OBJECTIVE: To analyze the RNA binding protein of Toxoplasma gondii (TgDDX39) using bioinformatics technology, and to evaluate the immunogenicity of TgDDX39, so as to provide insights into development of toxoplasmosis vaccines. METHODS: The amino acid sequences of TgDDX39 were retrieved from the ToxoDB database, and the physicochemical properties, transmembrane structure domain, signal peptide sites, post-translational modification sites, coils, secondary and tertiary structures, hydrophobicity, and antigenic epitopes of the TgDDX39 protein were predicted using online bioinformatics tools, incluiding ProtParam, TMHMM 2.0, SignalP 5.0, NetPhos 3.1, COILS, SOPMA, Phyre2, ProtScale, ABCpred, SYFPEITHI and DNA-STAR. RESULTS: TgDDX39 protein was predicted to be an unstable hydrophilic protein with the molecular formula of C₂₁₇₃H₃₄₅₈N₅₉₈O₆₆₁S₁₈, which contained 434 amino acids and had an estimated molecular weight of 49.1 kDa and a theoretical isoelectric point of 5.55. The protein was predicted to have an extremely low possibility of signal peptides, without transmembrane regions, and contain 27 phosphorylation sites. The β turn and random coils accounted for 39.63% of the secondary structure of the TgDDX39 protein, and a coiled helix tended to produce in one site. In addition, the TgDDX39 protein contained multiple B and T cell antigenic epitopes. CONCLUSIONS Bioinformatics analyses predict that TgDDX39 protein has high immunogenicity and contains multiple antigenic epitopes. TgDDX39 protein is a potential candidate antigen for vaccine development.
Humans ; Toxoplasma/metabolism* ; Toxoplasmosis/prevention & control* ; Vaccines ; Epitopes, T-Lymphocyte ; Computational Biology ; Protozoan Proteins/chemistry*

The sodium hydrogen exchanger 1 (NHE1), which functions in maintaining the ratio of Na+ and H+ ions, is widely distributed in cell plasma membranes. It plays a prominent role in pH balancing, cell proliferation, differentiation, adhesion, and migration. However, its exact subcellular location and biological functions in Toxoplasma gondii are largely unclear. In this study, we cloned the C-terminal sequence of T. gondii NHE1 (TgNHE1) incorporating the C-terminal peptide of NHE1 (C-NHE1) into the pGEX4T-1 expression plasmid. The peptide sequence was predicted to have good antigenicity based on the information obtained from an immune epitope database. After induction of heterologous gene expression with isopropyl-b-D-thiogalactoside, the recombinant C-NHE1 protein successfully expressed in a soluble form was purified by glutathione sepharose beads as an immunogen for production of a rabbit polyclonal antiserum. The specificity of this antiserum was confirmed by western blotting and immunofluorescence. The antiserum could reduce T. gondii invasion into host cells, indicated by the decreased TgNHE1 expression in T. gondii parasites that were pre-incubated with antiserum in the process of cell entry. Furthermore, the antiserum reduced the virulence of T. gondii parasites to host cells in vitro, possibly by blocking the release of Ca2+. In this regard, this antiserum has potential to be a valuable tool for further studies of TgNHE1.
Animals ; Cell Line ; Immune Sera/genetics/immunology/*metabolism ; Male ; Mice ; Protozoan Proteins/genetics/*metabolism ; Rabbits ; Recombinant Proteins/immunology ; Sheep ; Sodium-Hydrogen Antiporter/genetics/immunology/*metabolism ; Toxoplasma/genetics/immunology/*metabolism ; Toxoplasmosis/parasitology/prevention & control

Endoplasmic Reticulum ; genetics ; metabolism ; Plasmodium berghei ; genetics ; metabolism ; Plasmodium falciparum ; genetics ; metabolism ; Protozoan Proteins ; genetics ; metabolism

Meiotic recombination is carried out through a specialized pathway for the formation and repair of DNA double-strand breaks (DSBs) made by the Spo11 protein. The present study shed light on the functional role of cyclin, CYC2, in Tetrahymena thermophila which has transcriptionally high expression level during meiosis process. Knocking out the CYC2 gene results in arrest of meiotic conjugation process at 2.5-3.5 h after conjugation initiation, before the meiosis division starts, and in company with the absence of DSBs. To investigate the underlying mechanism of this phenomenon, a complete transcriptome profile was performed between wild-type strain and CYC2 knock-out strain. Functional analysis of RNA-Seq results identifies related differentially expressed genes (DEGs) including SPO11 and these DEGs are enriched in DNA repair/mismatch repair (MMR) terms in homologous recombination (HR), which indicates that CYC2 could play a crucial role in meiosis by regulating SPO11 and participating in HR.
Cell Cycle Checkpoints ; Cyclins ; genetics ; metabolism ; DNA Breaks, Double-Stranded ; DNA Mismatch Repair ; DNA Repair ; Endodeoxyribonucleases ; genetics ; metabolism ; Homologous Recombination ; Meiosis ; Microscopy, Fluorescence ; Phenotype ; Protozoan Proteins ; genetics ; metabolism ; Real-Time Polymerase Chain Reaction ; Sequence Analysis, RNA ; Tetrahymena thermophila ; genetics ; metabolism ; Transcriptome

