Molecular analysis and characterization of a protein involved in the replication of intracellular Toxoplasma gondii

Previous studies in our laboratory have identified a cytoplasmic protein (p97) of T. gondii that is involved in the process of intracellular parasite replication. Monoclonal antibody inhibits parasite replication in vitro and recognizes a protein of approximate 97 kDa by Western blot analysis. Using biotinylation, we demonstrate that p97 is not expressed on the surface of the tachyzoite. Polyclonal sera raised against the purified native protein was used to isolate a cDNA of 3.3 kb from a library. The product of this gene expresses a protein of approximate M_r 97 kDa that is reactive to the antibody (1B8) raised against the native antigen. The protein sequence of this product suggests that it is within the cytoplasm as suggested by the lack of a signal sequence or hydrophobic trans-membrane domain. This protein fails to dissociate into a monomer in the presence of non-ionic detergents as shown by gel filtration and density gradient. Southern blot analysis demonstrates a homologous gene sequence in two closely related Apicomplexa, Neospora caninum and Besnoitia jellisoni suggesting this protein is conserved among certain species of the Sarcocystidae.     

A pteridine reductase gene ptr1 contiguous to a P-glycoprotein confers resistance to antifolates in Trypanosoma cruzi

We have isolated the pteridine reductase-1 gene (ptr1), from Trypanosoma cruzi (Y strain), located contiguous to the Trypanosoma cruzi P-glycoprotein-2 (tcpgp2). The gene encodes a member of the family of short-chain dehydrogenases, enzymes that are involved in several oxidoreduction reactions. One member of the family, pteridine reductase-1 (PTR1) has been previously described in Leishmania as being involved in antifolate resistance. The ptr1 gene from T. cruzi presents an 828 bp open reading frame, coding for a 276 amino acid protein with a predicted molecular mass of 30 kDa. The deduced amino acid sequence exhibited a remarkable homology with the ptr1 genes of Leishmania major and Leishmania tarentolae. Southern blot analysis using a specific probe indicated that T. cruzi PTR1 is encoded by a single copy gene located in two chromosomes of about 0.9 and 1.2 Mb. Western blot analysis using a polyclonal antiserum against recombinant PTR1 revealed that the protein is only expressed in the epimastigote forms of the parasite; we did not detect the protein either in the amastigote or trypomastigote forms. Purified recombinant PTR1 exhibits a NADPH-dependent pteridine reductase activity comparable with those described in Leishmania. Gene transfection experiments using the pTEX expression vector show that, under the conditions tested, T. cruzi PTR1 is involved in resistance to the methotrexate, aminopterin and trimethoprim antifolates.     

Characterization of <Emphasis Type="Italic">p</Emphasis>24 Gene of <Emphasis Type="Italic">Spodoptera litura</Emphasis> Multicapsid Nucleopolyhedrovirus

Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) p24 gene is 753 bp long, potentially encoding 244 amino acids with a predicted molecular weight of 27.3 kDa. Homology analysis indicated that SpltMNPV P24 has 20–36% amino acid identity with that of other known baculoviruses. RT-PCR results showed that the p24 gene is transcribed actively at the late stage of infection and the mRNA start site was mapped within a consensus baculovirus late promoter sequence (ATAAG). Western blot analysis of extracts from SpltMNPV-infected S. litura cells detected a specific 28 kDa protein, and this protein was not N-glycosylated. Structural localization revealed that SpltMNPV P24 was associated with the nucleocapsid of occlusion-derived virus (ODV) as a complex form of 83 kDa.     

Molecular cloning and characterization of a T24-like protein in Echinococcus multilocularis

One tetraspanin, designated as E24, was cloned from a full-length enriched vector-capping cDNA library of Echinococcus multilocularis metacestode. The amino acid sequence and phylogenetic analysis suggested that E24 is a T24-like protein. The crucial, functional large extracellular loop (LEL) domain of E24 was expressed and characterized using a polyclonal antiserum by Western blot and immunohistochemistry. The results showed that anti-recombinant-E24 (anti-recE24) antibody can specifically recognize approximately 25 kDa recombinant protein and 25 kDa cyst-extracted antigen; the germinal layer of both the protoscolex-free and protoscolex-formed cysts were intensely labeled by immunofluorescent antibody. This study revealed that E24 is an antigenic, germinal layer-located protein of E. multilocularis metacestode, implying for its potential in diagnostic and vaccine development.     

