Toxoplasma gondii is a single-celled parasite that infects nearly all warm-blooded animals, including humans (Montoya and Liesenfeld, 2004). It occurs worldwide and can persist for a lifetime in mammals. Humans get infected by eating undercooked meat of animals containing the tissue cysts of this parasite. In immune-competent individuals, T. gondii infection usually does not cause significant clinical symptoms, whereas in pregnant or immunocompromised individuals, T. gondii infection (toxoplasmosis) can cause more serious problems like abortion and even death (Dunn et al., 1999; Wang et al., 2017). A combination of pyrimethamine and sulfadiazine is usually used to treat toxoplasmosis, although it is generally inefficient and causes side effects (Alday and Doggett, 2017). Worse still, there is a lack of vaccines to prevent T. gondii infection in humans or animals.
Toxoplasma/enzymology* ; Animals ; Humans ; Toxoplasmosis ; Mitochondria/enzymology* ; Protozoan Proteins/metabolism*

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

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

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

This report describes the molecular characterization of the Tc8.2 gene of Trypanosoma cruzi. Both the Tc8.2 gene and its encoded protein were analyzed by bioinformatics, while Northern blot and RT-PCR were used for the transcripts. Besides, immunolocalization of recombinant protein was done by immunofluorescence and electron microscopy. Analysis indicated the presence of a single copy of Tc8.2 in the T. cruzi genome and 2-different sized transcripts in epimastigotes/amastigotes and trypomastigotes. Immunoblotting showed 70 and 80 kDa polypeptides in epimastigotes and trypomastigotes, respectively, and a differential pattern of immunolocalization. Overall, the results suggest that Tc8.2 is differentially expressed during the T. cruzi life cycle.
Amino Acid Sequence ; Chagas Disease/*parasitology ; Gene Expression Regulation, Developmental ; Humans ; Life Cycle Stages ; Molecular Sequence Data ; Protozoan Proteins/*genetics/metabolism ; Sequence Alignment ; Trypanosoma cruzi/*genetics/*growth & development/isolation & purification/metabolism

The parasite Plasmodium falciparum causes severe malaria and is the most dangerous to humans. However, it exhibits resistance to their drugs. Farnesyltransferase has been identified in pathogenic protozoa of the genera Plasmodium and the target of farnesyltransferase includes Ras family. Therefore, the inhibition of farnesyltransferase has been suggested as a new strategy for the treatment of malaria. However, the exact functional mechanism of this agent is still unknown. In addition, the effect of farnesyltransferase inhibitor (FTIs) on mitochondrial level of malaria parasites is not fully understood. In this study, therefore, the effect of a FTI R115777 on the function of mitochondria of P. falciparum was investigated experimentally. As a result, FTI R115777 was found to suppress the infection rate of malaria parasites under in vitro condition. It also reduces the copy number of mtDNA-encoded cytochrome c oxidase III. In addition, the mitochondrial membrane potential (DeltaPsim) and the green fluorescence intensity of MitoTracker were decreased by FTI R115777. Chloroquine and atovaquone were measured by the mtDNA copy number as mitochondrial non-specific or specific inhibitor, respectively. Chloroquine did not affect the copy number of mtDNA-encoded cytochrome c oxidase III, while atovaquone induced to change the mtDNA copy number. These results suggest that FTI R115777 has strong influence on the mitochondrial function of P. falciparum. It may have therapeutic potential for malaria by targeting the mitochondria of parasites.
Antimalarials/*pharmacology ; Enzyme Inhibitors/*pharmacology ; Farnesyltranstransferase/*antagonists & inhibitors/genetics/*metabolism ; Humans ; Malaria, Falciparum/drug therapy/*parasitology ; Mitochondria/*drug effects/metabolism ; Plasmodium falciparum/drug effects/*enzymology/genetics ; Protozoan Proteins/*antagonists & inhibitors/genetics/metabolism ; Quinolones/*pharmacology

