medium at 37 degrees C for 48 h is sufficient to commit the parasites to the transformation process. After 72 h of incubation in fresh MEMTAU medium, 90% of the metacyclic parasites differentiate into forms that are morphologically indistinguishable from normal amastigotes. SDS-PAGE, Western blot and PAABS analyses indicate that the transformation of axenic metacyclic trypomastigotes to amastigotes is associated with protein, glycoprotein and antigenic modifications. These data suggest that (a) T. cruzi amastigotes can be obtained axenically in large amounts from metacyclic trypomastigotes, and (b) the amastigotes thus obtained are morphological, biological and antigenically similar to intracellular amastigotes. Consequently, this experimental system may facilitate a direct, in vitro assessment of the mechanisms that enable T. cruzi metacyclic trypomastigotes to transform into amastigotes in the cells of mammalian hosts.     

Role of inflammatory cells in Chagas' disease. III. Kinetics of human eosinophil activation upon interaction with parasites (Trypanosoma cruzi)

The kinetics of human eosinophil activation and granule secretion initiated by interaction with Trypanosoma cruzi amastigotes was studied by using a monoclonal IgG1 antibody (termed EG2) that is specific for an epitope present only in the secreted forms of both eosinophil cationic protein (ECP) and the eosinophil protein X (EP-X), and hence not detectable in unstimulated resting eosinophils. Studies were carried out by using electron microscopy and indirect immunofluorescence. In the electron microscopy studies, deposits of protein A-gold particles in parasite-containing eosinophils that had been incubated previously with EG2 antibody were first detected 4 hr after initiation of the eosinophil-amastigote interaction. Control tests performed with a monoclonal IgG1 unreactive with eosinophils showed no deposition of protein A-gold particles. EG2 antibody binding was confined to the crystalloid granule matrix, where ECP and EP-X are known to be stored. A similar kinetic pattern of ECP/EP-X solubilization and secretion was confirmed by the results of the indirect immunofluorescence experiments also showing the binding of EG2 antibody after 4 hr of cell-parasite interaction. The kinetics of ECP/EP-X solubilization and secretion paralleled the kinetics of destruction of internalized amastigotes, suggesting a role for these basic proteins in parasite killing. Consistent with this notion was the detection of ECP/EP-X in the fluid of phagocytic vacuoles containing amastigotes and associated with the ingested organisms at the same time as the parasites began to show structural alterations. These results outlined the kinetics of eosinophil activation in terms of the time required for mobilization of two basic proteins associated with eosinophil secretion that are known to be biologically active.     

Solid-phase assay for the detection of low-abundance enzymes, and antibodies to enzymes in immune reactions, using acid sphingomyelinase as a model

The development of a solid-phase immunosorbent assay, suitable for use with enzyme antigens, is described. Acid sphingomyelinase and a mouse monoclonal anti-sphingomyelinase antibody have been used to determine optimal conditions for the assay. The assay involves immobilization of a second antibody (anti-mouse IgG) in the wells of a polyvinyl microtiter plate. Soluble immune complexes of first antibody (monoclonal anti-sphingomyelinase) and antigen (sphingomyelinase), incubated in separate vials, are then reacted in the anti-mouse IgG-coated assay wells, and the extent of the cross-reaction between antibody and antigen is measured by direct assay of enzyme retained in the well. A necessary condition of the assay is that antibody must not inhibit enzyme activity, which makes it especially suitable for monoclonal antibodies. The assay finds useful application in hybridoma fluid screening, equivalence point determination, and demonstration of cross-reacting enzyme from various tissue sources.     

Trypanosoma cruzi: a specific surface marker for the amastigote form

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.     

Purification of tissue forms (Amastigotes) of Trypanosoma cruzi by immunoaffinity chromatography

A rapid and simple method for the purification of amastigotes of Trypanosoma cruzi from spleens of infected mice is described. A protein A-Sepharose 4B immunoadsorbent column bound with antisera to epimastigotes of T. cruzi was used to purify the tissue forms of this parasite. Host cells and debris are not retained, and parasites can be eluted in high yields and purity. Studies of surface glycoproteins and glycolipids of the purified amastigotes with 18 lectins of various specificities revealed the presence on the parasites of receptors for N-acetylglucosamine, N-acetylgalactosamine, D-galactose, and D-mannose binding lectins.     

