Parasite Polypeptides Research Articles

Genomic organization and expression profile of the mucin-associated surface protein (masp) family of the human pathogen Trypanosoma cruzi

A novel large multigene family was recently identified in the human pathogen Trypanosoma cruzi, causative agent of Chagas disease, and corresponds to ∼6% of the parasite diploid genome. The predicted gene products, mucin-associated surface proteins (MASPs), are characterized by highly conserved N- and C-terminal domains and a strikingly variable and repetitive central region. We report here an analysis of the genomic organization and expression profile of masp genes. Masps are not randomly distributed throughout the genome but instead are clustered with genes encoding mucin and other surface protein families. Masp transcripts vary in size, are preferentially expressed during the trypomastigote stage and contain highly conserved 5′ and 3′ untranslated regions. A sequence analysis of a trypomastigote cDNA library reveals the expression of multiple masp variants with a bias towards a particular masp subgroup. Immunofluorescence assays using antibodies generated against a MASP peptide reveals that the expression of particular MASPs at the cell membrane is limited to subsets of the parasite population. Western blots of phosphatidylinositol-specific phospholipase C (PI-PLC)-treated parasites suggest that MASP may be GPI-anchored and shed into the medium culture, thus contributing to the large repertoire of parasite polypeptides that are exposed to the host immune system.

Infection of bovine cells by the protozoan parasite Theileria annulata modulates expression of the ISGylation system

The apicomplexan parasite, Theileria annulata, dedifferentiates and induces continuous division of infected bovine myeloid cells. Re-expression of differentiation markers and a loss of proliferation occur upon treatment with buparvaquone, implying that parasite factors actively maintain the altered status of the infected cell. The factors that induce this unique transformation event have not been identified. However, parasite polypeptides (TashAT family) that are located in the infected leucocyte nucleus have been postulated to function as modulators of host cell phenotype. In this study differential RNA display and proteomic analysis were used to identify altered mRNA and polypeptide expression profiles in a bovine macrophage cell line (BoMac) transfected with TashAT2. One of the genes identified by differential display was found to encode an ubiquitin-like protease (bUBP43) belonging to the UBP43 family. The bUBP43 gene and the gene encoding its ubiquitin-like substrate, bISG15, were expressed at a low level in T. annulata-infected cells. However, infected cells were refractory to induction of elevated bISG15 expression by lipopolysaccharide or type 1 interferons while TashAT2-transfected cells showed no induction when treated with camptothecin. Modulation of the ISGylation system may be of relevance to the establishment of the transformed infected host cell, as ISGylation is associated with resistance to intracellular infection by pathogens, stimulation of the immune response and terminal differentiation of leukaemic cells.

Identification of Leishmania major cysteine proteinases as targets of the immune response in humans

In this study, we report the identification of two parasite polypeptides recognized by human sera of patients infected with Leishmania major. Isolation and sequencing of the two genes encoding these polypeptides revealed that one of the genes is similar to the L. major cathepsin L-like gene family CPB, whereas the other gene codes for the L. major homologue of the cysteine proteinase a (CPA) of L. mexicana. By restriction enzyme digestion of genomic DNA, we show that the CPB gene is present in multiple copies in contrast to the cysteine proteinase CPA gene which could be unique. Specific antibodies directed against the mature regions of both types expressed in Escherichia coli were used to analyze the expression of these polypeptides in different stages of the parasite's life cycle. Polypeptides of 27 and 40 kDa in size, corresponding to CPA and CPB respectively, were detected at higher level in amastigotes than in stationary phase promastigotes. Purified recombinant CPs were also used to examine the presence of specific antibodies in sera from either recovered or active cases of cutaneous leishmaniasis patients. Unlike sera from healthy uninfected controls, all the sera reacted with recombinant CPA and CPB. This finding indicates that individuals having recovered from cutaneous leishmaniasis or with clinically apparent disease have humoral responses to cysteine proteinases demonstrating the importance of these proteinases as targets of the immune response and also their potential use for serodiagnosis.

Tc45, a dimorphic Trypanosoma cruzi immunogen with variable chromosomal localization, is calreticulin.

We demonstrate that Tc45, a polypeptide described as an immunogenetically restricted Trypanosoma cruzi antigen in mice, is calreticulin, a dimorphic molecule encoded by genes with variable chromosomal distribution. Previously we showed that IgG from A.SW (H2s) mice immunized with T. cruzi trypomastigotes or epimastigotes and sera from infected humans recognize Tc45, a 45 kD parasite polypeptide. Herein we describe the cloning, sequencing, and expression of the Tc45 gene. A 98% homology in the deduced amino acid sequence was found with a T. cruzi calreticulin-like molecule and 41% with Leishmania donovani and human calreticulin. In the T. cruzi CL Brener clone and in the Tulahuén strain, the gene is located in two and four chromosomes, respectively. Calreticulin was detected in several T. cruzi clones, in the Tulahuén strain, and in T. rangeli, displaying alternative 43 and 46 kD forms.

