Construction of luciferase-expressing Neospora caninum and drug screening.

BACKGROUND: Neospora caninum is an apicomplexan parasite that is particularly responsible for abortions in cattle and neuromuscular disease in dogs. Due to the limited effectiveness of currently available drugs, there is an urgent need for new therapeutic approaches to control neosporosis. Luciferase-based assays are potentially powerful tools in the search for antiprotozoal compounds, permitting the development of faster and more automated assays. The aim of this study was to construct a luciferase-expressing N. caninum and evaluate anti-N. caninum drugs. METHODS: Luciferase-expressing N. caninum (Nc1-Luc) was constructed using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR/Cas9). After testing the luciferase expression and phenotype of the Nc1-Luc strains, the drug sensitivity of Nc1-Luc strains was determined by treating them with known positive or negative drugs and calculating the half-maximal inhibitory concentration (IC50). The selective pan-rapidly accelerated fibrosarcoma (pan-RAF) inhibitor TAK-632 was then evaluated for anti-N. caninum effects using Nc1-Luc by luciferase activity reduction assay and other in vitro and in vivo studies. RESULTS: The phenotypes and drug sensitivity of Nc1-Luc strains were consistent with those of the parental strains Nc1, and Nc1-Luc strains can be used to determine the IC50 for anti-N. caninum drugs. Using the Nc1-Luc strains, TAK-632 showed promising activity against N. caninum, with an IC50 of 0.6131 µM and a selectivity index (SI) of 62.53. In vitro studies demonstrated that TAK-632 inhibited the invasion, proliferation, and division of N. caninum tachyzoites. In vivo studies showed that TAK-632 attenuated the virulence of N. caninum in mice and significantly reduced the parasite burden in the brain. CONCLUSIONS: In conclusion, a luciferase-expressing N. caninum strain was successfully constructed, which provides an effective tool for drug screening and related research on N. caninum. In addition, TAK-632 was found to inhibit the growth of N. caninum, which could be considered as a candidate lead compound for new therapeutics for neosporosis.

Identification of specific antigens between Toxoplasma gondii and Neospora caninum and application of potential diagnostic antigen TgGRA54.

Toxoplasma gondii is a zoonotic parasite that is very common in livestock. Meat products from livestock infected with T. gondii are one of the important transmission routes of toxoplasmosis. Rapid and reliable diagnosis is a prerequisite for the prevention and control of toxoplasmosis. Neospora caninum and T. gondii are similar in morphology and life history, and there are a large number of cross antigens between them, making clinical diagnosis of toxoplasmosis more difficult. In this study, immunoprecipitation-mass spectrometry (IP-MS) was used to screen for T. gondii-specific antigens, and the specific antigen was cloned and expressed in Escherichia coli. The specific antigen was then used to establish an indirect ELISA diagnostic method. A total of 241 specific antigens of T. gondii and 662 cross antigens between T. gondii and N. caninum were screened by IP-MS. Through bioinformatics analysis and homology comparison, seven proteins were selected for gene cloning and prokaryotic expression, and the most suitable antigen, TgGRA54, was selected to establish an indirect ELISA for T. gondii. Compared with the indirect immunofluorescent antibody test (IFAT), the positive coincidence rate of the ELISA based on rTgGRA54 was 100% (72/72) and the negative coincidence rate was 80.95% (17/21). The indirect ELISA method based on TgGRA54 recombinant protein was established to detect T. gondii antibodies in bovine sera, and the recombinant protein reacted well with T. gondii positive sera from sheep, mouse, and swine, indicating that the recombinant protein is a good diagnostic antigen for T. gondii.

Effects of different drugs and hormone treatment on Toxoplasma gondii glutathione S-transferase 2.