Attachment of Trypanosoma cruzi to host cells: a monoclonal antibody recognizes a trypomastigote stage-specific epitope on the gp 83 required for parasite attachment.

A set of monoclonal antibodies against the purified surface gp 83 of T. cruzi trypomastigotes was produced and the ability of these monoclonals to inhibit the attachment of trypomastigotes to heart myoblasts was investigated. Western blots of solubilized trypomastigotes, epimastigotes or amastigotes probed with this set of monoclonal antibodies show that the gp 83 is present in invasive trypomastigotes, but not in non-invasive epimastigotes or amastigotes. One monoclonal antibody (Mab 4A4) from this set inhibits the attachment of trypomastigotes to heart myoblasts, whereas the others (MAbs 2H6, 4B9, 2D11) do not. These results show that the Mab 4A4 recognizes an epitope on the gp 83 of invasive trypomastigotes required for parasite binding to host cells.     

Survival of Trypanosoma cruzi metacyclic trypomastigotes within Coxiella burnetii vacuoles: differentiation and replication within an acidic milieu

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.     

Trypanosoma cruzi adenylate cyclase activity. Purification and characterization.

Adenylate cyclase activity associated with Trypanosoma cruzi sedimentable fractions was solubilized by treatment with the non-ionic detergent Lubrol PX and 0.5 M-(NH4)2SO4. The following hydrodynamic and molecular parameters were established for a partially purified enzyme-detergent complex: sedimentation coefficient 6.2 S; Stokes radius 5.65 nm; partial specific volume 0.83 ml/g; Mr 244 000; frictional ratio 1.33. A Mr of about 124 000 was calculated for the detergent-free protein from these parameters. The pI of this enzyme activity was 6.2. A monoclonal antibody to T. cruzi adenylate cyclase was obtained, which inhibited cyclase activities from several lower eukaryotic organisms. The T. cruzi adenylate cyclase was further purified by using this antibody in immunoaffinity chromatographic columns. Fractions obtained after this chromatography showed, on SDS/polyacrylamide-gel electrophoresis, a main polypeptide band with an apparent Mr of about 56 000, which specifically reacted with the monoclonal antibody.     

Mercaptopyridine-N-oxide, an NADH-fumarate reductase inhibitor, blocks Trypanosoma cruzi growth in culture and in infected myoblasts

The enzyme NADH-fumarate reductase is not found in mammalian cells but it is present in several parasitic protozoa including Trypanosoma cruzi, the parasite that causes Chagas' disease. This study shows that the drug 2-mercaptopyridine-N-oxide (MPNO) inhibits NADH-fumarate reductase purified from T. cruzi (ID50 = 35 microM). When added to intact cells, MPNO inhibited the growth of T. cruzi epimastigotes in culture (ID50 = 0.08 microM) as well as the infection of mammalian myoblasts by T. cruzi trypomastigotes (ID50 = 20 microM). At a concentration of 2.4 microM, MPNO also inhibited the growth of amastigotes (intracellular dividing forms) in cultured mammalian myoblasts. Supplementation of culture media with 5 mM succinate, the product of fumarate reductase, partially protected against the inhibition of the growth of epimastigotes by MPNO. Moreover, MPNO inhibited the accumulation of succinate in cultures of epimastigotes, as measured by high performance liquid chromatography. Although MPNO may have other intracellular targets in addition to fumarate reductase, these results support the hypothesis that compounds which inhibit the enzyme fumarate reductase may be potential chemotherapeutic agents against Chagas' disease.     

A major cysteine proteinase is developmentally regulated in Trypanosoma cruzi

Epimastigotes of different stocks of Trypanosoma cruzi contain similar levels of proteinase activity on azocasein; amastigotes and trypomastigotes contain 10-fold lower levels of this proteolytic activity, which seems, therefore, to be developmentally regulated. The proteinase could be detected as a broad band, centered at about 60 kDa, which in some cases resolved into two close bands, in (a) SDS-polyacrylamide gels containing fibrinogen, and (b) Western blots probed with a polyclonal rabbit antiserum prepared against purified cysteine proteinase. No proteinase activity was observed at molecular weights lower than 55 kDa. The results show that the enzyme previously purified is the major cysteine proteinase present in epimastigotes of all stocks of T. cruzi tested.     

