A Cryptosporidium parvum sporozoite and oocyst lambda gt11 cDNA library was screened with a hyperimmune rabbit serum that was developed against insoluble fragments of ultrasonicated oocysts. A clone named Cp22.4.1 encoding a protein of 231 amino acids with 4 zinc-finger domains characterized by a Cys-X2-Cys-X4-His-X4-Cys motif was isolated and characterized. There was a complete match between the sequencing data of the coding region of Cp22.4.1 and the corresponding gene at chromosomal level. Cloning in a pBAD-TOPO-TA expression vector permitted to evaluate the antigenicity of the recombinant His-tagged antigen. This antigen was recognized by 2 out of 5 sera from Cryptosporidium immune calves and not by sera from parasite naive animals.
Amino Acid Sequence ; Animals ; Antibodies, Protozoan/*blood ; Antigens, Protozoan/chemistry/*immunology ; Base Sequence ; Cattle/*immunology ; Cryptosporidium parvum/*immunology ; Molecular Sequence Data ; Protozoan Proteins/chemistry/genetics ; Rabbits ; Recombinant Proteins ; Zinc Fingers/genetics/*immunology

Trypanosoma brucei, a protozoan parasite, causes sleeping sickness in humans and Nagana disease in domestic animals in central Africa. The trypanosome surface is extensively covered by glycosylphosphatidylinositol (GPI)-anchored proteins known as variant surface glycoproteins and procyclins. GPI anchoring is suggested to be important for trypanosome survival and establishment of infection. Trypanosomes are not only pathogenically important, but also constitute a useful model for elucidating the GPI biosynthesis pathway. This review focuses on the trypanosome GPI biosynthesis pathway. Studies on GPI that will be described indicate the potential for the design of drugs that specifically inhibit trypanosome GPI biosynthesis.
Animals ; Biosynthetic Pathways ; Glycosylphosphatidylinositols/*biosynthesis/chemistry ; Humans ; Protozoan Proteins/genetics/metabolism ; Trypanosoma brucei brucei/chemistry/genetics/*metabolism ; Trypanosomiasis, African/*parasitology

Trypanosoma brucei, a protozoan parasite, causes sleeping sickness in humans and Nagana disease in domestic animals in central Africa. The trypanosome surface is extensively covered by glycosylphosphatidylinositol (GPI)-anchored proteins known as variant surface glycoproteins and procyclins. GPI anchoring is suggested to be important for trypanosome survival and establishment of infection. Trypanosomes are not only pathogenically important, but also constitute a useful model for elucidating the GPI biosynthesis pathway. This review focuses on the trypanosome GPI biosynthesis pathway. Studies on GPI that will be described indicate the potential for the design of drugs that specifically inhibit trypanosome GPI biosynthesis.
Animals ; Biosynthetic Pathways ; Glycosylphosphatidylinositols/*biosynthesis/chemistry ; Humans ; Protozoan Proteins/genetics/metabolism ; Trypanosoma brucei brucei/chemistry/genetics/*metabolism ; Trypanosomiasis, African/*parasitology

BACKGROUNDPyruvate phosphate dikinase (PPDK) reversibly catalyzes the interconversion of phosphoenolpyruvate (PEP) and pyruvic acid, leading to catabolism and adenosine triphosphate (ATP) synthesis or gluconeogenesis and ATP consumption. Molecular modeling of PPDKs from divergent organisms demonstrates that the orientation of the phosphorylatable histidine residue within the central domain of PPDK determines whether this enzyme promotes catabolism or gluconeogenesis. The goal of this study was to determine whether PDDK from Giardia underwent adaptive evolution in order to produce more energy under anaerobic conditions.

METHODSA total of 123 PPDK sequences from protozoans, proteobacteria, plants, and algae were selected, based upon sequence similarities to Giardia lamblia PPDK and Zea mays PPDK. Three-dimensional (3-D) models were generated for PPDKs from divergent organisms and were used to compare the orientation of the phosphorylatable histidine residue within the central domain of PPDKs. These PPDKs were compared using a maximum-likelihood tree.

RESULTSFor PPDK from Giardia, as well as from other anaerobic protozoans, the central domain tilted toward the N-terminal nucleotide-binding domain, indicating that this enzyme catalyzed ATP synthesis. Furthermore, the orientation of this central domain was determined by interactions between the N- and C-terminal domains. Phylogenetic analysis of the N- and C-terminal sequences of PPDKs from different species suggested that PPDK has likely undergone adaptive evolution in response to differences in environmental and metabolic conditions.

