Cloning, sequencing and functional expression of dihydrolipoamide dehydrogenase from the human pathogen Trypanosoma cruzi.

This work presents the complete sequences of a cDNA and the two allelic genes of dihydrolipoamide dehydrogenase (LipDH) from Trypanosoma cruzi, the causative agent of Chagas' disease (American trypanosomiasis). The full-length cDNA has an ORF of 1431 bp and encodes a protein of 477 amino acid residues. LipDH is a homodimeric protein with FAD as prosthetic group. The calculated molecular mass of the subunit of the mature protein with bound FAD is 50,066. Comparison of the deduced amino acid sequence of LipDH from T. cruzi with that of Trypanosoma brucei and man shows identities of 81% and 50%, respectively. An N-terminal nonapeptide, not present in the mature enzyme, represents a mitochondrial targeting sequence so far found only in trypanosomatids. The gene lpd1 of T. cruzi LipDH was expressed without the targeting sequence in Escherichia coli JRG1342 cells which are deficient for LipDH. For this purpose an ATG codon was introduced directly upstream the codon for Asn10 which represents the N-terminus of the mature protein. This system allowed the synthesis of 1000 U T. cruzi LipDH/1 bacterial cell culture. The recombinant protein was purified to homogeneity by (NH4)2SO4-precipitation and affinity chromatography on 5' AMP-Sepharose. The K(m) values for NAD+, NADH, lipoamide and dihydrolipoamide are identical with those of the enzyme isolated from the parasite. LipDH is present in all major developmental stages of T. cruzi as shown by northern and western blot analyses. This finding is in agreement with the citric acid cycle being active throughout the whole life cycle of the parasite. In vitro studies on a mammalian LipDH revealed the ability of the flavoenzyme to catalyze the redoxcycling and superoxide anion production of nitrofuran derivatives including the antitrypanosomal drug Nifurtimox. For that reason T. cruzi LipDH is regarded as a promising target for the structure-based development of new antiparasitic drugs. The bacterial expression system for the parasite enzyme will now allow the study of the role of T. cruzi LipDH in drug activation and the crystallization of the protein.

Trypanosoma cruzi: a 52-kDa protein sharing sequence homology with glutathione S-transferase is localized in parasite organelles morphologically resembling reservosomes.

We have previously isolated and characterized a Trypanosoma cruzi cDNA encoding a polypeptide with a molecular mass of 52 kDa (Tc52) sharing significant homology to glutathione S-transferase. In the present study, by molecular and immunological approaches, we showed that Tc52 is preferentially expressed by dividing forms of the parasite: (e.g., epimatigotes and amastigotes). Moreover, we could identify the reactive antigen in different T. cruzi strains. A different pattern of reactivity on immunoblots was observed in the case of Trypanosoma rangeli. Furthermore, immunofluorescence assays using T. cruzi epimastigote culture forms revealed that the reactive antigen is localized within cytoplasmic organelles morphologically ressembling the structures previously designated as the reservosome found mostly at the posterior end of the parasite. Furthermore, the antibodies did not react against trypomastigotes which emerged from infected fibroblasts, whereas amastigotes showed polar fluorescence. Immunogold labeling and electron micrographs further revealed that the Tc52 protein is mainly associated with organelles composed of a large network of multivesicular structures, the latter being more abundant in epimastigotes. Taken together, these results demonstrated that Tc52 is associated with organelles composed of a multivesicular network and appears to be developmentally regulated, being fully expressed by parasite dividing forms.

Growth inhibition of Trypanosoma cruzi and Leishmania donovani infantum by different snake venoms: preliminary identification of proteins from Cerastes cerastes venom which interact with the parasites.

Venom from three different snake species was tested in vitro against the protozoan parasites Trypanosoma cruzi and Leishmania donovani infantum. Two of them, Cerastes cerastes and Naja haje, exerted a significant growth inhibition of T. cruzi and L. d. infantum parasites. Heating of the venoms abolished their activity, suggesting that the active factors are thermolabile. Incubation of parasites with 125I-labelled C. cerastes venom proteins allowed preliminary identification of components which interact preferentially with the pathogens.

Characterization of a Leishmania antigen associated with cytoplasmic vesicles resembling endosomal-like structure.