Suppressed expression of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) in an irradiation-attenuated Plasmodium berghei XAT strain

Plasmodium berghei XAT (XAT) is a non-reversible, non-lethal type malaria parasite strain derived from the highly virulent lethal P. berghei NK65 (NK65) by X-irradiation. The difference in polypeptide expression between NK65 and XAT was examined in this study. Western blot patterns of the parasite polypeptides showed that a 30-kDa polypeptide was not detected in XAT. In the present paper, we focused the study on the difference in the expression of the 30-kDa polypeptide between XAT and NK65. Although several other significant differences were noted in the spots shown by two-dimensional gel electrophoresis, the 30-kDa polypeptide was isolated by means of preparative 2D-gel electrophoresis followed by HPLC, and N-terminal amino acid sequence of the polypeptide was eventually determined. Complementary DNA clones encoding the 30-kDa polypeptide were isolated and characterized. Full-length cDNA clones from XAT encoded a protein of 231 amino acid residues with a 693-bp open reading frame. The deduced amino acid sequence exhibited 67% identity with that for P. falciparum hypoxanthine-guanine phosphoribosyltransferase (HGPRT; EC 2.4.2.8), suggesting that this protein is P. berghei HGPRT. Northern blot analysis revealed that expression of HGPRT in XAT was only one-eighth of that in NK65. This finding indicates that HGPRT gene expression is markedly suppressed in XAT. The amino acid sequence of HGPRT from NK65 was identical to that from XAT. This finding showed that the amino acid sequence of XAT-HGPRT was not mutated and had not undergone deletion.

Characterisation of PfSec61, a Plasmodium falciparum homologue of a component of the translocation machinery at the endoplasmic reticulum membrane of eukaryotic cells

Plasmodium falciparum secretes several proteins that cause changes in the erythrocyte membrane enabling it to survive within red blood cells. Little is known of the mechanisms involved in the secretion and targeting of parasite polypeptides to the various cell compartments. The P. falciparum gene homologous to the mammalian Sec61α gene, which encodes a component of the translocation pore in the endoplasmic reticulum of eukaryotic cells, was characterised to investigate the translocation process in the parasite. PfSec61 is present as a unique copy in the parasite genome and was mapped to chromosome 13. It encodes a 40 kDa polypeptide, as shown by immunoblotting and immunoprecipitation of [ 35S]methionine metabolically-labelled parasite extracts. The deduced amino acid sequence of PfSec61 is 87% similar to the mammalian polypeptide, and the two proteins give similar hydropathy plots. These results strongly suggest that PfSec61 has the same topological orientation and functional role as Sec61 α. Anti-PfSec61 antibodies were used to investigate the cellular location and kinetics of expression of the polypeptide in the parasite. Immunofluorescence confocal microscopy showed that PfSec61 was located in the parasite cytoplasm, close to the nucleus, in a position consistent with its being in the endoplasmic reticulum.

Differential sensitivity of New World Leishmania spp. promastigotes to complement-mediated lysis: Correlation with the expression of three parasite polypeptides

American tegumentary leishmaniasis is caused by Leishmania of the subgenera Leishmania and Viannia. In this paper, we demonstrate that promastigotes of these two subgenera display distinct characteristic patterns of complement sensitivity during growth in vitro. Using fresh normal human serum in lytic assays, we show that while promastigotes of two species of the subgenus Leishmania differentiate into forms that are more resistant to the lytic action of complement, promastigotes of three species of the subgenus Viannia remain sensitive to complement mediated lysis during all stages of their growth in vitro. Complement resistance of the subgenus Leishmania is temporary, reaching its peak at the beginning of the stationary phase of growth, and decreasing thereafter. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) we detected in L. amazonensis (subgenus Leishmania), but not in L. guyanensis (subgenus Viannia), three polypeptides whose expression parallels the resistance of promastigotes to complement-mediated lysis.

