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

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

Microsporidia are eukaryotic organisms that cause zoonosis and are major opportunistic pathogens in HIV-positive patients. However, there is increasing evidence that these organisms can also cause gastrointestinal and ocular infections in immunocompetent individuals. In Korea, there have been no reports on human infections with microsporidia to date. In the present study, we used real-time PCR and nucleotide sequencing to detect Encephalitozoon intestinalis infection in seven of 139 human diarrheal stool specimens (5%) and Encephalitozoon hellem in three of 34 farm soil samples (8.8%). Genotype analysis of the E. hellem isolates based on the internal transcribed spacer 1 and polar tube protein genes showed that all isolates were genotype 1B. To our knowledge, this is the first report on human E. intestinalis infection in Korea and the first report revealing farm soil samples as a source of E. hellem infection. Because microsporidia are an important public health issue, further large-scale epidemiological studies are warranted.
AIDS-Related Opportunistic Infections/parasitology ; Adolescent ; Adult ; Aged ; Agriculture ; Base Sequence ; Child ; Child, Preschool ; DNA, Intergenic/genetics ; DNA, Protozoan/genetics ; Encephalitozoon/*genetics/*isolation & purification ; Encephalitozoonosis/*epidemiology ; Feces/*parasitology ; Female ; Fungal Proteins/genetics ; Humans ; Infant ; Infant, Newborn ; Male ; Middle Aged ; Molecular Typing ; Real-Time Polymerase Chain Reaction ; Republic of Korea/epidemiology ; Sequence Alignment ; Sequence Analysis, DNA ; Soil/*parasitology ; Young Adult

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

Giardia is a major public health concern and considered as reemerging in industrialized countries. The present study investigated the prevalence of giardiosis in 202 sheltered dogs using PCR. The infection rate was 33.2% (67/202); Gyeongsangbuk-do and Daejeon showed 25.7% (39/152, P<0.0001) and 56% (28/50), respectively. The prevalence of infected female dogs (46.7%, P<0.001) was higher than in male dogs (21.8%). A higher prevalence (43.5%, P<0.0001) was observed in mixed breed dogs than purebred (14.1%). Although most of the fecal samples collected were from dogs of > or =1 year of age which showed only 27.4% positive rate, 61.8% (P<0.001) of the total samples collected from young animals (<1 year of age) were positive for G. intestinalis. A significantly higher prevalence in symptomatic dogs (60.8%, P<0.0001) was observed than in asymptomatic dogs (23.8%). Furthermore, the analysis of nucleotide sequences of the samples revealed that G. intestinalis Assemblages A and C were found in the feces of dogs from Gyeongsangbuk-do and Daejeon. Since G. intestinalis Assemblage A has been known to infect humans, our results suggest that dogs can act as an important reservoir of giardiosis in Korea. Hence, hygienic management should be given to prevent possible transmission to humans.
Animals ; Base Sequence ; Dog Diseases/epidemiology/*parasitology ; Dogs ; Feces/parasitology ; Female ; Genotype ; Giardia/classification/genetics/*isolation & purification ; Giardiasis/epidemiology/parasitology/*veterinary ; Male ; Molecular Sequence Data ; Protozoan Proteins/genetics ; Republic of Korea/epidemiology

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