Appearance of a stage-specific immunodominant glycoprotein in encysting Entamoeba invadens

The appearance of cyst-specific proteins in encysting Entamoeba invadens and their immunogenicity were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting using an axenic encystation system in vitro. A rabbit antiserum against trophozoites of E. invadens reacted with a number of proteins of cysts after 1–4 days of incubation. Thus, a number of cyst proteins remained antigenically unchanged as common antigens of the two forms after transformation from trophozoites to cysts. A rabbit antiserum against cysts also reacted with the trophozoite proteins as well as the cyst proteins. The most interesting result was that the rabbit anticyst serum reacted predominantly with an 88-kDa protein of cysts after 1 day of incubation. The 88-kDa protein reacted with the anticyst serum absorbed with trophozoite proteins and was thus cyst-specific. The reactivity of the 88-kDa protein of cysts with the absorbed anticyst serum decreased as encystation proceeded. When soluble and particulate fractions prepared from cysts after 1 day of incubation were examined by electrophoresis and immunoblotting, the 88-kDa protein that had reacted with the absorbed anticyst serum was found to be present in the particulate fraction, which was rich in cell-wall fragments, and stained with periodic acid-Schiff 's reagent, indicating that it is a glycoprotein. The results indicate that encystation is accompanied by appearance of the cyst-specific 88-kDa glycoprotein, which is immunodominant and most abundantly expressed in cysts after 1 day of incubation and appears to be associated with the cyst wall. Received: 31 May 1999 / Accepted: 20 July 1999     

Proteins of the Plasmodium falciparum two transmembrane Maurer’s cleft protein family, PfMC-2TM, and the 130?kDa Maurer’s cleft protein define different domains of the infected erythrocyte intramembranous network

Plasmodium falciparum Maurer’s clefts participate in the transport of macromolecules within the cytoplasm, including the transport of virulence proteins to the erythrocyte membrane surface. We identified a family of genes PfMC-2TM encoding transmembrane proteins located within the intramembranous network of the infected erythrocyte using monoclonal antibody SP1C1. The distribution of the PfMC-2TM protein family within domains of the network was investigated by colocalization and confocal microscopy studies using monoclonal antibody SP1C1 specific for PFMC-2TM and monoclonal antibody SP1A6 specific for the130 kDa Maurer’s cleft protein. Peptide-specific antibodies were prepared against six peptides from different domains of PfMC-2TM and used with the Mabs, as well as known antibodies specific to Maurer’s clefts proteins (ring-expressed protein and membrane-associated histidine-rich protein 1), the erythrocyte membrane protein 1 (PfEMP-1), and serine-rich antigen in colocalization studies. We show that PfMC-2TM is located in the Maurer’s clefts throughout the intracellular blood stage, and immunoelectron microscopy shows domains of PfMC-2TM localized in the parasitophorous vacuole and parasitophorous vacuole membrane. The distribution of the 130 kDa Maurer’s cleft protein changes from within the parasite to the clefts during intracellular development as the parasite matures from young trophozoite to segmented schizont.     

Differential sensitivity of erythrocytic stages of the rodent malaria parasite Plasmodium chabaudi chabaudi to dioncophylline B, a highly active naphthylisoquinoline alkaloid

Four-week-old OF1 mice, infected with synchronized Plasmodium chabaudi chabaudi blood forms, were intraperitoneally injected with the naphthylisoquinoline alkaloid dioncophylline B (10 mg kg⁻¹ day⁻¹) at three consecutive days. The respective groups were treated when rings, trophozoites, and schizonts were predominant. Microscopical observations of thin blood smears were made every two hours after the start of the experiment. A clear dependency of the effectiveness of dioncophylline B treatments on the timing of drug administration was demonstrated. Based upon the evolution of total parasitaemia and the survival rates, it was concluded that ring stages are insensitive to dioncophylline B, while the drug is highly effective when given at the trophozoite stage and partially effective when given at the schizont stage. Dioncophylline B seems to act by inhibiting the haemozoin degradation, as indicated by pigment clumping, and by impairing the segmentation of schizonts. Accepted: 15 March 1999     

Rhop-3 protein conservation among Plasmodium species and induced protection against lethal P. yoelii and P. berghei challenge