Characterization of a Mis12 homologue in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The centromere/kinetochore represents an important complex on chromosomes that contains a large number of proteins and facilitates accurate chromosome segregation during cell division. Fission yeast Mis12 and its human homologue hMis12 have been identified as essential kinetochore components. Although homologues have been suggested to exist in plants, their function remains to be determined. In this study the full-length cDNA of the Mis12 homologue from Arabidopsis thaliana (AtMIS12) was successfully cloned by RACE-and RT-PCR and the DNA sequence determined. The 238 amino acid sequence deduced from the cDNA contains two conserved blocks and a coiled-coil motif, despite the poor overall similarity to fission yeast and human Mis12. The antibody raised against a partial peptide of AtMIS12 recognized a 27-kDa protein corresponding to the predicted molecular weight. Immunofluorescence labeling using the antibody revealed that AtMIS12 localizes at centromeric regions, like the centromeric histone H3 variant HTR12, throughout the cell cycle. These results indicate that AtMIS12 is a constitutive component of Arabidopsis kinetochores.     

Purification,characterization, and immunolocalization of paramyosin from the adult stage of<Emphasis Type="Italic"> Fasciola hepatica</Emphasis>

Paramyosin, a vaccine candidate in different helminthiases, was purified from the adult liver fluke Fasciola hepatica using two different procedures. The first started with a crude extraction of paramyosin in high-salt buffer followed by gel filtration chromatography and two precipitation-solubilization cycles; in the second, anion exchange chromatography replaced the gel filtration step. In both cases, the apparent molecular weight of the purified protein determined by sodium dodecyl sulfate gel electrophoresis under reducing and non-reducing conditions was 97 kDa and 200 kDa, respectively. The molecular weights were consistent with the presence of a dimeric protein linked by disulfide bridges. Western blot analysis showed that the dimeric and monomeric forms were both recognized by an antiserum raised against the F. hepatica 97 kDa band (-FhPmy), and by an anti-Schistosoma mansoni paramyosin immune serum. Immunohistochemistry using -FhPmy demonstrated the localization of paramyosin within the subtegumental muscle and in muscle cells surrounding the gut of adult parasites. We also observed labeling of extramuscular structures like testes, surface lamellae of the gut and the tegument of adult flukes.     

The Gene Product Specified by a Double Stranded RNA in the Flax Rust (<Emphasis Type="Italic">Melampsora lini</Emphasis>) is Expressed during Rust Growth

Strain SP6 of the flax rust (Melampsora lini) contains 11 double-stranded RNAs (dsRNAs) of unknown function. A large open-reading frame (B3ORF1) in dsRNA B3 encodes a polypeptide of 614 amino acids, and using an antiserum raised against the B3ORF1-glutathione S-transferase fusion protein prepared from a bacterial expression system, we have detected the presence of a 67 kDa polypeptide in rust urediospores. This polypeptide, identical in size to that of the predicted translation product of B3ORF1, was not detected in spores from either a fungal strain lacking the B3 dsRNA or an isogenic strain containing no dsRNA. These data indicate that B3ORF1 present in the flax rust B3 dsRNA is expressed in vivo which warrants farther investigation in search for its function during rust development.     

Cloning and sequencing of a cDNA fragment fromPlasmodium chabaudi chabaudi that contains repetitive sequences coding for a potentially lysine-rich aspartic acid-rich protein

Screening of a cDNA library (prepared in gt11) of the blood stages ofPlasmodium chabaudi chabaudi (AS) with immune serum has revealed an antigen that elicits a strong antibody response in infected mice. The clone (clone 6) expressing that antigen contains a 0.7 kb insert and produces a -galactosidase fusion protein of about 150 kDa. In Western blot analysis performed on parasite extracts, monoclonal antibodies and polyclonal sera prepared against the fusion protein revealed that the fusion protein contains part of a malarial protein of 93 kDa. Northern hybridization with clone 6 insert as probe detected a plasmodial RNA of about 3.2 kb, which could well code for a protein of this size. The insert hybridized to a singleEcoRI fragment and a singleHindIII fragment in genomic Southern blotting, suggesting that the gene is present in one copy in theP. chabaudi genome. The DNA sequence of clone 6 insert predicts a hydrophilic, acidic polypeptide consisting of seven repeats of 23–34 amino acids rich in lysine (24%) and aspartic acid (17.5%).     