In the present study we have used antibodies to Leishmania major promastigote antigens which were eluted from a glutathione-agarose column (LmGbp) and could identify several parasite components among different Leishmania species by using immunoprecipitation and Western blot techniques. The results also showed that some of LmGbp are present among the molecules released into the culture medium. Moreover, immunofluorescence assays clearly demonstrated that LmGbp are expressed by intracellular amastigotes. The electron micrographs of thawed cryosections of L. major-infected cells revealed that the antigens were associated with the membrane of the phagocytic vacuole. Moreover, the Western blot technique allowed us to identify, using other Leishmania species extracts and anti-LmGbp antibodies, a major polypeptide of an apparent molecular mass of 66 kDa. Immunofluorescence studies suggested that the 66 kDa polypeptide is associated with intracytoplasmic vesicles. Cryosections of Leishmania promastigotes improved the fine structure preservation of the organelles and enabled a number of features to be seen, particularly the structures considered as vesicles, which appeared as a complex tubulo-vesicular structure resembling mammalian cell endosomes and Leishmania organelles previously named 'megasomes'. Further studies using antibodies against the native 66 kDa protein will be needed to investigate the localization of the protein at the ultrastructural level and to follow its intracellular vesicular traffic.

Trypanosoma cruzi glutathione-binding proteins: immunogenicity during human and experimental Chagas' disease.

Following purification by affinity chromatography, three glutathione-binding proteins (TcGBP) of 45, 30, and 25 kDa were co-purified from Trypanosoma cruzi epimastigotes. Using 1-chloro-2,4 dinitrobenzene as substrate, a glutathione S-transferase activity of 70 nmol/min/mg of proteins was detected in the GSH binding fraction. An increased expression of TcGBP and total GST activity was observed upon incubation of parasites with phenobarbital, which is an inducer of GST synthesis. Immunofluorescence and electron microscopic experiments demonstrated that TcGBP were expressed by all developmental stages of the parasite, including infective forms. The expression of these proteins by intracellular dividing amastigotes could be in favour of a potential defensive role of these molecules against host attack. Results obtained by immunoprecipitation of in vitro translation products using anti-TcGBP antisera suggested that these three polypeptides are not glycosylated. In addition, antibodies directed against the TcGBP were found in a high proportion of T. cruzi-infected chronic chagasic patients' sera and in sera of chronically infected BALB/c mice. In contrast, acute chagasic patients' sera and acute-phase mouse sera were found to be poorly reactive with these proteins. Our results identify a new class of potential target antigens, which may be essential for the development of T. cruzi in its host. Their protective role in experimental models deserves to be investigated.

Trypanosoma cruzi: a carbohydrate epitope defined by a monoclonal antibody as a possible marker of the acute phase of human Chagas' disease.

An IgM monoclonal antibody (MAb) against a carbohydrate epitope present in Trypanosoma cruzi trypomastigote excretory-secretory antigens and expressed by different developmental stages of the parasite (epimastigote, trypomastigote and intracellular amastigote) was linked to a solid phase matrix and used as an antigen-capture antibody. Human serum complexes containing the epitope were then detected by using specific secondary antibodies against human immunoglobulin isotypes. Results of detection of IgM, IgG, and IgA serum complexes (SC) containing a T. cruzi polypeptide epitope showed that SC could be detected in 69% of the 13 Chagasic acute phase sera studied with IgG, in 84% with IgM, and in 75% with IgA. Only 16% (IgG-SC), 8% (IgM-SC), and 10% (IgA-SC) of chronic sera from 50 patients were positive. No patients with toxoplasmosis or rheumatoid factor were positive. Of the 11 leishmaniasis sera studied, four had IgG-SC, two had IgA-SC, and five had IgM-SC. Of the eight Yanomamo Indians infected by Onchocerca volvulus, three were found to have IgG-SC, two had IgM-SC, and two had IgA-SC. Thirteen sera from healthy individuals living in an endemic area were also studied. One subject had IgG IgM and IgA-SC. The results presented in this study show for the first time, the specific detection of IgM, IgG, and IgA immune complexes using a MAb against T. cruzi. The presence of the epitope in association with IgM antibodies in sera from patients with the acute phase of the disease suggests that this antigen(s) carrying the epitope that reacts with the MAb could be a marker(s) of active infection. In addition, the specificity of the serum complex capture assay allowed the detection of Chagas' disease in two different endemic areas (Argentina and Venezuela).

Trypanosoma cruzi: differential expression and distribution of an 85-kDa polypeptide epitope by in vitro developmental stages.

