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Protein phosphatases are post-translational regulators of Toxoplasma gondii proliferation, tachyzoite-bradyzoite differentiation and pathogenesis. Here, we identify the putative protein phosphatase 6 (TgPP6) subunits of T. gondii and elucidate their role in the parasite lytic cycle. The putative catalytic subunit TgPP6C and regulatory subunit TgPP6R likely form a complex whereas the predicted structural subunit TgPP6S, with low homology to the human PP6 structural subunit, does not coassemble with TgPP6C and TgPP6R. Functional studies showed that TgPP6C and TgPP6R are essential for parasite growth and replication. The ablation of TgPP6C significantly reduced the synchronous division of the parasite's daughter cells during endodyogeny, resulting in disordered rosettes. Moreover, the six conserved motifs of TgPP6C were required for efficient endodyogeny. Phosphoproteomic analysis revealed that ablation of TgPP6C predominately altered the phosphorylation status of proteins involved in the regulation of the parasite cell cycle. Deletion of TgPP6C significantly attenuated the parasite virulence in mice. Immunization of mice with TgPP6C-deficient type I RH strain induced protective immunity against challenge with a lethal dose of RH or PYS tachyzoites and Pru cysts. Taken together, the results show that TgPP6C contributes to the cell division, replication and pathogenicity in T. gondii., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Liang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Lysine malonylation is a post-translational modification (PTM), which regulates many cellular processes. Limited information is available about the level of lysine malonylation variations between Toxoplasma gondii strains of distinct genetic lineages. Yet, insights into such variations are needed to understand the extent to which lysine malonylation contributes to the differences in the virulence and repertoire of virulence factors between T. gondii genotypes. In this study, we profiled lysine malonylation in T. gondii using quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immuno-affinity purification. This analysis was performed on three T. gondii strains with distinctive pathogenicity in mice, including RH strain (type I), PRU strain (type II), and VEG strain (type III). In total, 111 differentially malonylated proteins and 152 sites were upregulated, and 17 proteins and 17 sites were downregulated in RH strain versus PRU strain; 50 proteins and 59 sites were upregulated, 50 proteins and 53 sites were downregulated in RH strain versus VEG strain; and 72 proteins and 90 sites were upregulated, and 7 proteins and 8 sites were downregulated in VEG strain versus PRU strain. Differentially malonylated proteins were involved in key processes, such as those mediating the regulation of protein metabolism, stress response, glycolysis, and actin cytoskeleton. These results reveal an association between lysine malonylation and intra-species virulence differences in T. gondii and offer a new resource for elucidating the contribution of lysine malonylation to energy metabolism and virulence in T. gondii., Competing Interests: The authors have declared that no competing interests exist.

Toxoplasma gondii is an important intracellular parasitic protozoan with a variety of hosts, including chickens, which poses a potential threat to public health. However, little is known regarding overall T. gondii infection in chickens in China. Herein, the prevalence and risk factors associated with T. gondii infection in chickens in China were investigated using a meta-analysis. Forty studies regarding the prevalence of T. gondii in chickens in China from 1993 to 2021 were identified using five databases (PubMed, Science Direct, CNKI, Wang Fang, and VIP). Quantitative and potential sources were analyzed through subgroup analysis and meta-regression in R v3.5.2. The overall prevalence of T. gondii in chickens in China was 13.4% (95% confidence interval (CI): 10.9-16.0). In the region subgroup, the lowest prevalence was presented in Northwestern China (6.0%, 95% CI: 3.2-9.5; P < 0.001). Seasonally, T. gondii prevalence was the highest in spring (17.9%, 95% CI: 7.7-30.9; P = 0.007). Among detection methods, the prevalence in the ELISA subgroup was the highest (22.8%, 95% CI: 17.1-29.1; P < 0.001). According to the farming mode, the prevalence of T. gondii in free-range chickens (19.5%, 95% CI: 15.4-23.9) was significantly higher than that in chickens raised by intensive farming (7.4%, 95% CI: 5.1-10.2; P < 0.001). We also estimated the relationships between region, sampling year, chicken age, chicken application, gender, sample classification, study quality, and T. gondii prevalence in chickens in China. Our study showed that region, season, and farming model played important roles in T. gondii infection of chickens. Integrated control measures should be undertaken to reduce the losses caused by T. gondii infection to the chicken industry., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Lysine crotonylation (Kcr) is an evolutionally conserved post-translational modification (PTM) on histone proteins. However, information about Kcr and its involvement in the biology and metabolism of Toxoplasma gondii is limited. In the present study, a global Kcr proteome analysis using LC-MS/MS in combination with immune-affinity method was performed. A total of 12,152 Kcr sites distributed over 2719 crotonylated proteins were identified. Consistent with lysine acetylation and succinylation in Apicomplexa, Kcr was associated with various metabolic pathways, including carbon metabolism, pyrimidine metabolism, glycolysis, gluconeogenesis, and proteasome. Markedly, many stage-specific proteins, histones, and histone-modifying enzymes related to the stage transition were found to have Kcr sites, suggesting a potential involvement of Kcr in the parasite stage transformation. Most components of the apical secretory organelles were identified as crotonylated proteins which were associated with the attachment, invasion, and replication of T. gondii. These results expanded our understanding of Kcr proteome and proposed new hypotheses for further research of the Kcr roles in the pathobiology of T. gondii infection.