Characterization of a profilin-like protein from <Emphasis Type="Italic">Schistosoma japonicum,</Emphasis> a potential new vaccine candidate

The tegumental membrane of platyhelminth parasites is of crucial importance for modulation of the host response and parasite survival. A complementary deoxyribonucleic acid (cDNA) containing an open reading frame of 390 bp, which encodes a profilin-like tegumental protein with a theoretical isoelectric point of 6.02 and a molecular weight of 14.42 kDa, had been identified by bioinformatic analysis. The coding region of the cDNA was cloned into the prokaryotic expression vector pGEX-4T-1 and expressed in Escherichia coli. The purified recombinant protein named rSj15 was immunogenic and could elicit a high titer of antibody in mice. Western blot analysis revealed that the protein was differentially expressed during the different growth stages of Schistosoma japonicum. Immunohistochemical analysis localized the protein to the tegument and underlying tissue of the S. japonicum adult worm. The rSj15 could induce the expressions of IL-12 in the cultured mouse dendritic cell.     

Molecular cloning and characterization of a novel immunoreactive ATPase/RNA helicase in human filarial parasite <Emphasis Type="Italic">Brugia malayi</Emphasis>

DEAD box proteins are putative RNA unwinding proteins found in organisms ranging from mammals to bacteria. We have identified a novel immunodominant cDNA clone, BmL3-helicase, encoding DEAD box RNA helicase by immunoscreening of a larval stage cDNA library of Brugia malayi. The cDNA sequence exhibited strong sequence homology to Caenorhabditis elegans and C. briggsae RNA helicase, a prototypic member of the DEAD (Asp-Glu-Ala-Asp) box protein family. The clone also showed similarity with RNA helicase of Wolbachia, an endosymbiotic bacterium of filarial parasite. It was overexpressed as ∼50 kDa His-tag fusion protein, and ATP hydrolysis assay of recombinant enzyme showed that either ATP or dATP was required for the unwinding activity, indicating BmL3-helicase as an ATP/dATP-dependent RNA helicase. The recombinant protein also demonstrated cross-seroreactivity with human bancroftian sera. The presence of BmL3-helicase in various life stages of B. malayi was confirmed by immunoblotting of parasite-life-cycle extracts with polyclonal sera against the BmL3-helicase, which showed high levels of expression in microfilaria, L_3, and adult (both male and female) stages. In the absence of an effective macrofilaricidal agent and validated anti-filarial drug targets, RNA helicases could be utilized as a rational drug target for developing agents against the human filarial parasite. Nucleotide sequence reported in this paper is available in the GenBank™, EMBL, and DDBJ databases under the accession number EF409381.     

A shared antigen among <Emphasis Type="Italic">Babesia</Emphasis> species: ribosomal phosphoprotein P0 as a universal babesial vaccine candidate

Babesia gibsoni ribosomal phosphoprotein P0 (BgP0) was previously identified as a cross-protective antigen against Babesia microti infection in mice. Interestingly, the same protein showed considerable antigenicity when tested with serum samples collected from Babesia-infected animals. Moreover, the polyclonal antibody raised against the recombinant BgP0 (rBgP0) recognized the P0 homologues from other Babesia species either by immunoblotting or by immunoscreening. The P0 genes from Babesia caballi, Babesia equi, and Babesia bigemina were then cloned and sequenced. The phylogenic analyses based on the amino acid sequences indicated that BgP0 has high identities with B. caballi P0 (88.1%), B. bigemina P0 (85.6%), Babesia bovis P0 (81.4%), and B. equi P0 (64.9%). Western blot analyses revealed that the corresponding native proteins ranged between 31 and 34 kDa, consistent with predicated molecular weight of Babesia P0. Furthermore, the immunogenic property of anti-rBgP0 IgG was evaluated against a B. bovis in vitro culture. The growth of B. bovis parasites was restricted by anti-rBgP0 IgG in a concentration-dependent manner, and significant reductions in parasitemia were observed only at 1 mg/ml in the culture. Taken together, these data suggest that P0 is a conserved protective antigen among Babesia species and might be a potentially universal vaccine candidate for babesiosis.     