BACKGROUND: Glutathione S-transferase (GST) in eukaryotic organisms has multiple functions such as detoxifying endogenous/exogenous harmful substances to protect cells from oxidative damage, participating in sterol synthesis and metabolism, and regulating signaling pathways. Our previous work identified an important GST protein in Toxoplasma that contributes to vesicle trafficking called TgGST2, the deletion of which significantly reduces the virulence of the parasite. Meanwhile, we considered that TgGST2 may also play a role in other pathways of parasite life activities. METHODS: The tertiary structures of TgGST2 as well as estradiol (E2) and progesterone (P4) were predicted by trRosetta and Autodock Vina software, the binding sites were analyzed by PyMol's GetBox Plugin, and the binding capacity was evaluated using Discovery Studio plots software. We examined the influence of E2 and P4 on TgGST2 via glutathione S-transferase enzyme activity and indirect immunofluorescence assay (IFA) and through the localization observation of TgGST2 to evaluate its response ability in different drugs. RESULTS: TgGST2 could bind to exogenous E2 and P4, and that enzymatic activity was inhibited by the hormones in a concentration-dependent manner. Upon P4 treatment, the localization of TgGST2 changed from Golgi and vesicles to hollow circles, leading to abnormal localization of the molecular transporter Sortilin (VPS10) and microneme proteins (M2AP and MIC2), which ultimately affect the parasite life activities, but E2 had no significant effect. Moreover, diverse types of drugs had divergent effects on TgGST2, among which treatment with antifungal agents (voriconazole and clarithromycin), anticarcinogens (KU-60019, WYE-132 and SH5-07) and coccidiostats (dinitolmide and diclazuril) made the localization of TgGST2 appear in different forms, including dots, circles and rod shaped. CONCLUSIONS: Our study shows that TgGST2 plays a role in sterol treatment and can be affected by P4, which leads to deficient parasite motility. TgGST2 exerts divergent effects in response to the different properties of the drugs themselves. Its responsiveness to diverse drugs implies a viable target for the development of drugs directed against Toxoplasma and related pathogenic parasites.

Depletion of Toxoplasma adenine nucleotide translocator leads to defects in mitochondrial morphology.

BACKGROUND: Adenine nucleotide translocase (ANT) is a protein that catalyzes the exchange of ADP/ATP across the inner mitochondrial membrane. Beyond this, ANT is closely associated with cell death pathways and mitochondrial dysfunction. It is a potential therapeutic target for many diseases. The function of the ANT in Toxoplasma gondii is poorly understood. METHODS: The CRISPR/CAS9 gene editing tool was used to identify and study the function of the ANT protein in T. gondii. We constructed T. gondii ANT transgenic parasite lines, including endogenous tag strain, knockout strain and gene complement strain, to clarify the function and location of TgANT. Mitochondrial morphology was observed by immunofluorescence and transmission electron microscopy. RESULTS: Toxoplasma gondii was found to encode an ANT protein, which was designated TgANT. TgANT localized to the inner mitochondrial membrane. The proliferation of the Δant strain was significantly reduced. More important, depletion of TgANT resulted in significant changes in the morphology and ultrastructure of mitochondria, abnormal apicoplast division and abnormal cytoskeletal daughter budding. In addition, the pathogenicity of the Δant strain to mice was significantly reduced. CONCLUSIONS: Altogether, we identified and characterized the ANT protein of T. gondii. Depletion of TgANT inhibited parasite growth and impaired apicoplast and mitochondrial biogenesis, as well as abnormal parasite division, suggesting TgANT is important for parasite growth.

Toxoplasma gondii glutathione S-transferase 2 plays an important role in partial secretory protein transport.

Toxoplasma gondii is an apicomplexan parasite, which has three unique secretory organelles: micronemes, rhoptries, and dense granules. Almost all the secreted proteins are transported through the endoplasmic reticulum (ER) and Golgi system to function in their respective destination by accurate targeting and packaging. Glutathione S-transferase (GST) is a supergene family enzyme that has multiple functions, which include regulation of cell proliferation and death signaling pathways, and participation in transportation and metabolism in mammal cells. However, the role of GST in Toxoplasma gondii has not been explained. In this study, we identified three GST proteins in T gondii, of which GST2 acts as a membrane protein that localizes to the Golgi-endosomal system and colocalizes with proteins involved in vesicle transport as well, including synaptobrevin, putative sortilin (VPS10), Rab5 and Rab6, which function as vesicle transport factors. Moreover, the loss of TgGST2 leads to Rab5 and Rab6 distribution of discrete puncta, and incorrect localization and decreased expression of several secretory proteins, and to significantly reduced invasion capacity and virulence to mice. Consistent with its relation to vesicle transport proteins, the distribution of TgGST2 relies on post-Golgi trafficking. Overall, our findings demonstrated that TgGST2 contributes to vesicle trafficking and plays a critical role in parasite lytic cycle.

Selenium deficiency inhibits differentiation and immune function and imbalances the Th1/Th2 of dendritic cells.