Purification and Preliminary Sequencing of Tc45, an Immunodominant Trypanosoma cruzi Antigen: Absence of Homology with Cruzipain, Cruzain, and a 46-Kilodalton Protein

In spite of being separated by more than 20 million years of evolution, the murine and human immune systems share extensive similarities. Thus, experimental results obtained with the murine model may have predictive value for human Chagas' disease. Challenge of the H-2 congenic mouse stains A.SW (H-2s) and A.CA (H-2f) with Trypanosoma cruzi yields different results. The A.CA animals die approximately 12 days postinfection, while A.SW mice survive indefinitely. A 45-kD protein (Tc45), an antigen differentially recognized by the A.SW strain, is present in cultured epimastigotes and blood trypomastigotes. We describe here its purification from epimastigotes. The presence of Tc45 was monitored and a single band was detected. Since the molecular weights of Tc45, cruzipain, cruzain, and a 46-kD parasite polypeptide are similar, it was important to determine if these molecules are related. A complete lack of homology was observed when the sequence of cruzain, cruzipain, and the 46-kD polypeptide were compared with the preliminary sequence of Tc45.

Rosetting Plasmodium falciparum-infected erythrocytes express unique strain-specific antigens on their surface.

Spontaneous binding of uninfected erythrocytes to Plasmodium falciparum-infected erythrocytes (rosetting) has been suggested to have a critical role in the induction of cerebral malaria. We report here that rosetting can be mediated by several molecular mechanisms involving parasite polypeptides with M(r)s of 22,000 or 28,000, termed rosettins. Antibodies to either polypeptide disrupt rosettes in a strain-specific fashion. Rosettes of five of the seven isolates examined thus far are more easily disrpted by anti-22,000-M(r) rosettin antibodies than by anti-28,000-M(r) rosettin antibodies. Polyclonal anti-22,000-M(r) rosettin antibodies raised in mice or rabbits strongly and strain specifically stain the surface of nonfixed erythrocytes infected with late asexual stages of rosetting P. falciparum. Simultaneous antibody staining and rosetting are seen when the anti-22,000-M(r) rosettin antiserum is diluted so that only partial disruption of rosettes is obtained, confirming that the fluorescence-labelled infected erythrocytes are involved in rosetting. The 22,000-M(r) rosettin is accessible for surface iodination on erythrocytes infected with strains of rosetting parasites sensitive to anti-22,000-M(r) rosettin antibodies, whereas no labelling occurred on either normal erythrocytes or nonrosetting-P. falciparum-infected erythrocytes. Purified anti-22,000-M(r) rosettin serum immunoglobulin G immunoprecipitated three parasite-derived polypeptides with M(r)s of 22,000, 45,000 (doublet), and 50,000 from lysates of [35S]methionine-labelled, parasite-infected erythrocytes. Our results suggest that rosetting is mediated by strain-specific, antigenically distinct, P. falciparum-derived polypeptides.

Development of ELISAs based on recombinant antigens for the detection of Toxoplasma gondii-specific antibodies in sheep and cats

ELISAs using recombinant parasite polypeptides as antigens were developed to measure Toxoplasma gondii-specific antibodies in the sera of sheep and cats. Compared with an ELISA based on traditional parasite antigen, the ELISA for sheep sera had a sensitivity of 79% and a negative predictive value of 80%, and the ELISA for cat sera had a sensitivity of 100% and a negative predictive value of 100%. Both ELISAs had specificities of 100% and positive predictive values of 100%. These ELISAs appear to be a useful cost-effective alternative to ELISAs based on traditional parasite antigen for the measurement of T. gondii-specific antibodies in the sera of sheep and cats.

Polypeptides reactive with antibodies eluted from the surface of Babesia bovis-infected erythrocytes.

A technique was sought that would enable identification of surface-exposed parasite antigens on Babesia bovis-infected erythrocytes (BbIE) that are not detectable by surface-specific immunoprecipitations. Antibodies which bind to the surface of BbIE were recovered from intact cells using a low pH wash procedure. The eluted antibodies were then used in conventional immunoprecipitation assays to identify parasite-synthesized polypeptides carrying epitopes that are exposed on the surface or are cross-reactive with such epitopes. The results of these experiments support our previous data, obtained using a surface-specific immunoprecipitation technique, in the identification of a repertoire of parasite-derived antigens on the surface of infected erythrocytes (Allred et al., 1991). In addition, two polypeptides of M(r) 68,000 and 185,000 were identified which react strongly with the eluted antibodies but which are not detected by surface-immunoprecipitation. These data illustrate the potential of this approach for identification of parasite polypeptides which carry epitopes exposed on, or cross-reactive with exposed epitopes of the infected erythrocyte surface.