Cloning and characterization of a gene coding for a protein (KAP) associated with the kinetoplast of epimastigotes and amastigotes of Trypanosoma cruzi

We have cloned and characterized a gene of Trypanosoma cruzi which encodes a protein, KAP (kinetoplasts-associated protein), expressed in the kinetoplasts of epimastigotes and amastigotes, the replicative stages of the parasite, but not in kinetoplasts of trypomastigotes. The single-copy gene is transcribed into a 3900-nt polyadenylated mRNA. Its trans-splicing acceptor site is preceded by a run of 15 adenosine residues. An open reading frame of 1052 codons is followed by a 3′ untranslated region containing short sequences characteristic of rapidly degradable RNAs. The potential translation product of the KAP gene contains a central region composed of four blocks of repeats of a 9-amino-acid motif. Rabbit antibodies raised against three synthetic peptides containing KAP sequence recognized a 175-kDa protein in epimastigotes and amastigotes which appears by indirect immunofluorescence to be associated with their kinetoplasts. The antibodies do not recognize the kinetoplast of trypomastigotes. The amino terminus of KAP contains features compatible with mitochondrial topogenic sequences.     

The use of an excreted superoxide dismutase in an ELISA and Western blotting for the diagnosis of <Emphasis Type="Italic">Leishmania</Emphasis> (<Emphasis Type="Italic">Leishmania</Emphasis>) <Emphasis Type="Italic">infantum</Emphasis> naturally infected dogs

An excreted iron superoxide dismutase of pI 3.75 and a molecular mass of approximately 25 kDa was partially purified by QAE Sephadex ion-exchange chromatography from the in vitro culture of Leishmania (Leishmania) infantum. This enzyme was detected by enzyme-linked immunosorbent assay and Western blot of anti-L. infantum antibodies in dog serum. For the determination of the sensitivity and specificity of this protein, the results using the complete-parasite antigen fraction were taken as references. For this, 39 sera were assayed in dogs from different Spanish provinces. By Western blot, at a dilution of 1:250, 82% of the sera were positive when superoxide dismutase excreted was used as the antigen, against 56.4% positivity when the complete parasite was used as the antigen. These findings support the results of a previous study, indicating that the superoxide dismutase excreted can be useful in diagnosing L. (L.) infantum.     

Detection and Localization of <Emphasis Type="Italic">Rice stripe virus</Emphasis> Gene Products <Emphasis Type="Italic">In Vivo</Emphasis>

The genome of the Tenuivirus, Rice stripe virus (RSV) comprises four RNAs, the smallest three of which each contain two open reading frames (ORFs) arranged in an ambisense manner. The expression of the ORFs from RNAs 2–4 in plants and the insect vector, Laodelphax striatellus, was studied using antisera raised against the gene products. In Western blotting of the proteins from infected plants, the molecular masses of p2, p3, pc3 (nucleocapsid protein, N) and p4 (major non-structural protein, NCP) were as expected; that of pc4 appeared larger than expected. Antisera to the N- and C-terminal parts of the complementary ORF on RNA 2, analogous to that encoding glycoproteins on genomes of bunyaviruses and tospoviruses, revealed banding patterns suggestive of processing of the product; the possible processing is discussed. Four types of inclusion bodies were identified by immunofluorescent and immunogold microscopy of thin sections of infected leaves. Most electron-dense amorphous semi-electron-opaque inclusion bodies (dASO) contained only p4 while some contained at least p2, pc2-N, p3, pc3 as well as p4. A ring-like structure containing at least pc2-N, p4 and pc4 was also identified in infected plant cells. Fibrillar amorphous semi-electron-opaque inclusion bodies (fASO) contained only p4. Filamentous electron-opaque inclusion bodies (FEO), which consist of pc2-N^.and p4, were found both in infected plant cells and in the mid-gut lumen and mid-gut epithelial cells of L. striatellus. This suggests an interaction between p4 and pc2-N and a function of pc2-N distinct from that of its-homologue in Bunyaviridae. Our results confirm the in vivo ambisense coding strategy of Tenuivirus RNA 2 and provide further evidence that RSV does not produce enveloped virions in infected rice plants.     

Alternative Expression of a Chitosanase Gene Produces Two Different Proteins in Cells Infected withChlorellaVirus CVK2

SeveralChlorellavirus CVK2 proteins had chitosanase and/or chitinase activities. A gene coding for an ORF of 328 amino acids (aa) with a predicted molecular mass of 36,769 Da was cloned from the viral genome. The predicted amino acid sequence of an N′-portion (174 aa) of this gene product (vChta-1) showed 22 to 25% identity with various bacterial chitosanases. A glutathioneS-transferase (GST)–vChta-1 fusion protein had strong chitosanase activity. Western blot analysis with antisera raised against the vChta-1 protein identified two proteins of 37 and 65 kDa in virus-infectedChlorellacells beginning at 240 min postinfection and continuing until cell lysis. The larger protein was packaged in the virion, while the smaller one remained in the cell lysate. Both chitosanase proteins were produced from the single gene,vChta-1,by a mechanism of alternative gene expression.     