Prevalence of antibodies to Trypanosoma cruzi in raccoons (Procyon lotor) from an urban area of northern Virginia

Raccoons (Procyon lotor) are reservoir hosts for Trypanosoma cruzi. A 3-yr-long serological survey was conducted to determine the prevalence of antibodies to this zoonotic parasite in raccoons collected from Fairfax County, Virginia, a suburban/urban area outside Washington, D.C. Serum samples from 464 raccoons were examined for T. cruzi antibodies at a 1:40 dilution with an indirect fluorescent antibody test (IFAT) and Brazil strain T. cruzi amastigotes and trypomastigotes as antigen. A positive IFAT test was found in 154 (33%) of the 464 samples. The yearly prevalence was 49 of 132 (37%) in 2000; 19 of 120 (16%) in 2001; and 86 of 212 (41%) in 2002. Our study indicates that raccoons in this area of Virginia are frequently exposed to T. cruzi.     

Immunological characterization of antigens released by Trypanosoma cruzi-infected cells.

Chagas' disease, caused by Trypanosorna cruzi, is characterized by the appearance of pathological lesions in the heart and other tissues during the chronic phase. The mechanisms responsible for such damage are still unclear. In the vertebrate host, T. cruzi replicates intracellularly before transforming from amastigotes into trypomastigotes. The infected host cell then lyses, releasing the cytoplasmic contents and the parasites that shed membrane glycoproteins soon after release. The sum of all these components we have termed released antigen (Rag). We characterized antigens, released in vitro by fibroblasts infected with T. cruzi, obtained by concentrating conditioned serum-free culture media. The results demonstrate that Rag contains a complex protein mixture including stage-specific T. cruzi antigens (Ssp-1, -2, -4), glucose-regulated protein (Grp) 78h, and peptides recognized by the monoclonal antibody 2B10. These peptides exhibit neuraminidase activity and are expressed by intracellular and 10-20% of released trypomastigotes. Additionally, Rag is recognized by sera from T. cruzi-infected mice and human chagasic patients. Rag also stimulates in vitro production of interferon-gamma by splenocytes from resistant C57B1/6 and susceptible BALB/c infected mice and interleukin-4 by splenocytes from BALB/c infected mice. Altogether these results indicate that Rag is immunologically relevant and could contribute to pathogenesis of T. cruzi infection.     

Purification of L-dopa decarboxylase from rat liver and production of polyclonal and monoclonal antibodies against it

L-DOPA decarboxylase [DDC, aromatic-L-amino acid carboxyl-lyase, EC 4.1.1.28] was purified 800-fold from rat liver by several column chromatographic steps. The enzyme (specific activity, about 6 mumol/min X mg protein) had a molecular weight of 100,000 and gave a single band with a molecular weight of 50,000 on SDS-polyacrylamide gel electrophoresis. Its isoelectric point was pH 5.7. The absorption spectrum in the visible region of the purified DDC showed maxima at 330 and 420 nm. Polyclonal and monoclonal antibodies against DDC were produced by using this purified protein as an antigen. Polyclonal anti-DDC serum immunoprecipitated the DDC activities of rat, guinea-pig and rabbit livers (about 1, 10, and more than 100 microliter of antiserum, respectively, were required for 50% precipitation of 2 nmol/min of activity of these enzymes). The monoclonal antibody, named MA-1, belonged to the IgG1 subclass and immunoprecipitated the DDC activities of rat and guinea-pig livers to the same extent (about 0.5 micrograms of IgG was required to immunoprecipitate 2 nmol/min activity of each enzyme), but it did not affect the rabbit enzyme. The antibody MA-1 detected DDC molecules of both the purified enzyme and crude homogenate of rat liver blotted onto a nitrocellulose sheet. Immunohistochemically this antibody also stained specific neurons in the substantia nigra, raphe nucleus and locus coeruleus of rat brain.     