CONCLUSIONThese results suggested that PPDK in anaerobic organisms is functionally adapted to generate energy more efficiently in an anaerobic environment.

Adenosine Triphosphate ; metabolism ; Evolution, Molecular ; Giardia lamblia ; enzymology ; Protozoan Proteins ; chemistry ; classification ; genetics ; Pyruvate, Orthophosphate Dikinase ; chemistry ; classification ; genetics

Plasmodium vivax malaria re-emerged in South Korea in 1993, and epidemics continue since then. We examined genetic variation in the region encompassing the apical membrane antigen-1 (PvAMA-1) of the parasites by DNA sequencing of the 22 re-emerging P. vivax isolates. The genotype of the PvAMA-1, which was based on sequence data previously reported for the polymorphic regions, showed that two haplotypes were present at one polymorphic site. Compared with reported data, the two types, SKOR type I and type II, were similar to Chinese CH-10A and CH-05A isolates, respectively. Thus, the present study showed that two genotypes of AMA-1 genes coexist in the re-emerging Korean P. vivax.
Adult ; Aged ; Amino Acid Sequence ; Animals ; *Antigens, Protozoan ; Base Sequence ; Child ; Female ; Genotype ; Human ; Korea ; Malaria, Vivax/*genetics ; Male ; Membrane Proteins/chemistry/*genetics ; Middle Aged ; Molecular Sequence Data ; Polymorphism (Genetics) ; Protozoan Proteins/chemistry/*genetics ; Support, Non-U.S. Gov't

Plasmodium vivax, a protozoan malaria parasite of humans, represents a major public health concern in the Republic of Korea (= South Korea). However, little is known about the genetic properties and population structures of the P. vivax isolates circulating in South Korea. This article reviews known polymorphic genetic markers in South Korean isolates of P. vivax and briefly summarizes the current issues surrounding the gene and population structures of this parasite. The critical genetic characteristics of major antigens of the parasite, such as circumsporozoite protein (CSP), merozoite surface protein 1 (MSP-1) and MSP-3, Duffy binding protein (DBP), apical membrane antigen 1 (AMA-1), and GAM-1, are also discussed.
Amino Acid Sequence ; Animals ; Antigens, Protozoan/chemistry/*genetics ; Base Sequence ; Humans ; Malaria, Vivax/*parasitology ; Molecular Sequence Data ; Plasmodium vivax/chemistry/*genetics/isolation & purification ; *Polymorphism, Genetic ; Protozoan Proteins/chemistry/*genetics ; Republic of Korea ; Sequence Alignment

Protozoan ciliates are a group of unicellular eukaryotes. The special characteristics of stop codons usage in termination of protein biosynthesis in ciliates cells makes them an ideal model to study the mechanism of stop codon recognition of polypeptides release factors. To localize the functional positions of biomolecules in ciliates cell, we constructed a macronuclear artificial chromosome containing a gene encoding red fluorescence protein (EoMAC_R) based on the structural characteristics of ciliates chromosome. Three factors, L11, eRF1a, and eRF3 that are involved in termination process of protein synthesis were colocalized in Euplotes octocarinatus cells by using novel EoMAC_R and the previously constructed EoMAC_G. The results indicated that protein synthesis mainly occurred inside the "C" shape macronucleus, suggesting that EoMAC could be a useful tool for localizing biomolecules in ciliates cell.
Chromosomes, Artificial ; Codon, Terminator ; metabolism ; Euplotes ; chemistry ; Peptide Termination Factors ; analysis ; genetics ; metabolism ; Peptides ; metabolism ; Protein Biosynthesis ; genetics ; Protozoan Proteins ; analysis ; genetics ; Ribosomal Proteins ; analysis ; genetics