The expression by Trypanosoma cruzi developmental stages of an 85-kDa polypeptide epitope defined by the 155D3 monoclonal antibody (mAb) has been investigated. Immunoprecipitation revealed the presence of an 85-kDa antigen in the NP-40 soluble extract of parasites freshly released from infected fibroblasts; this antigen was not found in epimastigote and Leishmania infantum promastigote. Indirect immunofluorescence revealed that the mAb 155D3 failed to react with trypomastigotes, whereas extracellular amastigotes were heavily stained. Positive organisms displayed either surface or polar fluorescence. Since the same mAb immunoprecipitated the 85-kDa antigen in both radioactive iodine- and methionine-labeled trypomastigote detergent soluble extracts, the reactive epitope is likely to be hidden in a cryptic site in trypomastigotes. An alternative explanation for the negative immunofluorescence on trypomastigotes and the positive immunoprecipitation is the presence, in the extracts, of a small population of parasites already expressing the 155D3 epitope. Immunoelectron microscopy revealed that the target epitope is heterogenously distributed among the populations of differentiating parasites. Two types of immunogold labeling were observed: (a) mAb revealed a high amount of reactive material associated with the periphery of the parasites and (b) a label was observed on the inner surface of peripheral vacuoles that might correspond to cross sections of inflated flagellar pockets and in association with vesicles which were released by the parasites. The surface expression of the epitope recognized by the 155D3 mAb was followed by fluorescence-activated cell-sorting analysis. The results showed that the epitope is increasingly accessible during trypomastigote differentiation in vitro. Taken together, these results suggest that the epitope reacting with the 155D3 mAb is heavily expressed on extracellular amastigotes after the transformation process and, thus, appears to be developmentally regulated.

Characterization of major surface and excretory-secretory immunogens of Trypanosoma cruzi trypomastigotes and identification of potential protective antigen.

The surface antigens of Trypanosoma cruzi trypomastigotes were identified by immunoprecipitation and were compared with metabolically labelled excretory-secretory products (ES) released by the parasites in vitro. A series of major immunogenic components in the ES antigens were revealed (160 kDa, 130 kDa and 80-110 kDa). The trypomastigote surface also bears the 130 kDa band and the 80-110 kDa complex. Competition experiments demonstrated the common antigenic structure of the ES and the surface antigens. Two-dimensional analysis of ES antigens immunoprecipitated by human Chagasic serum revealed several spots in the 80-110 kDa region with a wide range of isoelectric points (PI between 5.4 and 6.7). This reflects a charge heterogeneity of these polypeptides. The trypomastigote 85 kDa polypeptide was also identified in the ES antigens by using a monoclonal antibody against this antigen. Two-dimensional analysis of the 85 kDa proteins shed from the surface of trypomastigotes and immunoprecipitated by the monoclonal antibody 155D3 showed 2 major spots: a major part of the 85 kDa polypeptide was found at pH 6.5-6.6, whereas a substantial amount of the antigen was found at pH 5.7. An additional component with molecular weight of approximately 58 kDa and isoelectric points of 6.5 and 6.6, was also visualized. Detection of the 85 kDa polypeptide circulating in serum from patients with acute and chronic Chagas' disease was achieved using an enzyme-linked immunosorbent assay. In addition, the data obtained showed that a polyclonal antibody to the 85 kDa polypeptide could be used to passively induce a partial protection of Fischer rats against acute lethal infection. Thus, the antigens recognized by polyclonal antibody appear to play a role in the development of protective immunity against T. cruzi.

IgE receptor on human eosinophils (FcERII). Comparison with B cell CD23 and association with an adhesion molecule.

IgE FcR (FcERII) on human eosinophils was characterized and compared with FcERII present on B cells (CD23). Two mAb, BB10 (anti-eosinophil FcERII) and 135 (anti-CD23), bound to the major component of FcERII at 45,000 to 50,000 Mr, both on purified hypodense eosinophils and on a B cell line (WIL-2WT). The specific ligand, human myeloma IgE, was able to bind to the molecules immunoprecipitated by BB10. A cross-reactivity between BB10 and a mAb anti-Leishmania gp63, which is a "fibronectin (Fn)-like" molecule, containing the L-arginine-L-glycyl-L-aspartyl (RGD) cell attachment domain indicated the presence of such a sequence in the common structure present on eosinophil and B cell FcERII. The synthetic tetrapeptide RGDS as well as its inverted sequence (SDGR) reduced the binding of BB10 and anti-Fn mAb to eosinophils and B cells. Flow microfluorometry analysis revealed a variable binding of BB10 and anti-Fn mAb to eosinophils purified from different patients, results compatible with recent findings on the inducibility of FcERIIb. The significant inhibition of IgE-dependent cytotoxicity against parasite targets by preincubation of eosinophils with BB10, anti-Fn and anti-CD23 mAb, with anti-RGDS polyclonal antibodies or with the SDGR peptide suggested the requirement of this cell adhesion sequence for the function of low affinity FcERII. The presence of such a sequence in the C-terminal domain of B cell FcERII raised the possibility of its role in B cell adhesion or B cell growth.