Failure of Trypanosoma cruzi to trigger the respiratory burst of activated macrophages. Mechanism for immune evasion and importance of oxygen-independent killing

Activated macrophages are thought to kill Trypanosoma cruzi, which lack catalase, by the generation of hydrogen peroxide. We investigated triggering of the respiratory burst of activated macrophages induced by phagocytosis of virulent T. cruzi, bloodform trypomastigotes, amastigotes obtained from spleens, and tissue culture organisms; and of relatively nonvirulent epimastigotes. All stages of T. cruzi prompted the release of less than 10% of hydrogen peroxide released by activated macrophages when stimulated with PMA or Candida. Superoxide anion production was not stimulated by PMA or Candida in activated macrophages nor was there a significant qualitative reduction of nitroblue tetrazolium induced by ingestion of virulent T. cruzi. Opsonization of T. cruzi with specific antibody did not promote the release of hydrogen peroxide or the reduction of nitroblue tetrazolium. Similar results were observed with activated spleen macrophages. Incubation of activated macrophages with catalase, catalase and superoxide dismutase, sodium benzoate with or without catalase, and respiratory burst-exhausting PMA failed to inhibit the killing of T. cruzi in vitro. These results indicate that 1) virulent opsonized or unopsonized T. cruzi do not trigger a respiratory burst by activated macrophages and 2) oxygen-independent killing of T. cruzi is of prime importance.     

Monoclonal antibodies against Trypanosoma cruzi neuraminidase reveal enzyme polymorphism, recognize a subset of trypomastigotes, and enhance infection in vitro

The identification and characterization of two murine mAb (TCN-1 and TCN-2) that react with the neuraminidase of Trypanosoma cruzi is reported. The mAb were identified based on their ability to inhibit enzyme activity and recognize neuraminidase in crude enzyme preparations. TCN-1 and TCN-2 recognized Ag in tissue culture trypomastigotes but not in the amastigotes, epimastigotes, or metacyclic trypomastigotes using immunoblot assays and immunofluorescence. In addition, clones Y-H6, MV-13, and Silvio X-10/4 of T. cruzi revealed a unique banding pattern characteristic of each clone. In Silvio X-10/4, the mAb recognized four distinct bands ranging from 121,000 to 203,000 whereas in Y-H6 and MV-13 they identified bands ranging from 138,000 to 222,000. Characterization of neuraminidase by two-dimensional PAGE revealed the polypeptides that make up the enzyme to have isoelectrical points ranging from 6.55 to 7.30. Immunofluorescence and C-mediated lysis assays showed that the mAb reacted with a subset of trypomastigotes representing 28% of the total parasite population. Functional studies showed that the mAb enhanced infection of cultured cells by trypomastigotes. Our experiments confirm previous findings with polyclonal Ab and are in accordance with the hypothesis that neuraminidase modulates infection through a negative control mechanism.     

Distribution of α-Galactosyl-Containing Epitopes on Trypanosoma cruzi Trypomastigote and Amastigote Forms from Infected Vero Cells Detected by Chagasic Antibodies

Reactivity of different Trypanosoma cruzi developmental forms with purified Chagasic anti-α-galactosyl antibodies (anti-Gal) was studied using epimastigotes from axenic cultures, trypomastigotes and amastigotes from infected Vero cell cultures, and an immunogold labeling method as observed by electron microscopy. Epimastigotes were poorly labeled, whereas extracellular trypomastigotes and amastigotes bound heterogeneously to the antibody with many cells being intensely labeled at the cell surface, including the membrane lining the cell body, the flagellum and the flagellar pocket. Parasites with poor labeling at the cell surface generally had several gold particles within the cell, mostly in cytoplasmic vacuoles. The Golgi complex of trypomastigotes was strongly labeled. Intracellular parasites were labeled at the parasite cell surface or within vacuolar structures. The expression in T. cruzi -infected Vero cells of α-galactosyl antigenic structures acquired from the parasite was shown by moderate labeling with Chagasic anti-Gal of the membrane lining parasite-free outward cell projections. The reactivity with purified anti-Gal from healthy individuals at the same concentrations of Chagasic anti-Gal was poor, with gold particles appearing in the nucleus and cytoplasm but not at the cell surface. It paralleled the labeling with Bandeireae simplicifolia IB-4 lectin. The results provide a basis for autoimmune reactions involving anti-Gal from chronic Chagasic patients.