In the present study, Rhop-3 polymorphism among Plasmodium falciparum field and laboratory isolates and among rodent Plasmodium species was investigated and identified. The Rhop-3 gene was found in all Plasmodium species so far tested. The overall structure of the Rhop-3 protein was found conserved among P. falciparum, Plasmodium yoelii, and Plasmodium berghei. However, it was more conserved among rodent Plasmodium species than between P. falciparum and Plasmodium vivax. The most conserved regions of Rhop-3 are the second half of exon 6 (amino acid #548 to #665) and the beginning of exon 3 (amino acid #59 to #210). Recombinant C-terminal partial and full-length Rhop-3 proteins of P. yoelii and P. berghei were expressed in Escherichia coli and purified. Immunization-challenge experiments in mice using recombinant Rhop-3 proteins led to a delay in parasite development and protected mice from a homologous lethal challenge infection. In a group of eight outbred Carworth Farm White (CFW) mice immunized with P. yoelii C-terminal recombinant His-Y1412 protein, three mice (37.5%) were protected from a lethal P. yoelii challenge. In BALB/cJ mice one mouse (20%) survived the infection. Immunization of mice with P. berghei recombinant full-length Rhop-3 protein in BALB/cJ mice led to a 40% survival from lethal P. berghei challenge. CFW mice immunized with P. berghei recombinant full-length Rhop-3 protein showed a significant delay in parasite development with a heterologous P. yoelii challenge. The Rhop-3 protein is a promising candidate for an asexual stage malaria vaccine.Nucleotide sequence data reported in this paper have been submitted to GeneBank with the Accession numbers: AY044907-AY044914.     

Identification of the 150-kDa surface antigen of Entamoeba histolytica as a galactose- and N -acetyl-d-galactosamine-inhibitable lectin

A monoclonal antibody that reacts with a 150-kDa protein of Entamoeba histolytica on Western immunoblotting under nonreducing conditions inhibits the adherence and cytotoxicity of the ameba to mammalian cells in vitro. Affinity purification of solubilized trophozoites using the monoclonal antibody and electophoresis yielded three glycoproteins with molecular masses of 150, 170, and 260 kDa, suggesting the existence of either a common epitope or the close association of these proteins. The 260-kDa fraction was identified as the well-known galactose (Gal)- and N-acetyl-d-galactosamine (GalNAc)-inhibitable lectin. The 150- and 170-kDa fractions seemed to exist as part of a 380-kDa native protein with an isoelectric point of pH␣6.9. The N-terminal amino acid sequence of the 150-kDa protein was unique, indicating that the protein was not a degraded product of the 260-kDa lectin. By gel filtration, the 260-kDa lectin and the 150/170-kDa protein could be separated. When Chinese hamster ovary cells were pretreated with the fraction consisting of the 150/170-kDa protein the adherence of trophozoites to Chinese hamster ovary cells was competitively inhibited to a level equivalent to that observed for the 260-kDa lectin. The inhibitory effect was lost in the presence of Gal and GalNAc but was not influenced by the presence of glucose. These results demonstrate that the 150/170-kDa protein is a Gal/GalNAc-inhibitable lectin. The existence of a sugar-binding domain in the protein was confirmed by Gal-affinity chromatography. Received: 7 January 1998 / Accepted: 4 March 1998     

Naturally acquired IgG antibodies against the C-terminal part of Plasmodium falciparum sporozoite threonine-asparagine-rich protein in a low endemic area

Plasmodium falciparum sporozoite threonine–asparagine-rich protein (STARP), a 78-kDa surface protein, is considered a potential vaccine candidate. The C-terminal part of STARP has been evolved under positive selection, suggesting the presence of immunodominant epitopes. However, little is known about the immune responses against STARP among individuals upon natural malaria exposure. In this study, we have cloned and expressed in Escherichia coli the recombinant C-terminal part of STARP spanning 118 amino acids in order to examine the humoral immune response against this protein. Blood samples were randomly collected from 74 individuals living in a malaria endemic area of Thailand who were acutely infected with P. falciparum (n = 54) and with Plasmodium vivax (n = 20). Malaria-negative blood samples were also obtained from 27 individuals living in the same endemic area who had experienced prior infection with P. falciparum 6 months to 1 year before sample collection and 20 healthy subjects without history of malaria exposure. Western blot analysis revealed that IgG antibodies against this recombinant peptide were found in 23 of 54 serum samples (42.6%) from P. falciparum-infected individuals. All serum samples from P. vivax-infected cases, non-infected individuals, and those who experienced prior infection with P. falciparum gave negative results, indicating that naturally acquired IgG antibodies against the C-terminal part of STARP are species-specific and short-lived. Provided that antibodies against STARP could confer protection, it is likely that malaria vaccine derived from the C-terminal part of STARP could probably be boosted upon natural exposure to P. falciparum.     