In vitro inhibition of multiplication of Babesia bigemina by using monoclonal antibodies

A panel of 21 monoclonal antibodies (MAb) against the erythrocytic stages of Babesia bigemina was produced. The 21 MAb could be divided into four distinct immunofluorescence groups on the basis of their patterns of binding to B. bigemina-infected erythrocytes. Nine MAb positive in the indirect immunofluorescent antibody test with acetone-fixed B. bigemina-infected erythrocytes were selected for purification and further characterization. The MAb were species specific, since they did not cross-react with B. bovis, but they were not stage specific. The purpose of this study was to evaluate the biological activity of B. bigemina-specific MAb in vitro. Three MAb inhibited the multiplication of B. bigemina in vitro as assessed by a lowered percentage of parasitized erythrocytes and by a decreased incorporation of [3H]hypoxanthine. These MAb are now being utilized to purify the correspondent parasite polypeptides, either native or recombinant, in order to test them as subunit immunogens.

Sera from Cameroon recognize proteins of Plasmodium falciparum isolates from geographically diverse areas of the world

Immunological reactions of clinically-defined sera collected from 176 children and adults (3 to 63 years of age) living in malaria endemic regions of Cameroon were evaluated by ELISA, growth inhibition studies and immunoprecipitation assays using different parasite isolates from geographically diverse areas. The proportions of sera positive by ELISA and with positive growth inhibitory activity tended to increase with increased age. SDS-PAGE analyses of immunoprecipitates using [ 35S]methionine labelled parasite polypeptides revealed that a wide range of proteins was recognized by the sera. There were many similarities in the patterns of antigens immunoprecipitated in the different isolates, particularly when immune sera were used. Variability in response was more evident in sera collected from children. These findings suggest that strains may share components which generate protective immunity.

The interaction of a Trypanosoma cruzi surface protein with Vero cells and its relationship with parasite adhesion

Previous studies have shown that adhesion to fibroblastic cells of cell culture-derived trypomastigotes of Trypanosoma cruzi probably occurs through a ligand-receptor interaction. The results now obtained indicate that solubilization with a mild detergent (‘Chaps’, 0.8%) of 125I-surface proteins of trypomastigotes, followed by detergent removal and interaction of the solubilized proteins with a monolayer of intact Vero cells, brings about binding to the cells of a parasite surface protein, which exhibits a molecular weight of 83 000 and isoelectric point of 8.1–8.6 upon two-dimensional polyacrylamide gel electrophoresis. This polypeptide was detected in extracts of highly adherent, extracellularly incubated parasites, but not in extracts of poorly adhesive, recently released trypomastigotes. The detergent-free extracts of incubated trypomastigotes inhibit attachment of live parasites to Vero cells, while extracts of fresh trypomastigotes are nearly ineffective. Binding of the parasite polypeptide to the cells is stimulated by parasite trypsinization or activation in the presence of tunicamycin, and it is inhibited by the presence of mannan or by Vero cell trypsinization, thus showing a similar behaviour to that observed for parasite attachment to Vero cells under these conditions. These results suggest that the surface membranes of activated, highly adherent T. cruzi trypomastigotes contain an 83 kDa polypeptide which acts as a lectin-like protein that can interact with the surface of Vero fibroblasts, probably through mannose residues of a glycoprotein receptor of the host cell.

Clinical Trypanosoma rangeli infection as a complication of Chagas‘ disease

Laboratory studies on a group of 20 patients from the Rio Negro Valley, Colombia selected for detailed study showed that 14 gave antibody reactions on immunoassay consistent with Trypanosoma cruzi or T. rangeli infections. Four were diagnosed as having T. rangeli infection, 4 had mixed infections and 6 were infected with T. cruzi alone. Immunoprecipitation analysis showed that sera from T. cruzi-infected patients recognized a similar range of trypomastigote-derived polypeptides as sera from patients in Brazil, and all of the Colombian sera reacted with the 160 kiloDalton (kDa) polypeptide associated with active infection. Although sera from patients with T. rangeli infection alone gave a positive immunofluorescence or ELISA reaction with T. rangeli, they failed to bind to parasite polypeptides by either immunoprecipitation or Western blotting. Intriguingly, sera from patients with mixed infections consistently gave a stronger, but qualitatively similar, binding reaction in immunoprecipitation and Western blotting compared to sera from patients infected with T. cruzi alone.

Cloning of Leishmania donovani genes encoding antigens recognized during human visceral leishmaniasis

In this report we describe the construction and analysis of a genomic library of Leishmania donovani gene segments in the bacteriophage vector λgt11. This cloning vector permits the expression of parasite polypeptides as fusion products with Escherichia coli β-galactosidase. A group of 90 clones which express L. donovani antigens have been isolated from this library using various polyvalent antisera. Many of these clones appear to encode parasite membrane antigens some of which are recognized by sera from patients with visceral leishmaniasis (kala azar). Some clones reacted with specific subsets of kala azar sera while others reacted with all kala azar sera tested. In addition, some of the clones appear to encode conserved antigens that are recognized by sera from mice immunized with L. major. Preliminary characterization of five clones indicated that each one contains a distinct genetic element and that at least four encode different fusion peptides.

