PruΔcdpk2 Protects Pigs against Acute Toxoplasmosis Depending on T-Lymphocyte Subsets and Natural Killer Cell Responses.

Toxoplasma gondii is a widespread protozoan parasite approximately infecting one-third of the world population and can cause serious public health problems. In this study, we investigated the protective effect of the attenuated vaccine Pru:Δcdpk2 against acute toxoplasmosis and explored the underlying immune mechanisms of the protection in pigs. The systemic T-cell and natural killer (NK) cell responses were analyzed, including kinetics, phenotype, and multifunctionality (interferon [IFN]-γ, tumor necrosis factor [TNF]-α), and the IFN-γ levels were analyzed in PBMCs. Our results showed that T. gondii-specific antibodies were induced by Pru:Δcdpk2. After challenging with RH, the antibodies were able to respond quickly in the immunized group, and the expression level was significantly higher than that in the unimmunized group. The expression level of IFN-γ significantly increased after vaccination, and the CD3+ γδ-, NK, and CD3+ γδ+ cell subsets also significantly increased. At the same time, functional analysis indicated that these cells were polarized toward a Th1 phenotype, showing the ability to secrete IFN-γ and TNF-α. The CD4+CD8α-T cell population exhibited a higher frequency of IFN-γ+ producing cells compared with the CD4-CD8α+ and CD4+CD8α+ cell populations during the early days of vaccination. Our results indicated that the attenuated vaccine could induce the expression of NK, γδ, and CD3αß cells in pigs, and IFN-γ and TNF-α secreted by these cells are important for resistance to T. gondii infection.

Phosphoproteomic analysis reveals changes in A-Raf-related protein phosphorylation in response to Toxoplasma gondii infection in porcine macrophages.

BACKGROUND: Toxoplasma gondii is an obligate intracellular protozoan parasite that causes severe threats to humans and livestock. Macrophages are the cell type preferentially infected by T. gondii in vivo. Protein phosphorylation is an important posttranslational modification involved in diverse cellular functions. A rapidly accelerated fibrosarcoma kinase (A-Raf) is a member of the Raf family of serine/threonine protein kinases that is necessary for MAPK activation. Our previous research found that knockout of A-Raf could reduce T. gondii-induced apoptosis in porcine alveolar macrophages (3D4/21 cells). However, limited information is available on protein phosphorylation variations and the role of A-Raf in macrophages infected with T. gondii. METHODS: We used immobilized metal affinity chromatography (IMAC) in combination with liquid chromatography tandem mass spectrometry (LC-MS/MS) to profile changes in phosphorylation in T. gondii-infected 3D4/21 and 3D4/21-ΔAraf cells. RESULTS: A total of 1647 differentially expressed phosphorylated proteins (DEPPs) with 3876 differentially phosphorylated sites (DPSs) were identified in T. gondii-infected 3D4/21 cells (p3T group) when compared with uninfected 3D4/21 cells (pho3 group), and 959 DEPPs with 1540 DPSs were identified in the p3T group compared with infected 3D4/21-ΔAraf cells (p3KT group). Venn analysis revealed 552 DPSs corresponding to 406 DEPPs with the same phosphorylated sites when comparing p3T/pho3 versus p3T/p3KT, which were identified as DPSs and DEPPs that were directly or indirectly related to A-Raf. CONCLUSIONS: Our results revealed distinct responses of macrophages to T. gondii infection and the potential roles of A-Raf in fighting infection via phosphorylation of crucial proteins.

Toxoplasma gondii infection regulates apoptosis of host cells via miR-185/ARAF axis.