The present study determined and compared the genetic diversity of Plasmodium falciparum strains infecting children living in 2 areas from Gabon with different malaria endemicity. Blood samples were collected from febrile children from 2008 to 2009 in 2 health centres from rural (Oyem) and urban (Owendo) areas. Genetic diversity was determined in P. falciparum isolates by analyzing the merozoite surface protein-1 (msp1) gene polymorphism using nested-PCR. Overall, 168 children with mild falciparum malaria were included. K1, Ro33, and Mad20 alleles were found in 110 (65.5%), 94 (55.9%), and 35 (20.8%) isolates, respectively, without difference according to the site (P>0.05). Allelic families' frequencies were comparable between children less than 5 years old from the 2 sites; while among the older children the proportions of Ro33 and Mad20 alleles were 1.7 to 2.0 fold higher at Oyem. Thirty-three different alleles were detected, 16 (48.5%) were common to both sites, and 10 out of the 17 specific alleles were found at Oyem. Furthermore, multiple infection carriers were frequent at Oyem (57.7% vs 42.2% at Owendo; P=0.04) where the complexity of infection was of 1.88 (+/-0.95) higher compared to that found at Owendo (1.55+/-0.75). Extended genetic diversity of P. falciparum strains infecting Gabonese symptomatic children and high multiplicity of infections were observed in rural area. Alleles common to the 2 sites were frequent; the site-specific alleles predominated in the rural area. Such distribution of the alleles should be taken into accounts when designing MSP1 or MSP2 malaria vaccine.
Child ; Child, Preschool ; Female ; Gabon ; *Gene Frequency ; Genetic Variation ; Genotype ; Humans ; Infant ; Malaria, Falciparum/*parasitology ; Male ; Merozoite Surface Protein 1/*genetics/metabolism ; Plasmodium falciparum/*genetics/metabolism ; Protozoan Proteins/*genetics/metabolism ; Rural Population ; Urban Population

Toxoplasma gondii is a eukaryotic parasite of the phylum Apicomplexa, which infects all warm-blood animals, including humans. In the present study, we examined sequence variation in dense granule 20 (GRA20) genes among T. gondii isolates collected from different hosts and geographical regions worldwide. The complete GRA20 genes were amplified from 16 T. gondii isolates using PCR, sequence were analyzed, and phylogenetic reconstruction was analyzed by maximum parsimony (MP) and maximum likelihood (ML) methods. The results showed that the complete GRA20 gene sequence was 1,586 bp in length among all the isolates used in this study, and the sequence variations in nucleotides were 0-7.9% among all strains. However, removing the type III strains (CTG, VEG), the sequence variations became very low, only 0-0.7%. These results indicated that the GRA20 sequence in type III was more divergence. Phylogenetic analysis of GRA20 sequences using MP and ML methods can differentiate 2 major clonal lineage types (type I and type III) into their respective clusters, indicating the GRA20 gene may represent a novel genetic marker for intraspecific phylogenetic analyses of T. gondii.
Animals ; Base Sequence ; Brazil ; China ; Deer ; *Genetic Variation ; Genotype ; Goats ; Humans ; Molecular Sequence Data ; Phylogeny ; Protozoan Proteins/*genetics/metabolism ; Sheep ; Swine ; Toxoplasma/classification/*genetics/isolation & purification/parasitology/physiology ; Toxoplasmosis/*parasitology ; Toxoplasmosis, Animal/*parasitology ; United States

Toxoplasma gondii is an opportunistic protozoan parasite that can infect almost all warm-blooded animals including humans with a worldwide distribution. Micronemes play an important role in invasion process of T. gondii, associated with the attachment, motility, and host cell recognition. In this research, sequence diversity in microneme protein 6 (MIC6) gene among 16 T. gondii isolates from different hosts and geographical regions and 1 reference strain was examined. The results showed that the sequence of all the examined T. gondii strains was 1,050 bp in length, and their A + T content was between 45.7% and 46.1%. Sequence analysis presented 33 nucleotide mutation positions (0-1.1%), resulting in 23 amino acid substitutions (0-2.3%) aligned with T. gondii RH strain. Moreover, T. gondii strains representing the 3 classical genotypes (Type I, II, and III) were separated into different clusters based on the locus of MIC6 using phylogenetic analyses by Bayesian inference (BI), maximum parsimony (MP), and maximum likelihood (ML), but T. gondii strains belonging to ToxoDB #9 were separated into different clusters. Our results suggested that MIC6 gene is not a suitable marker for T. gondii population genetic studies.
Amino Acid Sequence ; Animals ; Base Sequence ; Cats ; Cell Adhesion Molecules/chemistry/*genetics/metabolism ; Deer ; *Genetic Variation ; Genotype ; Goats ; Humans ; Molecular Sequence Data ; Phylogeny ; Protozoan Proteins/chemistry/*genetics/metabolism ; Sequence Alignment ; Sheep ; Swine ; Toxoplasma/classification/*genetics/isolation & purification/physiology ; Toxoplasmosis/*parasitology ; Toxoplasmosis, Animal/*parasitology