Selenium (Se) deficiency inhibits immune cell differentiation, affects immune response, and leads to cellular and humoral immune dysfunction. However, the impact of Se deficiency on the differentiation and Th1/Th2 balance of dendritic cells is still unclear. In this study, we replicated a model of Se-deficient chickens by feeding the chickens with a low-Se diet (i.e., the content of Se is 0.008 mg per kg diet). On this basis, we explored the effect of Se deficiency on the differentiation of chicken dendritic cells by induction culture of peripheral blood monocyte cells. We induced chicken dendritic cells by incubating mononuclear cells with a 100 ng mL-1 recombinant chicken granulocyte-macrophage colony-stimulating factor and 20 ng mL-1 recombinant chicken IL-4 for total 7 days. The results showed that Se deficiency decreased the expression of cell-surface markers including CD11c, CD40, CD86, and MHC II. Furthermore, we analyzed the cytokine profiles using real-time quantitative PCR and ELISA. The results indicated that Se deficiency inhibited the expression of selenoproteins and changed the secretion of IL-10, IL-12p40, and IFN-γ. Additionally, Se deficiency weakened the ability of dendritic cells to stimulate the proliferation of mixed allogeneic lymphocytes. In conclusion, Se deficiency suppressed the differentiation and immune function of chicken dendritic cells by down-regulating the expression of CD11c, CD40, CD86, MHC II, and selenoproteins. The result also showed that the Th1/Th2 imbalance was induced by enhancing the secretion of Th1-type cytokine IL-12p40 and IFN-γ and reducing that of Th2-type cytokine IL-10. Our findings contribute to understanding the mechanism of Se deficiency in the differentiation and immune function of chicken dendritic cells.

Involvement of mitochondrial pathway in environmental metal pollutant lead-induced apoptosis of chicken liver: perspectives from oxidative stress and energy metabolism.

This study aimed to investigate the possible mechanisms of environmental metal pollutant lead (Pb)-induced apoptosis in chicken. Forty 8-day-old healthy chickens were randomly assigned to two groups (n = 20/group) after raising standard commercial diet and drinking water for 1 week: including control group and Pb group ((CH3COO)2Pb 350 mg/L of drinking water); the chickens were given euthanasia and collected livers at 90 days. A significant increase of apoptosis rate were found in Pb group and Pb induced obvious ultrastructural changes of chicken liver. The mRNA levels of glycometabolism key enzymes were significantly lower in Pb group than those in controls. Higher levels of malondialdehyde (MDA) and nitric oxide (NO) were observed in Pb group; the activities of antioxidant enzymes and ATPases were significantly lower in Pb group than those in controls, while the inducible nitric oxide synthase (iNOS) activity was on the contrary. The mRNA and protein levels of pro-apoptotic genes were all lower in Pb group than those in controls. Altogether, Pb-induced mitochondrial swelling and nuclear chromatin condensation, oxidative stress, energy metabolism disorder, thereby lead to apoptosis via mitochondrial pathway in chicken liver, suggesting that Pb-induced mitochondrial pathway apoptosis plays an important role in the mechanisms of Pb cytotoxicity to chicken liver.

Down-regulation of microRNA-155 promotes selenium deficiency-induced apoptosis by tumor necrosis factor receptor superfamily member 1B in the broiler spleen.

The aim of this work was to explore the microRNA profile and the effect of microRNA-155 on apoptosis in the spleen of selenium-deficient broilers. We replicated the splenic-apoptotic model in selenium-deficient broilers. In vitro, microRNA-155 oligonucleotides were transfected into lymphocytes and subsequently treated with H2O2. We observed that selenium deficiency altered the microRNA profile and decreased the expression of microRNA-155 in the broiler spleens. Tumor necrosis factor receptor superfamily member 1B was verified as a target of microRNA-155 in the splenocytes. Morphological changes, increased levels of tumor necrosis factor receptor superfamily member 1B, c-Jun N-terminal kinase, Bak, Bax, Cyt-c, caspase9 and caspase3 and decreased levels of Bcl-2 demonstrated that selenium deficiency induced apoptosis in the spleen tissues. In vitro, microRNA-155 m inhibited the levels of ROS and reduced apoptosis compared with microRNA-155i in the lymphocytes. These results suggested that the reduced levels of microRNA-155 due to selenium deficiency could promote oxidative stress-induced apoptosis by increased tumor necrosis factor receptor superfamily member 1B in splenic cells.

Selenium accelerates chicken dendritic cells differentiation and affects selenoproteins expression.