Antibody response to plasmodium falciparum malaria. Comparisons of immunoglobulin concentrations, antibody titres and the antigenicity of different asexual forms of the parasite

Antibody titres of children and adults living in a malaria endemic region were measured by the indirect fluorescent antibody technique, using fluorescein-conjugated monospecific immunoglobulin antisera. The antigenicity of Plasmodium falciparum trophozoites was compared with that of schizonts obtained by in vitro culture of infected blood. Malarial antibody was detected with antisera to IgG, IgM and, at low levels, to IgA but not with antisera to IgD or IgE. Higher titres were obtained with schizonts as the source of antigen than with trophozoites. Adults in whom IgM antibodies were detected had a mean IgM concentration significantly higher than that for individuals who were IgM antibody negative, and there was a positive correlation between IgG fluorescent antibody titres and total IgG in the children examined. Serial plasma samples from children with acute P. falciparum infections were examined to determine changes in immunoglobulin concentrations, antibody titres and the levels of serum antigens as a result of the infection.     

In vitro sensitivity pattern of chloroquine and artemisinin in Plasmodium falciparum

Artemisinin (ART) and its derivatives form the mainstay of antimalarial therapy. Emergence of resistance to them poses a potential threat to future malaria control and elimination on a global level. It is important to know the mechanism of action of drug and development of drug resistance. We put forwards probable correlation between the mode of action of chloroquine (CQ) and ART. Modified trophozoite maturation inhibition assay, WHO Mark III assay and molecular marker study for CQ resistance at K76T codon in Plasmodium falciparum CQ-resistant transporter gene were carried out on cultured P. falciparum. On comparing trophozoite and schizont growth for both CQ-sensitive (MRC-2) and CQ-resistant (RKL-9) culture isolates, it was observed that the clearance of trophozoites and schizonts was similar with both drugs. The experiment supports that CQ interferes with heme detoxification pathway in food vacuoles of parasite, and this may be correlated as one of the plausible mechanisms of ART.     

Characterization of a Plasmodium chabaudi gene encoding a protein with glutamate-rich tandem repeats

Several highly antigenic proteins containing tandem repeats rich in glutamic acid residues have been described in Plasmodium falciparum. However, relatively little information is available about analogous genes in rodent parasites. This report describes a 4.2-kb genomic DNA fragment from P. chabaudi with a deduced amino acid sequence that is predominantly glutamate-rich tandem repeats. Several different monoclonal antibodies raised against a 93-kDa P. chabaudi protein, which does not correspond to the cloned DNA fragment, recognize a recombinant protein expressed from the 4.2-kb DNA fragment. The only sequence similarities between these two genes are tandem repeats with a predominance of glutamate pairs followed by a hydrophobic residue. This repetitious-sequence motif may be the basis for the observed cross-reactivity. A similar motif has been demonstrated to be the basis for antibody cross-reactivity between glutamate-rich proteins of P. falciparum. The expression of multiple glutamate-rich proteins with cross-reacting epitopes may be a general phenomenon in Plasmodium species. Received: 3 March 1998 / Accepted: 7 July 1998     