Anti-Plasmodium falciparum antibodies acquired by residents in a holoendemic area of Liberia during development of clinical immunity.

Sera from 48 children and adolescents (2-15 years of age), residing in a malaria holoendemic area of Liberia were investigated for specificities and isotypes of anti-P. falciparum antibodies. No clear-cut relationship to the development of clinical immunity was found when the overall antibody activities to total parasite antigens were determined by enzyme-linked immunosorbent assay (ELISA). Although there was a certain rise of IgM, total IgG- and IgG2 antibody activities, this was most pronounced at ages when a clinical but nonsterile immunity is already present. When the sera were investigated by immunoprecipitation of 35S-methionine labeled parasite polypeptides, the total number of parasite antigens precipitated was similar at all ages. Analysis by indirect immunofluorescence (IFA), registering antibodies to intracellular parasite antigens, revealed no age-dependent changes in antibody titers. In contrast, when the sera were assayed by a novel IFA, specific for a restricted number of parasite antigens in the membrane of infected erythrocytes, the frequency of positive sera as well as the anti-P. falciparum titers rose in parallel with the development of clinical immunity. Thus, these antigens appeared to be important inducers of protective immune responses and may be suitable candidates for a vaccine against the asexual blood stages of P. falciparum.

Immunogenic compositions containing malarial parasite polypeptide extracts for preparation of antimalarial vaccines and in vitro diagnosis of malaria antibodies in serum

Immunogenic compositions containing malarial parasite polypeptide extracts for preparation of antimalarial vaccines and in vitro diagnosis of malaria antibodies in serum

Parasite polypeptides lost during schizogony and erythrocyte invasion by the malaria parasites, Plasmodium chabaudi and Plasmodium knowlesi

By biosynthetically labelling Plasmodium chabaudi and P. knowlesi stage-specific polypeptides and allowing continued development, schizogony and reinvasion in vivo or in vitro, we have identified parasite polypeptides not taken into the erythrocyte by the invading mezozoite. Three major and two minor parasite polypeptides synthesized by rings or mid-stage trophozoites of P. chabaudi were either degraded preferentially during further development, or lost during schizogony and reinvasion. For both P. chabaudi and P. knowlesi, a 250 000 mol. wt. polypeptide synthesized during maturation of trophozoites to schizonts and merozoites was not taken into the erythrocyte by the invading merozoite. The late stage synthesis of this polypeptide by P. chabaudi and its loss at schizogony and reinvasion was confirmed by immunofluorescence staining with a monoclonal antibody to this antigen. The importance of these antigens in the erythrocyte invasion process and in the induction and expression of immunity to malaria is discussed.

Monoclonal antibodies that protect in vivo against Plasmodium chabaudi recognize a 250,000-dalton parasite polypeptide.

Twenty monoclonal antibodies have been prepared to the erythrocytes from CBA/Ca mice infected with the rodent malaria Plasmodium chabaudi. By immunofluorescence, 15 of these antibodies recognized parasite antigens expressed only during the development of mature trophozoites to schizonts and merozoites, 2 recognized parasite antigens that were expressed throughout most of the intraerythrocytic cycle, and 3 recognized the membranes of all infected and uninfected erythrocytes. By immunoprecipitation of [35S]methionine-labeled, parasitized erythrocytes, parasite antigens recognized by all of the antiparasite antibodies were characterized. Eleven precipitated a 250,000-dalton parasite polypeptide which was synthesized and expressed late in the intraerythrocytic cell cycle and which appeared to be the major coat protein of the merozoites. In passive protection experiments, transfer of hyperimmune serum before infection with the parasite resulted in a delay in the rise of parasitemia, reduction in peak parasitemias, and a delay in the clearance of the parasitemia. Two monoclonal antibodies to the 250,000-dalton polypeptide had a similar but not as marked effect on parasitemia when given as a single dose before infection. When mixed and administered throughout the course of infection, their effects were greater. They had no influence on the course of Plasmodium berghei KSP11 parasitemia. Monoclonal antibodies to other parasite antigens and normal erythrocyte antigens failed to have a significant and reproducible effect on P. chabaudi parasitemia. The results suggest that this 250,000-dalton malaria parasite antigen may be important in the induction and expression of antibody-mediated immunity to malaria.