The increasing prevalence of Toxoplasma gondii infection in the human population in the Republic of Korea (= Korea) is due to various reasons such as an increase in meat consumption. However, the importance of cats in transmitting T. gondii infection through oocysts to humans has seldom been assessed. A total of 300 fecal samples of stray cats captured around Seoul from June to August 2013 were examined for T. gondii B1 gene (indicating the presence of oocysts) using nested-PCR. Fourteen (4.7%) of 300 cats examined were positive for B1 gene. Female cats (7.5%) showed a higher prevalence than male cats (1.4%). Cats younger than 3 months (5.5%) showed a higher prevalence than cats (1.5%) older than 3 months. For laboratory passage of the positive samples, the fecal suspension (0.2 ml) of B1 gene positive cats was orally inoculated into experimental mice. Brain tissues of the mice were obtained after 40 days and examined for the presence of tissue cysts. Two isolates were successfully passaged (designated KNIH-1 and KNIH-2) and were molecularly analyzed using the SAG5D and SAG5E gene sequences. The SAG5D and SAG5E gene sequences showed high homologies with the ME49 strain (less virulent strain). The results indicated the importance of stray cats in transmitting T. gondii to humans in Korea, as revealed by detection of B1 gene in fecal samples. T. gondii isolates from cats were successfully passaged in the laboratory for the first time in Korea.
Animals ; Cat Diseases/diagnosis/epidemiology/*parasitology ; Cats ; Feces/*parasitology ; Female ; Genotype ; Humans ; Male ; Mice ; Protozoan Proteins/genetics ; Seoul/epidemiology ; Toxoplasma/genetics/*isolation & purification ; Toxoplasmosis/epidemiology/parasitology/transmission ; Toxoplasmosis, Animal/diagnosis/epidemiology/*parasitology

Toxoplasma gondii, an obligate intracellular protozoan parasite of the phylum Apicomplexa, can infect all warm-blooded vertebrates, including humans, livestock, and marine mammals. The aim of this study was to investigate whether superoxide dismutase (SOD) of T. gondii can be used as a new marker for genetic study or a potential vaccine candidate. The partial genome region of the SOD gene was amplified and sequenced from 10 different T. gondii isolates from different parts of the world, and all the sequences were examined by PCR-RFLP, sequence analysis, and phylogenetic reconstruction. The results showed that partial SOD gene sequences ranged from 1,702 bp to 1,712 bp and A + T contents varied from 50.1% to 51.1% among all examined isolates. Sequence alignment analysis identified total 43 variable nucleotide positions, and these results showed that 97.5% sequence similarity of SOD gene among all examined isolates. Phylogenetic analysis revealed that these SOD sequences were not an effective molecular marker for differential identification of T. gondii strains. The research demonstrated existence of low sequence variation in the SOD gene among T. gondii strains of different genotypes from different hosts and geographical regions.
Amino Acid Sequence ; Animals ; Base Sequence ; Cats ; *Genetic Variation ; Goats ; Humans ; Molecular Sequence Data ; Phylogeny ; Protozoan Proteins/chemistry/*genetics/metabolism ; Sequence Alignment ; Sheep ; Superoxide Dismutase/chemistry/*genetics/metabolism ; Toxoplasma/classification/*enzymology/genetics/isolation & purification ; Toxoplasmosis/*parasitology ; Toxoplasmosis, Animal/*parasitology

The objective of this study was to genetically characterize isolates of Giardia duodenalis and to determine if zoonotic potential of G. duodenalis could be found in stray cats from urban and suburban environments in Guangzhou, China. Among 102 fresh fecal samples of stray cats, 30 samples were collected in Baiyun district (urban) and 72 in Conghua district (suburban). G. duodenalis specimens were examined using light microscopy, then the positive specimens were subjected to PCR amplification and subsequent sequencing at 4 loci such as glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi), beta-giardin (bg), and small subunit ribosomal RNA (18S rRNA) genes. The phylogenetic trees were constructed using obtained sequences by MEGA5.2 software. Results show that 9.8% (10/102) feline fecal samples were found to be positive by microscopy, 10% (3/30) in Baiyun district and 9.7% (7/72) in Conghua district. Among the 10 positive samples, 9 were single infection (8 isolates, assemblage A; 1 isolate, assemblage F) and 1 sample was mixed infection with assemblages A and C. Based on tpi, gdh, and bg genes, all sequences of assemblage A showed complete homology with AI except for 1 isolate (CHC83). These findings not only confirmed the occurrence of G. duodenalis in stray cats, but also showed that zoonotic assemblage A was found for the first time in stray cats living in urban and suburban environments in China.
Animals ; Cat Diseases/*parasitology ; Cats ; China ; Cluster Analysis ; DNA, Protozoan/chemistry/genetics ; DNA, Ribosomal/chemistry/genetics ; Feces/parasitology ; Giardia lamblia/*classification/cytology/genetics/*isolation & purification ; Giardiasis/parasitology/*veterinary ; Microscopy ; Molecular Sequence Data ; Phylogeny ; Protozoan Proteins/genetics ; RNA, Ribosomal, 18S/genetics ; Sequence Analysis, DNA

