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*

Plasmodium vivax merozoite surface protein-1 (PvMSP1) gene codes for a major malaria vaccine candidate antigen. However, its polymorphic nature represents an obstacle to the design of a protective vaccine. In this study, we analyzed the genetic polymorphism and natural selection of the C-terminal 42 kDa fragment within PvMSP1 gene (Pv MSP142) from 77 P. vivax isolates, collected from imported cases of China-Myanmar border (CMB) areas in Yunnan province and the inland cases from Anhui, Yunnan, and Zhejiang province in China during 2009–2012. Totally, 41 haplotypes were identified and 30 of them were new haplotypes. The differences between the rates of non-synonymous and synonymous mutations suggest that PvMSP142 has evolved under natural selection, and a high selective pressure preferentially acted on regions identified of PvMSP133. Our results also demonstrated that PvMSP142 of P. vivax isolates collected on China-Myanmar border areas display higher genetic polymorphisms than those collected from inland of China. Such results have significant implications for understanding the dynamic of the P. vivax population and may be useful information towards China malaria elimination campaign strategies.
China ; Genetic Variation* ; Haplotypes ; Malaria ; Merozoite Surface Protein 1* ; Merozoites* ; Myanmar ; Plasmodium vivax* ; Plasmodium* ; Polymorphism, Genetic ; Selection, Genetic* ; Silent Mutation

Vivax malaria reemerged in Korea in 1993 and the outbreak has been continued with fluctuating numbers of annual indigenous cases. Understanding the nature of the genetic population of Plasmodium vivax circulating in Korea is beneficial for the knowledge of the nationwide parasite heterogeneity and in the implementation of malaria control programs in the country. Previously, we analyzed polymorphic nature of merozoite surface protein-1 (MSP-1) and MSP-3α in Korean P. vivax population and identified the Korean P. vivax population has been diversifying rapidly, with the appearance of parasites with new genetic subtypes, despite the recent reduction of the disease incidence. In the present study, we developed simple PCR-RFLP methods for rapid subtyping of MSP-1 and MSP-3α of Korean P. vivax isolates. These PCR-RFLP methods were able to easily distinguish each subtype of Korean P. vivax MSP-1 and MSP-3α with high accuracy. The PCR-RFLP subtyping methods developed here would be easily applied to massive epidemiological studies for molecular surveillance to understand genetic population of P. vivax and to supervise the genetic variation of the parasite circulating in Korea.
Epidemiologic Studies ; Genetic Variation ; Incidence ; Korea ; Malaria ; Malaria, Vivax ; Merozoite Surface Protein 1 ; Parasites ; Plasmodium vivax* ; Plasmodium* ; Population Characteristics

Malaria is an infectious disease affecting humans, which is transmitted by the bite of Anopheles mosquitoes harboring sporozoites of parasitic protozoans belonging to the genus Plasmodium. Despite past achievements to control the protozoan disease, malaria still remains a significant health threat up to now. In this study, we cloned and characterized the full-unit Plasmodium yoelii genes encoding merozoite surface protein 1 (MSP1), circumsporozoite protein (CSP), and Duffy-binding protein (DBP), each of which can be applied for investigations to obtain potent protective vaccines in the rodent malaria model, due to their specific expression patterns during the parasite life cycle. Recombinant fragments corresponding to the middle and C-terminal regions of PyMSP1 and PyCSP, respectively, displayed strong reactivity against P. yoelii-infected mice sera. Specific native antigens invoking strong humoral immune response during the primary and secondary infections of P. yoelii were also abundantly detected in experimental ICR mice. The low or negligible parasitemia observed in the secondary infected mice was likely to result from the neutralizing action of the protective antibodies. Identification of these antigenic proteins might provide the necessary information and means to characterize additional vaccine candidate antigens, selected solely on their ability to produce the protective antibodies.
Animals ; Anopheles ; Antibodies ; Clone Cells ; Coinfection ; Communicable Diseases ; Culicidae ; Humans ; Immunity, Humoral ; Life Cycle Stages ; Malaria ; Merozoite Surface Protein 1* ; Mice ; Mice, Inbred ICR ; Parasitemia ; Parasites ; Plasmodium yoelii* ; Plasmodium* ; Rodentia ; Sporozoites ; Vaccines

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

Merozoite surface proteins (MSPs) of malaria parasites play critical roles during the erythrocyte invasion and so are potential candidates for malaria vaccine development. However, because MSPs are often under strong immune selection, they can exhibit extensive genetic diversity. The gene encoding the merozoite surface protein-3 (MSP-3) of Plasmodium falciparum displays 2 allelic types, K1 and 3D7. In Thailand, the allelic frequency of the P. falciparum msp-3 gene was evaluated in a single P. falciparum population in Tak at the Thailand and Myanmar border. However, no study has yet looked at the extent of genetic diversity of the msp-3 gene in P. falciparum populations in other localities. Here, we genotyped the msp-3 alleles of 63 P. falciparum samples collected from 5 geographical populations along the borders of Thailand with 3 neighboring countries (Myanmar, Laos, and Cambodia). Our study indicated that the K1 and 3D7 alleles coexisted, but at different proportions in different Thai P. falciparum populations. K1 was more prevalent in populations at the Thailand-Myanmar and Thailand-Cambodia borders, whilst 3D7 was more prevalent at the Thailand-Laos border. Global analysis of the msp-3 allele frequencies revealed that proportions of K1 and 3D7 alleles of msp-3 also varied in different continents, suggesting the divergence of malaria parasite populations. In conclusion, the variation in the msp-3 allelic patterns of P. falciparum in Thailand provides fundamental knowledge for inferring the P. falciparum population structure and for the best design of msp-3 based malaria vaccines.
Antigens, Protozoan/*genetics ; *Gene Frequency ; *Genetic Variation ; Genotype ; Humans ; Malaria, Falciparum/epidemiology/*parasitology ; Plasmodium falciparum/classification/*genetics/isolation & purification ; Polymorphism, Genetic ; Protozoan Proteins/*genetics ; Thailand/epidemiology

The relationship between anti-Plasmodium vivax circumsporozoite protein (CSP) antibody levels and the prevalence of malaria in epidemic areas of South Korea was evaluated. Blood samples were collected from inhabitants of Gimpo-si (city), Paju-si, and Yeoncheon-gun (county) in Gyeonggi-do (province), as well as Cheorwon-gun in Gangwon-do from November to December 2004. Microscopic examinations were used to identify malaria parasites. ELISA was used to quantitate anti-circumsporozoite protein (CSP) antibodies against P. vivax. A total of 1,774 blood samples were collected. The overall CSP-ELISA-positive rate was 7.7% (n=139). The annual parasite incidences (APIs) in these areas gradually decreased from 2004 to 2005 (1.09 and 0.80, respectively). The positive rate in Gimpo (10.4%, 44/425) was the highest identified by CSP-ELISA. The highest API was found in Yeoncheon, followed by Cheorwon, Paju, and Gimpo in both years. The positive rates of CSP-ELISA were closely related to the APIs in the study areas. These results suggest that seroepidemiological studies based on CSP may be helpful in estimating the malaria prevalence in certain areas. In addition, this assay can be used to establish and evaluate malaria control and eradication programs in affected areas.
Adolescent ; Adult ; Antibodies, Protozoan/*blood ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Incidence ; Malaria, Vivax/*blood/*epidemiology/parasitology ; Male ; Middle Aged ; Plasmodium vivax/*immunology/physiology ; Prevalence ; Protozoan Proteins/immunology ; Republic of Korea/epidemiology ; Seroepidemiologic Studies ; Young Adult