Selenium (Se) promotes immune cell differentiation and improves immune response. Antigen-presenting cells such as dendritic cells (DCs) play an important role in immune system, however, the impact of Se on DCs is still unclear. In this study, we successfully induced and cultured chicken DCs from peripheral blood mononuclear cells by incubating mononuclear cells with 50 ng/mL recombinant chicken granulocyte-macrophage colony stimulating factor and 10 ng/mL recombinant chicken interleukin-4 for total 9 days. In + Se group, we added 10-7 mol/L sodium selenite from the first day of cell culture. The results showed that Se supplementation expedited and increased the expression of cell surface markers including CD11c, CD40, CD86, and MHC II. Principal component analysis showed that the expression of selenoproteins SelW, SelK, Dio3, GPX1, GPX2, SelN, SelS, SelH in chicken DCs was highly correlated, and SelW had highest correlation with the cell surface markers MHC II and CD11c. In conclusion, Se accelerates the differentiation and maturation of chicken DCs. Se regulates the differentiation and maturation of chicken DCs by selenoproteins. Selenoproteins has closely correlated to surface markers of chicken DCs.

Cadmium-induced endoplasmic reticulum stress in chicken neutrophils is alleviated by selenium.

Cadmium (Cd) decreases immune function and induces apoptosis of immune cells. Selenium (Se) can antagonize some metal element toxicity including Cd. To evaluate the cytotoxicity of Cd and the chemoprotective role of Se on bird neutrophils in vitro, we incubated chicken neutrophils cells with Cadmium chloride (CdCl2) (10-6M), Sodium selenite (Na2SeO3) (10-7M), and with a mixture of Na2SeO3 (10-7M) and CdCl2 (10-6M) for 12, 24, 36, and 48h. We found that Interleukin 1ß (IL-1ß), Interleukin 10 (IL-10), and interferon gamma (IFN-γ) increased and interleukin 17 (IL-17), interleukin 4 (IL-4) decreased significantly in the chicken neutrophils of the Cd treatment groups. Cd significantly increased the mRNA expression levels of nuclear factor kappaB (NF-κB), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor (TNF-α), and prostaglandin E2 (PGE2) and the nitric oxide (NO) content. In addition, we demonstrated that Cd induced the apoptosis of chicken neutrophils and increased mRNA level of Bak, Cysteine-aspartic protease (Caspase)-3, Caspase-9, Caspase-12, glucose-regulated protein 78 (GRP78) and activating transcription factor 6 (ATF6), decreased mRNA level of Bcl-xl, and Ca/calmodulin-dependent protein (CaM). Moreover, the expression of NF-κB and Caspase-12 protein increased significantly in the Cd treatment groups. Se pretreatment significantly protected neutrophils against Cd-caused alterations. Our work suggested that Cd-induced immune suppression, inflammatory response, and apoptosis via endoplasmic reticulum stress (ERS). Moreover, these factors played critical roles in Se-mediated chemoprevention against Cd-induced immunotoxicity.

Alleviation Mechanisms of Selenium on Cadmium-Spiked Neutrophil Injury to Chicken.

To determine the negative effects of cadmium (Cd) exposure and the protective role of selenium (Se) on Cd-spiked neutrophils of chicken, forty-eight 28-day-old Isa Brown male chickens were divided randomly into four groups. Group I (control group) was fed with the basic diet containing 0.2 mg/kg Se. Group II (Se-treated group) was fed with the basic diet supplemented with Na2SeO3, and the total Se content was 2 mg/kg. Group III (Se/Cd-treated group) was fed with the basic diet supplemented with Na2SeO3; the total Se content was 2 mg/kg and supplemented with 150 mg/kg CdCl2. Group IV (Cd-treated group) was fed with the basic diet supplemented with 150 mg/kg CdCl2. Analyses of inflammatory factors, cytokines, and heat shock protein (Hsp) messenger RNA (mRNA) expression were detected by real-time PCR (RT-PCR). Additionally, we evaluated the phagocytic rate of neutrophils in peripheral blood. First, we observed that Cd significantly induced the mRNA expression levels of inflammatory factors NF-κB, iNOS, COX-2, and TNF-α, while Se/Cd treatment reduced their mRNA expression, although these expression levels remained higher than that of the control group. In addition, the mRNA expression levels of cytokines (IL-2, IL-4, and IL-10) for the Se-treated group exhibited significant differences between the Se/Cd-treated group and the Cd-treated group. Furthermore, the mRNA expression levels of Hsps demonstrated that the Se/Cd-treated group and the Cd-treated group were significantly higher (P < 0.05) than the control group and the Se-treated group. These results demonstrated that Se presented partial protection on Cd-spiked neutrophils of chicken with Hsps being involved in the process of the Cd-spiked toxic effects in chicken peripheral blood neutrophils.