BACKGROUND: Toxoplasmosis is a zoonosis with a worldwide presence that is caused by the intracellular parasite Toxoplasma gondii. Active regulation of apoptosis is an important immune mechanism by which host cells resist the growth of T. gondii or avoid excessive pathological damage induced by this parasite. Previous studies found that upregulated expression of microRNA-185 (miR-185) during T. gondii infection has a potential role in regulating the expression of the ARAF gene, which is reported to be associated with cell proliferation and apoptosis. METHODS: The expression levels of miR-185 and the ARAF gene were evaluated by qPCR and Western blot, respectively, in mice tissues, porcine kidney epithelial cells (PK-15) and porcine alveolar macrophages (3D4/21) following infection with the T. gondii ToxoDB#9 and RH strains. The dual luciferase reporter assay was then used to verify the relationship between miR-185 and ARAF targets in PK-15 cells. PK-15 and 3D4/21 cell lines with stable knockout of the ARAF gene were established by CRISPR, and then the apoptosis rates of the cells following T. gondii infection were detected using cell flow cytometry assays. Simultaneously, the activities of cleaved caspase-3, as a key apoptosis executive protein, were detected by Western blot to evaluate the apoptosis levels of cells. RESULTS: Infection with both the T. gondii ToxoDB#9 and RH strains induced an increased expression of miR-185 and a decreased expression of ARAF in mice tissues, PK-15 and 3D4/21 cells. MiR-185 mimic transfections showed a significantly negative correlation in expression levels between miR-185 and the ARAF gene. The dual luciferase reporter assay confirmed that ARAF was a target of miR-185. Functional investigation revealed that T. gondii infection induced the apoptosis of PK-15 and 3D4/21 cells, which could be inhibited by ARAF knockout or overexpression of miR-185. The expression levels of cleaved caspase-3 protein were significantly lower in cells with ARAF knockout than in normal cells, which were consistent with the results of the cell flow cytometry assays. CONCLUSIONS: Toxoplasma gondii infection could lead to the upregulation of miR-185 and the downregulation of ARAF, which was not related to the strain of T. gondii and the host cells. Toxoplasma gondii infection could regulate the apoptosis of host cells via the miR-185/ARAF axis, which represents an additional strategy used by T. gondii to counteract host-cell apoptosis in order to maintain survival and reproduce in the host cells.

Tandem mass tag-based quantitative proteomics analyses of a chicken-original virulent and its attenuated Histomonas meleagridis strain in China.

Introduction: Histomonas meleagridis can cause histomonosis in poultry. Due to the prohibition of effective drugs, the prevention and treatment of the disease requires new strategies. Questions about its pathogenic mechanisms and virulence factors remain puzzling. Methods: To address these issues, a tandem mass tag (TMT) comparative proteomic analysis of a virulent strain and its attenuated strain of Chinese chicken-origin was performed. Results: A total of 3,494 proteins were identified in the experiment, of which 745 proteins were differentially expressed (fold change ≥1.2 or ≤0.83 and p < 0.05), with 192 up-regulated proteins and 553 down-regulated proteins in the virulent strain relative to the attenuated strain. Discussion: Surface protein BspA like, digestive cysteine proteinase, actin, and GH family 25 lysozyme were noted among the proteins up regulated in virulent strains, and these several proteins may be directly related to the pathogenic capacity of the histomonad. Ferredoxin, 60S ribosomal protein L6, 40S ribosomal protein S3, and NADP-dependent malic enzyme which associated with biosynthesis and metabolism were also noted, which have the potential to be new drug targets. The up-regulation of alpha-amylase, ras-like protein 1, ras-like protein 2, and involucrin in attenuated strains helps to understand how it is adapted to the long-term in vitro culture environment. The above results provide some candidate protein-coding genes for further functional verification, which will help to understand the molecular mechanism of pathogenicity and attenuation of H. meleagridis more comprehensively.

Global MicroRNAs Expression Profile Analysis Reveals Possible Regulatory Mechanisms of Brain Injury Induced by Toxoplasma gondii Infection.

Toxoplasma gondii (T. gondii) is an obligate intracellular parasitic protozoan that can cause toxoplasmosis in humans and other endotherms. T. gondii can manipulate the host gene expression profile by interfering with miRNA expression, which is closely associated with the molecular mechanisms of T. gondii-induced brain injury. However, it is unclear how T. gondii manipulates the gene expression of central nervous system (CNS) cells through modulation of miRNA expression in vivo during acute and chronic infection. Therefore, high-throughput sequencing was used to investigate expression profiles of brain miRNAs at 10, 25, and 50 days post-infection (DPI) in pigs infected with the Chinese I genotype T. gondii strain in this study. Compared with the control group 87, 68, and 135 differentially expressed miRNAs (DEMs) were identified in the infected porcine brains at 10, 25, and 50 DPI, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that a large number significantly enriched GO terms and KEGG pathways were found, and were mostly associated with stimulus or immune response, signal transduction, cell death or apoptosis, metabolic processes, immune system or diseases, and cancers. miRNA-gene network analysis revealed that the crucial connecting nodes, including DEMs and their target genes, might have key roles in the interactions between porcine brain and T. gondii. These results suggest that the regulatory strategies of T. gondii are involved in the modulation of a variety of host cell signaling pathways and cellular processes, containing unfolded protein response (UPR), oxidative stress (OS), autophagy, apoptosis, tumorigenesis, and inflammatory responses, by interfering with the global miRNA expression profile of CNS cells, allowing parasites to persist in the host CNS cells and contribute to pathological damage of porcine brain. To our knowledge, this is the first report on miRNA expression profile in porcine brains during acute and chronic T. gondii infection in vivo. Our results provide new insights into the mechanisms underlying T. gondii-induced brain injury during different infection stages and novel targets for developing therapeutic agents against T. gondii.