Antigenicity of Trichomonas vaginalis heat-shock proteins in human infections

Patients infected with Trichomonas vaginalis mount humoral and cellular immune responses that often do not protect against reinfection. The oxidative stressors produced by leukocytes may trigger a heat-shock-like response in T. vaginalis trophozoites, helping the parasite to survive host immune defenses. The antigenicity of T. vaginalis heat-shock proteins (HSPs) was examined by immunoprecipitation of T. vaginalis heat-induced proteins with sera from infected patients and controls. When T. vaginalis was heat-shocked, HSPs of 169–167 and 140–137 kDa were specifically recognized by sera from infected male and female patients. However, the majority of T. vaginalis HSPs were also immunoprecipitated by control sera; all sera recognized 72- to 71-kDa, 47- to 45-kDa, 38- to 37-kDa, 35-kDa, and 31-kDa heat-induced proteins. At least 15 proteins from non-heat-shocked T. vaginalis were immunoprecipitated by sera from infected patients and controls, indicating that natural or cross-reacting antibodies could participate in host responses to trichomoniasis. Molecules of 158, 135, 89, and 74–72 kDa were immunoprecipitated from some non-heat-shocked parasites only by patients' sera. A 38-kDa T. vaginalis protein was immunoprecipitated only by sera from infected females and may be specific for infection in women. Received: 26 August 1999 / Accepted: 15 September 1999     

A merozoite-specific 22-kDa rhoptry protein of the coccidium Eimeria nieschulzi (Sporozoa,Coccidia) is exocytosed in the parasitophorous vacuole upon host cell invasion

A monoclonal antibody (Mab D12A4) was used to follow the genesis and fate of rhoptries from early first-generation merogony through second-generation merozoites of the rat coccidium Eimeria nieschulzi. The epitope recognized by Mab D12A4 belongs to a 22-kDa protein which can be localized first in developing meronts 25 h post-infection. Early rhoptries appear as distinct granules in the cytoplasm of developing meronts and elongate into mature organelles before merozoite release. The 22-kDa protein is found in the parasitophorous vacuole after host cell invasion. Western blotting and immunofluorescence showed that the 22-kDa rhoptry protein is expressed in schizonts and merozoites but not in sporozoites. Received: 9 August 1997 / Accepted: 14 October 1997     

Stage-specific protein synthesis by asexual blood stage parasites of Plasmodium knowlesi

P. knowlesi parasites with a maximum age distribution of 3 h were metabolically labelled with [³⁵S]methionine during 9 sequential non-overlapping intervals, from young rings to mature segmented schizonts. The proteins synthesised at the different stages were compared using sodium dodecyl sulphate-polyacrylamide gel electrophoresis; more than 40 polypeptides (M_r 20 000 to over 200 000) were identified in the different parasite preparations. The major polypeptides synthesised by rings and trophozoites of different ages were similar, but differences in minor polypeptides could always be recognised. At the onset of schizogony ring and trophozoite specific proteins ceased to be synthesised and proteins specific to schizogony emerged. In general, schizont-specific proteins were of higher molecular weight than ring stage proteins. Details of the morphological changes which occurred during the metabolic labelling episode permits correlation between parasite structure and synthesis of particular polypeptides. Comparison of parasite components metabolically labelled with [³H]glucosamine during defined periods of development also revealed stage-specific synthesis of glycoproteins. The extent to which proteins are altered after synthesis has been investigated by pulse-chase experiments.     

Molecular genetic characterization and subcellular localization of Theileria annulata mitochondrial heat-shock protein 70

A Theileria annulata mitochondrial heat-shock protein of the 70-kDa family (Tamthsp70) was isolated by screening of the cDNA library of a T. annulata-infected bovine lymphoblastoid cell line with an antibody raised against T. annulata schizonts. The Tamthsp70 coding sequence was found to be most closely related to a previously reported mitochondrial hsp70 gene of Eimeria tenella exhibiting a similarity of 67% with mitochondrial hsp70 genes of eukaryotic plants (Pisum sativum, Phaseolus vulgaris) and with dnaK proteins of prokaryotes (Rhizobium meliloti, Agrobacterium tumefaciens). The Tamthsp70 mRNA is expressed within the sporozoite, schizont, and merozoite stages of the parasite, which suggests that it is constitutively transcribed throughout the life cycle. The gene encodes a polypeptide of 681 amino acids and exhibits a mitochondrial targeting sequence and several sequence motifs common to mitochondrial hsp70 and prokaryotic dnaK proteins. The protein level of the Tamthsp70 protein after heat shock decreased slightly during the exposure of infected cells to a temperature of 42 °C in comparison with cells cultured at 37 °C. By immunofluorescence the protein was located in the area in which the schizonts reside within infected cells. Immunoelectron microscopy showed that the hsp70 protein was predominantly localized in the mitochondria of the parasites. However, it was also found in small amounts in the cytoplasm of the parasite and host cell. This indicates (1) that Tamthsp70 is very probably translated in the parasite cytoplasm and then transported across the mitochondrial membrane into the mitochondrial matrix and (2) that it is transported across the parasite membrane into the host-cell cytoplasm. Received: 23 December 1999 / Accepted: 10 January 2000     