Recombinant antigenic proteins of Toxoplasma gondii are alternative source of antigens which are easily obtainable for serodiagnosis of toxoplasmosis. In this study, highly antigenic secretory organellar proteins, dense granular GRA2 and GRA3, rhoptrial ROP2, and micronemal MIC2, were analyzed by bioinformatics approach to express as water-soluble forms of antigenic domains. The transmembrane region and disorder tendency of 4 secretory proteins were predicted to clone the genes into pGEX-4T-1 vector. Recombinant plasmids were transformed into BL21 (DE3) pLysS E. coli, and GST fusion proteins were expressed with IPTG. As a result, GST fusion proteins with GRA225-105, GRA339-138, ROP2324-561, and MIC21-284 domains had respectively higher value of IgG avidity. The rGST-GRA225-105 and rGST-GRA339-138 were soluble, while rGST-ROP2324-561 and rGST-MIC21-284 were not. GRA231-71, intrinsically unstructured domain (IUD) of GRA2, was used as a linker to enhance the solubility. The rGST-GRA231-71-ROP2324-561, a chimeric protein, appeared to be soluble. Moreover, rGST-GRA231-71-MIC21-284 was also soluble and had higher IgG avidity comparing to rGST-MIC21-284. These 4 highly expressed and water-soluble recombinant antigenic proteins may be promising candidates to improve the serodiagnosis of toxoplasmosis in addition to the major surface antigen of SAG1.
Animals ; Antibodies, Protozoan/immunology ; Antibody Affinity ; Antigens, Protozoan/chemistry/*diagnostic use/genetics/immunology ; *Gene Expression ; Immunoglobulin G/blood/immunology ; Mice, Inbred BALB C ; Recombinant Proteins/chemistry/*diagnostic use/genetics/immunology ; Serologic Tests/methods ; Solubility ; Toxoplasma/genetics/immunology/*metabolism ; Toxoplasmosis/diagnosis

OBJECTIVETo identify and clone a new full ORF gene of PyDyn (Plasmodium yoelii dynamin-like protein), and examine the protection of their expression products.

METHODUsing the P. yoelii Genome technology and RT-PCR.

RESULTSThe full ORF gene of PyDyn was amplified from mRNA of the erythrocytic stage of P. yoelii., three domains of PyDyn were expressed in E. coli., and the fairly positive immunogenicity of them was showed by IFA. The full ORF gene of PyDyn was 2,433 bp and encode 811 amino acids. Its Gene Bank access number is AF458071. PyDyn belongs to the dynamin-like protein family according to its property.

CONCLUSIONThe new full ORF gene of PyDyn is obtained and identified; their expressed domains are probably new candidates for malaria vaccine.

Amino Acid Sequence ; Animals ; Base Sequence ; Cloning, Molecular ; Dynamins ; genetics ; immunology ; Escherichia coli ; genetics ; Genes, Protozoan ; genetics ; immunology ; Malaria Vaccines ; immunology ; Molecular Sequence Data ; Plasmodium yoelii ; chemistry ; genetics ; Protozoan Proteins ; genetics ; immunology ; Vaccines, Synthetic ; immunology

Cryptosporidium and Giardia are 2 protozoan parasites responsible for waterborne diseases outbreaks worldwide. In order to assess the prevalence of these protozoans in drinking water samples in the northern part of Portugal and the risk of human infection, we have established a long term program aiming at pinpointing the sources of surface water, drinking water, and environmental contamination, working with the water-supply industry. Total 43 sources of drinking water samples were selected, and a total of 167 samples were analyzed using the Method 1623. Sensitivity assays regarding the genetic characterization by PCR and sequencing of the genes, 18S SSU rRNA, for Cryptosporidium spp. and beta,-giardin for G. duodenalis were set in the laboratory. According to the defined criteria, molecular analysis was performed over 4 samples. Environmental stages of the protozoa were detected in 25.7% (43 out of 167) of the water samples, 8.4% (14 out of 167) with cysts of Giardia, 10.2% (17 out of 167) with oocysts of Cryptosporidium and 7.2% (12 out of 167) for both species. The mean concentrations were 0.1-12.7 oocysts of Cryptosporidium spp. per 10 L and 0.1-108.3 cysts of Giardia duodenalis per 10 L. Our results suggest that the efficiency in drinking water plants must be ameliorated in their efficiency in reducing the levels of contamination. We suggest the implementation of systematic monitoring programs for both protozoa. To authors' knowledge, this is the first report evaluating the concentration of environmental stages of Cryptosporidium and Giardia in drinking water samples in the northern part of Portugal.
Animals ; Cryptosporidium/*isolation & purification ; Cytoskeletal Proteins/genetics ; DNA, Protozoan/chemistry/genetics ; DNA, Ribosomal/chemistry/genetics ; Genes, rRNA ; Giardia lamblia/*isolation & purification ; Humans ; Molecular Sequence Data ; Polymerase Chain Reaction ; Portugal ; Protozoan Proteins/genetics ; RNA, Protozoan/genetics ; RNA, Ribosomal, 18S/genetics ; Risk Assessment ; Sequence Analysis, DNA ; Water/*parasitology