gp 58/68, a parasite component that contributes to the escape of the trypomastigote form of T. cruzi from damage by the human alternative complement pathway.

A glycoprotein of apparent molecular weight 58,000 (unreduced)/68,000 (in its reduced form) (gp 58/68), which is one of the fibronectin/collagen receptors of Trypanosoma cruzi, was purified to homogeneity from the trypomastigote forms of the Tehuantepec and Y strains of the parasite. Purified gp 58/68 inhibited formation of cell-bound and fluid-phase alternative pathway C3 convertase in a dose-dependent fashion, as assessed using purified human complement components. Gp 58/68 differed from the human regulatory proteins H, DAF, MCP and CR1 and from previously reported regulatory proteins on the parasite membrane in that it was unable to enhance decay-dissociation of preformed alternative pathway C3 convertase sites, did not serve as a co-factor for I-mediated cleavage of C3b and had no inhibitory activity on the classical pathway convertases. The inhibitory effect of gp 58/68 was most likely dependent on an interaction of the protein with factor B rather than with C3b. Gp 58/68 provides trypomastigotes with an additional potential mechanism for escaping complement lysis by the human alternative pathway.

Identification and isolation of Trypanosoma cruzi trypomastigote collagen-binding proteins: possible role in cell-parasite interaction.

We have shown here that collagen type I bound efficiently to the trypomastigote surface. In addition, monoclonal and polyclonal antibodies against collagen types I and III inhibited the infection of fibroblasts by the parasite. These results suggested the presence of collagen-binding protein(s) on the parasite surface. This protein was identified from trypomastigote surface antigens using affinity chromatography on a Gelatin Ultrogel column (denatured form of collagen). These collagen-binding proteins were revealed as a low-affinity gelatin binding protein (LAG Bp) of 98 kDa, and a high-affinity binding protein (HAG Bp) of 58 and 68 kDa under non-reducing and reducing conditions respectively. In addition, HAG Bp and LAG Bp bound to collagen type I. The 58/68 kDa protein was purified to homogeneity on a wheat germ agglutinin Sepharose column. A polyclonal antibody to this glycoprotein, as well as a monoclonal antibody (McAb) 155D3 produced against the HAG Bp, immunoprecipitated two parasite surface antigens of 160 and 58 kDa under non-reducing conditions which migrated at a position of 80-85 and 68 kDa when reduced. However, only the 80-85 kDa component could be precipitated from [35S] methionine-labelled trypomastigote antigens under reducing conditions. The antibodies to the 58/68 kDa glycoprotein as well as McAb 155D3 diminished the invasion of fibroblasts by parasites. Taken together these results suggest that the same receptor binds fibronectin and/or collagen and that both the 80-85 and 58/68 kDa glycoproteins form part of the same receptor. These trypomastigote surface molecules may interact with the host cell fibronectin and/or collagen during the initial phase of parasite-cell recognition.

Role of the RGD sequence in parasite adhesion to host cells.

For many protozoan parasites, one of the first events in the process of infection is attachment to the surface of host cells. This adhesion phase usually involves ligand-receptor interactions, and has stimulated interest in the biochemical characterization of those host cell and parasite surface components involved. In this article, Ali Ouaissi discusses the strategy employed by pathogens such as Trypanosoma cruzi, Trichomonas, Leishmania and Treponema pallidum, in binding to their host cells' fibronectin receptors. Two systems appear available - to bind to the dimeric cell surface fibronectin through the Arginine-Glycine-Aspartic acid (RGD) sequence that is not occupied by the host cell surface receptors, or to present a surface antigen representing a 'fibronectin-like' molecule containing the RGD sequence directly to the host cell fibronectin receptors.

The major surface protein of Leishmania promastigotes is a fibronectin-like molecule.