Specificities of antibodies that inhibit merozoite dispersal from malaria-infected erythrocytes

When malaria schizont-infected erythrocytes are cultured with immune serum, antibodies prevent dispersal of merozoites, resulting in the formation of immune clusters of merozoites (ICM) and inhibition of parasite growth. Antigens recognized by these antibodies were identified by probing two dimensional immunoblots of Plasmodium falciparum antigens with antibodies dissociated from immune complexes present at the surface of merozoites in ICM. Total immune serum recognized 88 of the 135 protein spots detected by colloidal gold staining, but antibodies dissociated from immune complexes recognized only 15 protein spots attributable to no more than eight distinct antigens. Antigens recognized by antibodies that inhibit merozoite dispersal include the precursor to the major merozoite surface antigens (gp195), a 126-kDa serine-repeat antigen (SERA), the 130-kDa protein that appears to bind to glycophorin (GBP130), and the approx. 45-kDa merozoite surface antigen. One other antigen (230/215-kDa doublet) was identified by using antibodies affinity purified from recombinant expression proteins. The identities of the other three antigens (150 kDa, 127 kDa and less than 30 kDa) were not determined. This approach provides a strategy for identifying epitopes accessible at the merozoite surface which may be important components of a multivalent vaccine against blood stages of P. falciparum.     

Regulation by Ca2+-calmodulin of the actin-bundling activity ofPhysarum 210-kDa protein

Summary From the plasmodia of a lower eukaryote,Physarum polycephalum, we have previously purified a 210-kDa protein that showed similar properties to those of smooth muscle caldesmon. Further characterization of the 210-kDa protein revealed that it bundled actin filaments. This bundling activity was inhibited by calmodulin in the presence of Ca²⁺. Unlike smooth muscle caldesmon, the 210-kDa protein bundled actin filaments whether or not a reducing agent, such as dithiothreitol, was present. The protein was shown to have two (or more) different actin-binding sites which were classified into salt-sensitive and salt-insensitive sites. Electron microscopy revealed that the 210-kDa protein was an elongated molecule (mean length, 97 ± 25 nm) which was bent in the middle. The Stokes radius and sedimentation coefficient of the 210-kDa protein were 130 Å and 2.9 S, respectively. An immunofluorescence study revealed that the 210-kDa protein colocalized with the bundles of actin filaments in thin-spread preparations ofPhysarum plasmodia, suggesting that the 210-kDa protein was regulating the appearance and disappearance of the actin bundles that are associated with the contraction-relaxation cycle of the plasmodia.     

The 140/130/105 kilodalton protein complex in the rhoptries of Plasmodium falciparum consists of discrete polypeptides

Four monoclonal antibodies (MAbs) recognise an antigen localised in the rhoptries of Plasmodium falciparum merozoites using both indirect immunofluorescence assay and immunoelectron microscopy with immunogold labeling. All MAbs immunoprecipitated bands at 140, 130 and 105 kDa from [35S]methionine-labeled parasites; however, one MAb immunoblotted only the 130 kDa protein and another MAb immunoblotted the 105 kDa protein. The affinity purified antigen complex consisted of proteins of 140, 130, 105 and 98 kDa. The individual proteins were subjected to peptide mapping with Staphylococcus aureus V8 protease; the 98 kDa protein was a degradation product of the 105 kDa protein and the 140, 130, and 105 kDa proteins were found to be unrelated. The antigen complex was synthesised at the mid trophozoite stage and was considered to be soluble as judged by release from mature schizonts by freeze/thaw lysis. One of the MAbs inhibited parasite growth and/or merozoite invasion of erythrocytes, in vitro, to a small but significant extent.     

