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Difficulties of in vitro culture and genetic manipulation of Eimeria tenella have hindered the screening of virulence factors in this parasite. In this study, the E. tenella rhoptry protein 30 (EtROP30) was expressed in Toxoplasma gondii (RH∆Ku80-EtROP30), and its effect on the proliferation and virulence of parasites was investigated. The results revealed that the expression of EtROP30 had no impact on the invasion and egress processes. However, the RH∆Ku80-EtROP30 strain formed larger plaques compared to the RH∆Ku80, indicating that the EtROP30 expression promotes T. gondii proliferation. Furthermore, the RH∆Ku80-EtROP30 strain exhibited greater pathogenicity, resulting in earlier mortality and shorter overall survival time compared to RH∆Ku80. These results imply that EtROP30 expression facilitates parasite intracellular proliferation and virulence in mice, suggesting that EtROP30 might be a candidate virulence factor of E. tenella., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

MORN proteins play a key role in the cytoskeletal structure of eukaryotes and are essential for the close arrangement of the endoplasmic reticulum and plasma membrane. A gene with nine MORN motifs (TGGT1_292120, named Tg MORN2 ) was identified in the Toxoplasma gondii genome; it was presumed to belong to the MORN protein family and to have the function of forming the cytoskeleton, which affects the survival of T. gondii . However, the genetic deletion of MORN2 did not noticeably affect parasite growth and virulence. Using adjacent protein labeling techniques, we identified a network of TgMORN2 interactions, which mainly included endoplasmic reticulum stress (ER stress)-related proteins. In exploring these data, we found that the pathogenicity of the KO-Tg MORN2 strain was significantly reduced in the case of tunicamycin-induced ER stress. Reticulon TgRTN (TGGT1_226430) and tubulin β-Tubulin were identified as interaction proteins of TgMORN2. Collectively, TgMORN2 plays a role in ER stress, which lays a foundation for further research on the function of the MORN protein in T. gondii .

Toxoplasma gondii is an intracellular protozoan parasite that infects most warm-blooded animals, including humans, and causes toxoplasmosis. In this study, the pathogenicity of RH strain of T. gondii in broiler chicks was investigated and the susceptible tissues of chicks were clarified. Fourteen-day-old broiler chicks were injected intraperitoneally with 1 × 10 7 , 1 × 10 6 , and 1 × 10 5 tachyzoites of T. gondii, respectively. The results showed that the spleen and lungs were frequently positive for T. gondii DNA. Moreover, in chicks, infection with only 1 × 10 7 tachyzoites resulted in clinical symptoms, with lower weight gain, higher body temperature, anorexia and apathy, indicating that chicks are insusceptible to T. gondii infection. Then, this study investigated the relationship between T. gondii survival and chick body temperature. In the experiment, the invasion and proliferation of T. gondii were evaluated at 37 °C and 41 °C, respectively, and the survival of the parasites was significantly inhibited at 41 °C. In conclusion, broiler chicks are insusceptible to T. gondii infection, and the higher body temperature compared to other susceptible animals is a key factor in the reduction of T. gondii pathogenicity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

We had shown in our previous study that TgUrm1 (ubiquitin-related Modifier 1) was involved in the regulation of anti-oxidant stress in Toxoplasma gondii by conjugating with TgAhp1. It is generally believed that Urm1 binds to target proteins through a mechanism involving Uba (ubiquitin-like activator protein). Here, we identified the TgUrm1-exclusive ubiquitin-like activator-TgUba1, which was located in the cytoplasm of Toxoplasma . TgUba1 contained three domains, including the atrophin-1 domain (ANT1), the E1-like domain (AD), and the rhodanese homology domain (RHD). We explored the interaction of TgUba1 with TgUrm1, and the AD domain was essential for the interaction of the two proteins. The TgUba1 knockout and complementary mutants were obtained based on CRISPR/Cas9 gene editing technology. The knockout of TgUba1 attenuated parasite proliferation and virulence in mice, but not invasion and egress processes, revealing the pivotal role played by TgUba1 in T. gondii survival. Meanwhile, the conjugate band of TgUrm1 was significantly reduced under oxidative stress stimulation without TgUba1, indicating that TgUba1 enhanced the targeted conjugation ability of TgUrm1 in response to oxidative stress, especially under diamide (Dia) stimulation. Furthermore, eleven TgUba1-interacting proteins were identified by proximity-based protein labeling techniques, relating them to ubiquitin-like modifications, anti-oxidative stress and metabolic regulation processes. In conclusion, TgUba1 was essential for T. gondii survival and might be a potential ubiquitin-like activator protein for TgUrm1.