The major surface glycoprotein of Leishmania chagasi promastigotes showed crossreactivity with fibronectin (Fn), a large glycoprotein that is a major constituent of the extracellular matrix of most mononuclear cells. Polyclonal and monoclonal antibodies against Fn precipitated two molecules of 63-58 kDa from the lysates of both 125I and [35S]methionine-labeled promastigotes. In addition, a monoclonal antibody against a 15-kDa fragment of Fn containing the Arg-Gly-Asp-Ser (RGDS) sequence and several polyclonal monospecific mouse antibodies against a synthetic RGDS peptide also recognized the above two molecules. The attachment of Leishmania promastigotes to mouse peritoneal macrophages in vitro was partially inhibited when promastigotes were treated with F(ab')2 fragment of an anti-Fn IgG. Identical results were obtained by saturating the Fn receptors on macrophages using different peptides containing the RGDS sequence. Moreover, antigen preparations rich in glycoprotein 63 could efficiently promote the attachment and spreading of 3T3 mouse fibroblasts to surfaces coated with the antigen. These results clearly suggest that the gp63 of L. chagasi promastigotes is an Fn-like molecule that shares certain biological and molecular characteristics with Fn.

Trypanosoma cruzi: fibronectin promotes uptake of epimastigote culture forms by human neutrophils and monocytes.

Treatment of human neutrophils and monocytes with human plasma fibronectin (Fn) enhanced their association with Trypanosoma cruzi epimastigote culture forms, a stage of parasite which activates the alternative complement pathway, and this related to the concentration of Fn in the culture medium. By increasing the incubation time, the parasite interiorization by phagocytic cells was observed. An enhancing effect of this latter phenomenon was obtained in the presence of Fn, while the addition of anti-Fn antibodies exerted an inhibitory effect. Moreover, the velocity of phagocytosis of complement-coated epimastigotes in the presence of Fn appeared greater than that observed using non-coated epimastigotes or parasites preincubated in the presence of heat-inactivated C6-deficient rabbit serum. In addition, as a consequence of cell-Fn parasite interaction, cell activation was also seen. This has been demonstrated by a chemiluminescence assay. Using radiolabeled epimastigotes (3H-uridine), we demonstrated that a proportion of ingested parasites in the presence of Fn were killed. When neutrophils were used as effector cells, the cytotoxicity was greater than that observed with monocytes. This finding of increased trypanosome uptake by phagocytic cells in the presence of fibronectin suggests that this glycoprotein could act as a ligand or cofactor to mediate parasite-cell interaction.

[Identification in Plasmodium falciparum of a protein specifically binding human fibronectins]. / Identification chez Plasmodium falciparum d'une protéine se liant spécifiquement à la fibronectine humaine.

A fibronectin binding protein (FnBp) was identified in 3H isoleucine labeled P. falciparum schizonts using affinity chromatography on human fibronectin (Fn) coupled to Sepharose 4B. After incubation of Nonidet-P 40 parasite lysate with Fn-Sepharose, elution was performed with SDS-PAGE buffer. Analysis of FnBp by SDS-PAGE demonstrated a major band which migrated with an apparent Mr of 70,000 under reducing conditions. This band was not found when human or rabbit IgG coupled Sepharose 4B were used instead of Fn as control.

Trypanosoma cruzi infection inhibited by peptides modeled from a fibronectin cell attachment domain.

The mechanism by which Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, becomes attached to mammalian cells is not well understood. Fibronectin is thought to participate in the attachment, and in this study the region of fibronectin that interacts with the surface receptors of T. cruzi trypomastigotes was investigated by testing the binding of the amino acid sequence Arg-Gly-Asp-Ser, corresponding to the cell attachment site of fibronectin to T. cruzi trypomastigotes. Peptides with the sequence Arg-Gly-Asp-Ser, but not Arg-Phe-Asp-Ser, Arg-Phe-Asp-Ser-Ala-Ala-Arg-Phe-Asp, Ser-Lys-Pro, Glu-Ser-Gly, or Ala-Lys-Thr-Lys-Pro, bound to the parasite surface and inhibited cell invasion by the pathogen. Monoclonal antibodies to the cell attachment domain of fibronectin also inhibited cell infection by the parasite. The immunization of BALB/c mice with tetanus toxoid-conjugated peptide induced a significant protection against T. cruzi. The data support the notion that the sequence Arg-Gly-Asp-Ser of cell surface fibronectin acts as a recognition site for attachment of the parasites.