The objective of this study was to genetically characterize isolates of Giardia duodenalis and to determine if zoonotic potential of G. duodenalis could be found in stray cats from urban and suburban environments in Guangzhou, China. Among 102 fresh fecal samples of stray cats, 30 samples were collected in Baiyun district (urban) and 72 in Conghua district (suburban). G. duodenalis specimens were examined using light microscopy, then the positive specimens were subjected to PCR amplification and subsequent sequencing at 4 loci such as glutamate dehydrogenase (gdh), triose phosphate isomerase (tpi), beta-giardin (bg), and small subunit ribosomal RNA (18S rRNA) genes. The phylogenetic trees were constructed using obtained sequences by MEGA5.2 software. Results show that 9.8% (10/102) feline fecal samples were found to be positive by microscopy, 10% (3/30) in Baiyun district and 9.7% (7/72) in Conghua district. Among the 10 positive samples, 9 were single infection (8 isolates, assemblage A; 1 isolate, assemblage F) and 1 sample was mixed infection with assemblages A and C. Based on tpi, gdh, and bg genes, all sequences of assemblage A showed complete homology with AI except for 1 isolate (CHC83). These findings not only confirmed the occurrence of G. duodenalis in stray cats, but also showed that zoonotic assemblage A was found for the first time in stray cats living in urban and suburban environments in China.
Animals ; Cat Diseases/*parasitology ; Cats ; China ; Cluster Analysis ; DNA, Protozoan/chemistry/genetics ; DNA, Ribosomal/chemistry/genetics ; Feces/parasitology ; Giardia lamblia/*classification/cytology/genetics/*isolation & purification ; Giardiasis/parasitology/*veterinary ; Microscopy ; Molecular Sequence Data ; Phylogeny ; Protozoan Proteins/genetics ; RNA, Ribosomal, 18S/genetics ; Sequence Analysis, DNA

The aim of the study was to explore the possible molecular markers of chloroquine resistance in Plasmodium vivax isolates in Thailand. A total of 30 P. vivax isolates were collected from a malaria endemic area along the Thai-Myanmar border in Mae Sot district of Thailand. Dried blood spot samples were collected for analysis of Pvmdr1 and Pvcrt-o polymorphisms. Blood samples (100 mul) were collected by finger-prick for in vitro chloroquine susceptibility testing by schizont maturation inhibition assay. Based on the cut-off IC50 of 100 nM, 19 (63.3%) isolates were classified as chloroquine resistant P. vivax isolates. Seven non-synonymous mutations and 2 synonymous were identified in Pvmdr1 gene. Y976F and F1076L mutations were detected in 7 (23.3%) and 16 isolates (53.3%), respectively. Analysis of Pvcrt-o gene revealed that all isolates were wild-type. Our results suggest that chloroquine resistance gene is now spreading in this area. Monitoring of chloroquine resistant molecular markers provide a useful tool for future control of P. vivax malaria.
Amino Acid Substitution ; Antimalarials/*pharmacology ; Chloroquine/*pharmacology ; *Drug Resistance ; Humans ; Inhibitory Concentration 50 ; Malaria, Vivax/*parasitology ; Membrane Transport Proteins/*genetics ; Multidrug Resistance-Associated Proteins/*genetics ; Mutation, Missense ; Myanmar ; Parasitic Sensitivity Tests ; Plasmodium vivax/*drug effects/genetics ; Protozoan Proteins/*genetics ; Thailand

The aim of the study was to explore the possible molecular markers of chloroquine resistance in Plasmodium vivax isolates in Thailand. A total of 30 P. vivax isolates were collected from a malaria endemic area along the Thai-Myanmar border in Mae Sot district of Thailand. Dried blood spot samples were collected for analysis of Pvmdr1 and Pvcrt-o polymorphisms. Blood samples (100 mul) were collected by finger-prick for in vitro chloroquine susceptibility testing by schizont maturation inhibition assay. Based on the cut-off IC50 of 100 nM, 19 (63.3%) isolates were classified as chloroquine resistant P. vivax isolates. Seven non-synonymous mutations and 2 synonymous were identified in Pvmdr1 gene. Y976F and F1076L mutations were detected in 7 (23.3%) and 16 isolates (53.3%), respectively. Analysis of Pvcrt-o gene revealed that all isolates were wild-type. Our results suggest that chloroquine resistance gene is now spreading in this area. Monitoring of chloroquine resistant molecular markers provide a useful tool for future control of P. vivax malaria.
Amino Acid Substitution ; Antimalarials/*pharmacology ; Chloroquine/*pharmacology ; *Drug Resistance ; Humans ; Inhibitory Concentration 50 ; Malaria, Vivax/*parasitology ; Membrane Transport Proteins/*genetics ; Multidrug Resistance-Associated Proteins/*genetics ; Mutation, Missense ; Myanmar ; Parasitic Sensitivity Tests ; Plasmodium vivax/*drug effects/genetics ; Protozoan Proteins/*genetics ; Thailand