Toxoplasma gondii infects almost all warm-blooded animals, and toxoplasmosis is a common zoonotic parasitic disease worldwide. A nested PCR with high specificity and sensitivity was developed in this study based on the data collected on the infection rate of toxoplasmosis in chickens in Shandong province, and the effect of low temperature on the infectivity of tachyzoites was investigated. The sampling data showed that the total prevalence of T. gondii in Shandong province was 12.3%, and the positive rate varied in different regions, ranging from 6.7% to 21%. Chickens were infected with T. gondii under laboratory conditions, and positive chicken hearts were stored under various cold conditions to infect mice for reinfection evaluation. The results demonstrated that the parasites maintained high infectivity in mice even after 6 h of storage at -20 °C ambient temperature, indicating that short-term cryopreservation is not effective in reducing the risk of T. gondii transmission. These results form the basis for assessing the risk of toxoplasmosis contamination in consumed chicken products and provide information on the prevention of parasite transmission from animals to humans., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Ubiquitin-related modifier 1 (Urm1) is a ubiquitin-like molecule (UBL) with the ability to act as a posttranslational protein modifier. Here, we characterized the Toxoplasma gondii homolog of URM1 located in the tachyzoite cytoplasm. The total loss of the TgURM1 resulted in a significant reduction in parasite invasion, replication, and virulence in mice, revealing that TgURM1 plays a pivotal role in T. gondii survival. For TgURM1, urmylation was significantly induced by oxidative stress, and mutations of the C-terminal glycine-glycine motif of TgURM1 blocked the urmylation process. Furthermore, the TgURM1 knockout strain was intolerant to oxidative stress, suggesting that TgURM1 is involved in the oxidative stress process. TgAHP1, an alkyl hydroperoxide reductase, was screened via proximity-based protein labeling techniques and proteomics and was shown to interact with TgURM1 under oxidative stress conditions. In conclusion, TgURM1 is a UBL protein involved in the response of Toxoplasma to oxidative stress. IMPORTANCE T. gondii has an intricate life cycle which involves multiple morphologically and physiologically distinct stages, and posttranslational modifications (PTMs) may be key regulators of protein expression at relevant life cycle stages. In recent years, ubiquitin-like proteins with modification functions have been discovered and studied, including Sumo, Rub1, ATG8, and ATG12. Ubiquitin-related modifier 1 (Urm1) is a ubiquitin-like molecule (UBL), which is considered to be the oldest ubiquitin-like system. In this study, we identified the Urm1 gene in Toxoplasma and explored that the urmylation of Urm1 was significantly induced by oxidative stress. Fewer studies have been conducted on ubiquitin-like proteins of parasites, and our results provide theoretical support for the research of metabolic regulation and antioxidative stress processes in T. gondii.

Toxoplasma gondii is a protozoan parasite that is widely parasitic in the nucleated cells of warm-blooded animals. Bioinformatic analysis of alkyl hydroperoxide reductase 1 (AHP1) of T. gondii is a member of the Prxs family and exhibits peroxidase activity. Cys 166 was certified to be a key enzyme active site of TgAHP1, indicating that the enzyme follows a cysteine-dependent redox process. TgAHP1 was present in a punctate staining pattern anterior to the T. gondii nucleus. Oxidative stress experiments showed that the ∆Ahp1 strain was more sensitive to tert-butyl hydroperoxide (tBOOH) than hydrogen peroxide (H 2 O 2 ), indicating that tBOOH may be a sensitive substrate for TgAHP1. Under tBOOH culture conditions, the ∆Ahp1 strain was significantly less invasive, proliferative, and pathogenic in mice. This was mainly due to the induction of tBOOH, which increased the level of reactive oxygen species in the parasites and eventually led to apoptosis. This study shows that TgAHP1 is a peroxisomes protein with cysteine-dependent peroxidase activity and sensitive to tBOOH., Competing Interests: The authors declare that there was no conflict of interest during the development of this research., (Copyright © 2021 Jinwen Wang et al.)