Characterization of an immunodominantGiardia lamblia protein antigen related to alpha giardin

The trophozoites ofGiardia lamblia possess several protein antigens, predominant among them a protein of 32,000 Da. In the present study, we used monospecific antibodies that recognize this protein to demonstrate its presence on a variety ofG. lamblia isolates from human and animal sources. Immune electron microscopy was used to localize 32-kDa antigen on the trophozoite membrane and disk. Immunofluorescent assays employing monospecific antibodies confirmed the presence of 32-kDa antigen on the membrane and disk and its absence on flagella or nuclei. The N-terminal 17 amino acids of the 32-kDa antigen are identical to -1-giardin, a protein component of microribbons on the ventral disk. These results suggest that the 32-kDa immunodominant trophozoite antigen is -1-giardin.     

A novel cDNA clone encoding a specific excretory/secretory antigen of larval Trichinella pseudospiralis

A cDNA library for Trichinella pseudospiralis was constructed to study the expression of specific antigens. Four positive clones were identified using antibodies against the excretory/secretory (ES) products of the nematode as probe. Sequence analysis showed that they contained identical cDNA inserts of 606 bp, including a 5′ non-translated region of 96 bp, a core translated segment of 408 bp and a poly(A)⁺ 3′ terminus. It encoded a novel 136-amino-acid polypeptide. Southern blot analysis indicated that the cDNA did not cross-hybridize to the genomic digests of T. spiralis, mouse, or rat. A single copy only of its complementary sequence was found in the genome of T. pseudospiralis. Using the lambda ZAP expression system, the cDNA was induced to express a 23-kDa β-galactosidase-fusion protein which did not cross-react with polyclonal and monoclonal antibodies against T. spiralis, heat shock proteins, or four heterologous species of nematodes. The antiserum against the fusion protein recognized a 15-kDa band from the ES products of T. pseudospiralis in immunoblotting. Immunocytolocalization demonstrated that the anti-fusion protein serum only recognized an epitope in the stichosome of T. pseudospiralis and not in T. spiralis. The protein can therefore serve as a specific antigen for the differential diagnosis of trichinellosis. Received: 22 December 1998 / Accepted: 17 February 1999     

Identification and characterisation of proteins associated with the rhoptry organelles of Plasmodium falciparum merozoites

We describe antigens of Plasmodium falciparum recognised by murine monoclonal antibodies which by immunofluorescence react with the rhoptry organelles of the extracellular merozoite stage. Immunoblotting shows that the antibodies recognise two major parasite antigens of M_r 82 and 65 kilodaltons (kDa). Immunoprecipitations from detergent extracts of [³⁵S]-methioninelabelled parasites show that the 82-kDa and 65-kDa antigens are parasite proteins. Pulse-chase experiments on synchronous parasite cultures show that the 82-kDa protein is synthesised during early schizogony and is later processed into the 65-kDa antigen in segmenting schizonts. In Nonidet P-40, these antigens are non-covalently associated with two other proteins of 40 kDa and 42 kDa. The 40/42-kDa doublet is synthesised in parallel with the 82 kDa antigen and persists, apparently unchanged, till the end of the cell cycle.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycolbis-(aminoethylether) tetraacetic acid - PMSF phenylmethylsulphonylfluoride - TLCK tosyl-L-lysine chloromethyl ketone - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - M _r relative molecular mass     

Protection of hamsters from amebic liver abscess formation by a monoclonal antibody to a 150-kDa surface lectin of Entamoeba histolytica

We examined the effects of passive immunization with a monoclonal antibody (EH3015) that recognizes a 150-kDa surface lectin of Entamoeba histolytica on amebic liver-abscess formation in hamsters. The hamsters were inoculated i.p with 0.1, 1.0, or 10 mg of EH3015 at 24 h prior to an intrahepatic challenge with 10⁵ trophozoites of E. histolytica. In hamsters treated with 1.0 and 10 mg of EH3015 the incidence of liver abscesses was significantly reduced. These results demonstrate that monoclonal antibody EH3015 can prevent the development of amebic liver abscesses and that the 150-kDa lectin may be a protective antigen on the surface of E. histolytica. Received: 5 May 1998 / Accepted: 28 May 1998