Purification and characterization of culture-derived exoantigens of Plasmodium falciparum

An 83 kDa glycoprotein and a 100 kDa glycoprotein have been purified from the supernatant fluid of in vitro cultures of Plasmodium falciparum by conventional cation-exchange liquid chromatography, size exclusion high performance liquid chromatography, and anion-exchange high performance liquid chromatography. Both proteins exist as dimers in the native state and have been identified as parasite antigens by Western immunoblotting and by their specific reactivity in the indirect enzyme-linked immunosorbent assay. The N-terminal amino acid sequence of these two proteins has been determined and they are at least 90% homologous. The use of monospecific rabbit antisera raised against the individual pure proteins confirm their cross-reactivity. We postulate that the 83 kDa protein is a specific processing product of the larger 100 kDa protein. The presence of these proteins in the culture supernatant suggests they could both be derived from the merozoite surface coat and are potential protective antigens.     

A western blot characterization of Mycobacterium bovis antigens recognized by cattle sera

The immune response to Mycobacterium bovis in cattle was assessed by Western blot. The antibody recognition pattern to M. bovis whole cell extracts and culture supernatant antigens was studied by using sera from M. bovis-infected (n=62) and healthy (n=38) cattle. Although the recognition patterns were highly variable, some proteins were regularly detected, mainly those with molecular masses of 17, 23, 28, 42, 66, 71 and 80 kDa in cellular extracts, and with molecular masses of 23 and 33 kDa in supernatants. Whole cell extract antigens were more frequently recognized than culture supernatant antigens. Healthy controls produced only a week antibody response.The antibody response was variable, depending on tuberculosis stage. In early stages very few antibodies were detected. A response against the 66-kDa stress protein was mounted in intermediate tuberculosis and remained stable in more advanced disease. In late diseases, the preferentially recognized antigens were a 28-kDa cellular protein and supernatant antigens.The 28-kDa protein was studied in some detail. As determined by using monoclonal antibodies, the 28-kDa protein is different from superoxide dismutase. This protein aggregated in stored cell extracts and was not totally transferred to nitrocellulose.The principal conclusions of this work are: (i) whole cell extract proteins are more frequently recognized than the secreted proteins and (ii) a 28-kDa protein is a major antigen in late disease.     

Cloning and expression of a chitinase gene from the hyperparasitic fungus Aphanocladium album

Summary Recombinant clones from a cDNA library of an Aphanocladium album chitinase-overproducing mutant strain were isolated by screening with antiserum against a 39 kDa chitinase purified from this hyperparasitic fungus. Analysis of the isolated positive clones indicated that most of them carried the same cDNA. A cDNA from this group was used as a hybridization probe to isolate an 8 kb DNA fragment from a genomic library of the wild-type strain. The chitinase 1 gene was mapped to this fragment by two independent approaches. Its partial DNA sequence was in perfect agreement with an amino-terminal peptide sequence obtained by sequencing 23 amino acids of the 39 kDa chitinase. Its transfer in Fusarium oxysporum resulted in a transformant producting both a protein of about 39 kDa that cross-reacted with the chitinase antiserum and a chitinase activity that was inhibited by the same antiserum. Northern blot analysis indicates that the cloned chitinase gene was subject to catabolite repression and appeared inducible by chitin.     

Antigenic cross reactivity between p195 and a distinct protein of 100 kDa in Plasmodium falciparum

A monoclonal antibody raised against merozoites of Plasmodium falciparum clone T9/96 was shown to react with an extremely strain specific epitope on a 195 kDa protein synthesized only by late trophozoites and schizonts. This protein was shown to exhibit all of the characteristics attributed to the molecule known variously as merozoite surface protein precursor, polymorphic schizont antigen and p195. The monoclonal antibody also identified a cross-reactive epitope on a distinct protein of 100 kDa in ring stage parasites which was shown to be synthesized throughout the asexual cycle and was not a processing product of p195. One-dimensional peptide mapping studies suggested that these two proteins share a degree of common sequence or structure.