Lysine 2-hydroxyisobutyrylation (K hib ) is a recently discovered and evolutionarily conserved form of protein post-translational modification (PTM) found in mammalian and yeast cells. Previous studies have shown that K hib plays roles in the activity of gene transcription and K hib -containing proteins are closely related to the cellular metabolism. In this study, a global K hib -containing analysis using the latest databases (ToxoDB 46, 8322 sequences, downloaded on April 16, 2020) and sensitive immune-affinity enrichment coupled with liquid chromatography-tandem mass spectrometry was performed. A total of 1078 K hib modification sites across 400 K hib -containing proteins were identified in tachyzoites of Toxoplasma gondii RH strain. Bioinformatics and functional enrichment analysis showed that K hib -modified proteins were associated with various biological processes, such as ribosome, glycolysis/gluconeogenesis, and central carbon metabolism. Interestingly, many proteins of the secretory organelles (e.g., microneme, rhoptry, and dense granule) that play roles in the infection cycle of T. gondii were found to be K hib -modified, suggesting the involvement of K hib in key biological process during T. gondii infection. We also found that histone proteins, key enzymes related to cellular metabolism, and several glideosome components had K hib sites. These results expanded our understanding of the roles of K hib in T. gondii and should promote further investigations of how K hib regulates gene expression and key biological functions in T. gondii.

Toxoplasma gondii and Neospora caninum are two closely related protozoan parasites which can cause abortion and significant economic losses in sheep and goats. However, it is yet to know whether black-bone sheep and goats are infected with T. gondii and N. caninum in China. In the present investigation, the seroprevalence and risk factors of T. gondii and N. caninum infections in black-boned sheep and goats were investigated in Yunnan Province, subtropical southwest China between July and August of 2017. A total of 481 serum samples were tested for T. gondii antibodies using the Modified Agglutination Test (MAT), and 468 serum samples were examined for N. caninum antibodies by indirect Enzyme-Linked Immunosorbent Assay (iELISA). The overall seroprevalence of T. gondii in black-boned sheep and goats was 36.80% (177/481, 95% CI 32.49-41.11), and 40 out of 468 serum samples were N. caninum-seropositive (8.55%, 95% CI 6.02-11.08). There was significant difference in the seroprevalence of T. gondii infection in different regions (χ 2  = 19.869, df = 2, P<0.01). As for the seroprevalence of N. caninum infection, region (χ 2  = 8.558, df = 2, P<0.05), age (χ 2  = 16.631, df = 3, P < 0.01), gender (χ 2  = 11.219, df = 1, P < 0.01) and species (χ 2  = 8.673, df = 1, P < 0.01) were the risk factors. In addition, the seroprevalence of coinfection of T. gondii and N. caninum in black-boned sheep and goats was 3.63% (17/468, 95% CI 1.94-5.32). To our knowledge, this is the first report of T. gondii and N. caninum seroprevalence in black-boned sheep and goats in China, which provided base-line data for the execution of control strategies and measures against T. gondii and N. caninum infection in black-boned sheep and goats., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Infection with the apicomplexan protozoan parasite Toxoplasma gondii is an ongoing public health problem. The parasite's ability to invade and replicate within the host cell is dependent on many effectors, such as dense granule proteins (GRAs) released from the specialized organelle dense granules, into host cells. GRAs have emerged as important determinants of T. gondii pathogenesis. However, the functions of some GRAs remain undefined. In this study, we used CRISPR-Cas9 technique to disrupt 17 GRA genes ( GRA11, GRA12 bis, GRA13, GRA14, GRA20, GRA21, GRA28-31, GRA33-38 , and GRA40 ) in the virulent T. gondii RH strain. The CRISPR-Cas9 constructs abolished the expression of the 17 GRA genes. Functional characterization of single Δ GRA mutants was achieved in vitro using cell-based plaque assay and egress assay, and in vivo in BALB/c mice. Targeted deletion of these 17 GRA genes had no significant effect neither on the in vitro growth and egress of the mutant strains from the host cells nor on the parasite virulence in the mouse model of infection. Comparative analysis of the transcriptomics data of the 17 GRA genes suggest that GRAs may serve different functions in different genotypes and life cycle stages of the parasite. In sum, although these 17 GRAs might not be essential for RH strain growth in vitro or virulence in mice, they may have roles in other strains or parasite stages, which warrants further investigations.

Toxoplasma gondii can infect nearly all warm-blooded animals including birds. However, limited information on the molecular epidemiology and genotypes of T. gondii infecting poultry is available in China. Therefore, the present study characterized T. gondii genotypes in poultry meat in eastern China. During August 2015 and September 2016, muscle tissue samples collecting from 414 poultries (257 chickens, 115 ducks and 42 geese) in Shandong provinces were used to detect the T. gondii B1 gene by a semi-nested PCR, and the positive samples were genotyped at 10 nuclear loci (i.e., SAG1, alternative SAG2, 5'- and 3'-SAG2, SAG3, L358, BTUB, c22-8, GRA6, c29-2, PK1) and an apicoplast locus Apico by multi-locus PCR-RFLP technology. Thirty-two (7.37%) samples from 414 poultry meat were T. gondii B1 gene positive. Chicken had the highest T. gondii prevalence (8.17%), followed by ducks (7.83%) and geese (4.76%). Furthermore, only one genotype (ToxoDB#9) was identified. This is the first genetic characterization of T. gondii isolates from poultry meat in Shandong province, eastern China and also the first report of genotype ToxoDB#9 was found in poultry in China, which provide basic data for the surveillance and control of T. gondii infection in poultry, other animals and humans., (Copyright © 2017. Published by Elsevier B.V.)