Evaluation of Origanum vulgare Essential Oil and Its Active Ingredients as Potential Drugs for the Treatment of Toxoplasmosis.

Toxoplasma gondii is a serious hazard to public health and animal husbandry. Due to the current dilemma of treatment of toxoplasmosis, it is urgent to find new anti-T. gondii drugs to treat toxoplasmosis. In this study, the anti-T. gondii activity of Origanum vulgare essential oil (Ov EO) was firstly studied, and then, carvanol (Ca), the main ingredient of Ov EO was evaluated using the MTT assay on human foreskin fibroblast (HFF) cells in vitro. The cytotoxicity was evaluated using the MTT assay on HFF cells. The CC50 of Ov EO and Ca was 134.9 and 43.93 µg/ml, respectively. Both of them exhibited anti-parasitic activity, and inhibited the growth of T. gondii in a dose-dependent manner. For the inhibition effect, Ca was better than Ov EO at the same concentration, the IC50 of Ov EO and Ca was 16.08 and 7.688 µg/ml, respectively. In addition, treatment with Ca, was found to change the morphology of T. gondii tachyzoites and made their shapes curl up. These results showed that Ca was able to inhibit the proliferation of T. gondii by reducing invasion, which may be due to its detrimental effect on the mobility of tachyzoites. Our results indicated that Ca could be a potential new and effective drug for treating toxoplasmosis.

In Vitro Evaluation of Lavandula angustifolia Essential Oil on Anti-Toxoplasma Activity.

The current methods of treating toxoplasmosis have a number of side effects, and these therapies are only effective against the acute stage of the disease. Thus, development of new low toxicity and efficient anti-Toxoplasma drugs is extremely important. Natural products are important sources for screening new drugs; among them, essential oils (EOs) have efficacy in anti-bacterial, anti-inflammatory, anti-insect, and other aspects. In this study, 16 EOs were screened for their anti-T. gondii activity. Lavandula angustifolia essential oil (La EO)was found to have an anti-parasitic effect on T. gondii. The cytotoxicity of La EO was firstly evaluated using the MTT assay on human foreskin fibroblast (HFF) cells, and then the anti-T. gondii activity was evaluated by plaque assay. Finally, the invasion experiment and electron microscope observation were used to study the mechanism of La EO in anti-toxoplasma activity. The results indicated that the CC50 of La EO was 4.48 mg/ml and that La EO had activity against T. gondii and the inhibition was in a dose-dependent manner under safe concentrations. La EO was able to reduce T. gondii invasion, which may be due to its detrimental effect on changes of the morphology of tachyzoites. These findings indicated that La EO could be a potential drug for treating toxoplasmosis.

In vitro Anti-parasitic Activity of Pelargonium X. asperum Essential Oil Against Toxoplasma gondii.

Toxoplasmosis is a global zoonotic disease, and one-third of the human population is chronically infected by Toxoplasma gondii. Due to the limited effectiveness and prominent side effects of the existing drugs, there is a dire need for the discovery of new therapeutic options in the treatment of toxoplasmosis. In this study, five essential oils (EO) were screened for their anti-parasitic activity against T. gondii. The cytotoxicity of essential oils was evaluated using the MTT assay on human foreskin fibroblast cells. The CC50 values of Eucalyptus globulus EO, Cupressus sempervirens EO, Citrus aurantifolia EO, Melaleuca alternifolia EO, and Pelargonium X. asperum (Pa) EO were found to be 22.74, 7.25, 15.01, 6.26, and 4.77 mg/mL, respectively. Only PaEO exhibited anti-parasitic activity, and inhibited the growth of T. gondii in a dose-dependent manner. In addition, treatment with PaEO, was found to reduce the volume of T. gondii tachyzoites and make their membrane surfaces rough. These results showed that PaEO was able to inhibit the growth of T. gondii by reducing invasion, which may be due to its detrimental effect on the ability of tachyzoites to move. These findings suggest that PaEO could be a potential anti-T. gondii drug, which may facilitate the development of new and effective treatments against toxoplasmosis.