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Toxoplasma gondii infection evokes a strong Th1-type response with interleukin (IL)-12 and interferon (IFN)-γ secretion. Recent studies suggest that the infection of pregnant mice with T. gondii may lead to adverse pregnancy results caused by subversion of physiological immune tolerance at maternofetal interface rather than direct invasion of the parasite. Genotype-associated dense granule protein GRA15II tends to induce classically activated macrophage (M1) differentiation and subsequently activating NK, Th1, and Th17 cells whereas rhoptry protein ROP16I/III drives macrophages to alternatively activated macrophage (M2) polarization and elicits Th2 immune response. Unlike the archetypal strains of types I, II, and III, type Chinese 1 strains possess both GRA15II and ROP16I/III, suggesting a distinct pathogenesis of Toxoplasma -involved adverse pregnancies. We constructed T. gondii type Chinese 1 strain of WH3Δ rop16 based on CRISPR/Cas9 technology to explore the ROP16I/III-deficient/GRA15II-dominant parasites in induction of trophoblast apoptosis in vitro and abnormal pregnant outcomes of mice in vivo . Our study showed that Toxoplasma WH3Δ rop16 remarkably induced apoptosis of trophoblasts. C57BL/6 pregnant mice injected with the tachyzoites of WH3Δ rop16 presented increased absorptivity of fetuses in comparison with the mice infected with WH3 wild type (WH3 WT) parasites although no remarkable difference of virulence to mice was seen between the two strains. Additionally, the mice inoculated with WH3Δ rop16 tachyzoites exhibited a notable expression of both IL-17A and IFN-γ, while the percentage of CD4+CD25+FoxP3 [T regulatory cells (Tregs)] were diminished in splenocytes and placenta tissues compared to those infected with WH3 WT parasites. Accordingly, expressions of IL-4, IL-10, and transforming growth factor beta 1, the pivotal cytokines of Th2 and Tregs response, were significantly dampened whereas IFN-γ and IL-12 expressions were upregulated in WH3Δ rop16 -infected mice, which gave rise to more prominent outcomes of abnormal pregnancies. Our results indicated that the WH3Δ rop16 parasites with gra15II background of T. gondii type Chinese 1 strains may cause miscarriage and stillbirth due to subversion of the maternal immune tolerance and system immunity of the animals and the GRA15II effector contributes to the process of adverse pregnant consequences.

Toxoplasma gondii is an obligate intracellular opportunistic protozoan parasite. T. gondii invasion disturbs the balance between reactive oxygen species (ROS) production and antioxidant capacity in the host, triggering the oxidative stress response. Thioredoxin reductase (TR) of T. gondii helps to escape ROS-induced damage in the host, whereas T. gondii rhoptry protein 16 (ROP16) downregulates host innate immunity to suppress excessive inflammation and inhibit ROS production. However, whether TR and ROP16 synergistically promote resistance to ROS-induced damage remains unclear. Here, we used the CRISPR/Cas9 technology to successfully obtain a double TR and ROP16 gene knockout T. gondii strain. The double deletion of TR and ROP16 in T. gondii weakened its growth ability in vitro and decreased its virulence in vivo. Moreover, the double deletion of TR and ROP16 resulted in a lower antioxidant capacity, higher degree of lipid oxidation, and elevated ROS levels in the parasite and host cells. Interestingly, the deletion of the TR and ROP16 genes in T. gondii synergistically increased IL-12 levels, which triggered host cells to produce more ROS to resist T. gondii infection. These results show that TR and ROP16 in T. gondii play a synergistic role, facilitating resistance to ROS damage incurred by host immune cells through different pathways.

Toxoplasma gondii is a threat for immunocompromized individuals, and no treatment is available for enhancing immunity against infection. Molecular adjuvants may improve the efficacy of DNA vaccine-induced T cell immunity. Here, we report that cocktailed DNA immunization with ROP5 and ROP18 boosted immune responses induced by a single DNA immunization with ROP5 or ROP18, but also that co-administration of molecular adjuvant IL-33 enhanced immune efficacy induced by this cocktailed DNA vaccination. These improved immune responses were characterized by higher Toxoplasma -specific IgG2a titers, Th1 responses associated with the production of IFN-γ, IL-2, IL-12, as well as cell-mediated activity with higher frequencies of CD8+ and CD4+ T cells. More importantly, this enhanced immunity has the ability to confer remarkable protection against a high dose lethal challenge of the T. gondii RH strain and thus against chronic infection with the T. gondii PRU strain. These data show that IL-33 is a promising immunoadjuvant to facilitate humoral as well as cellular immunity in a vaccine setting against T. gondii , and suggest that it should be evaluated in strategies against other apicomplexan parasites. Toxoplasma gondii est une menace pour les individus immunodéprimés et aucun traitement n’est disponible pour renforcer l’immunité contre l’infection. Les adjuvants moléculaires peuvent améliorer l’efficacité de l’immunité des cellules T induite par un vaccin à ADN. Ici, nous rapportons que l’immunisation par le cocktail d’ADN de ROP5 et ROP18 a stimulé les réponses immunitaires induites par une seule immunisation par l’ADN de ROP5 ou ROP18, mais aussi que la co-administration de l’adjuvant moléculaire IL-33 a amélioré l’efficacité immunitaire induite par cette vaccination par cocktail d’ADN. Ces réponses immunitaires améliorées ont été caractérisées par des titres d’IgG2a spécifiques à Toxoplasma plus élevés, des réponses Th1 associées à la production d’IFN-γ, IL-2, IL-12 ainsi qu’une activité à médiation cellulaire où les fréquences des cellules T CD8+ et CD4+ étaient plus élevées. Plus important encore, cette immunité renforcée a la capacité de conférer une protection remarquable contre une provocation létale par haute dose de la souche RH de T. gondii et donc contre une infection chronique par la souche PRU de T. gondii . Ces données montrent qu’IL-33 est un immunoadjuvant prometteur pour faciliter l’immunité humorale et cellulaire dans un contexte de vaccination contre T. gondii et suggèrent qu’il devrait être évalué dans des stratégies contre d’autres parasites apicomplexes.

B abesia orientalis , a protozoan parasite transmitted by the tick Rhipicephalus haemaphysaloides , holds significant economic importance along the Yangtze River. Key factors in the host invasion process include rhoptry neck proteins (RON2, RON4, and RON5) and apical membrane antigen 1 (AMA1). However, the intricacies of the interaction between AMA1 and RONs remain incompletely elucidated in B. orientalis . To better understand these crucial invasion components, the RON4 gene of B. orientalis (BoRON4) was cloned and sequenced. RON4 is 3468 base pairs long, encodes 1155 amino acids, and has a predicted molecular weight of 130 kDa. Bioinformatics analysis revealed a unique region (amino acid residues 109–452) in BoRON4, which demonstrates higher sensitivity to epitope activity. The BoRON4 gene was strategically truncated, amplified, and cloned into the pGEX-6p-1 vector for fusion expression. We successfully used the mouse polyclonal antibody to identify native BoRON4 in B. orientalis lysates. Furthermore, the corresponding BoRON4 protein band was detected in the water buffalo serum infected with B. orientalis , while no such band was observed in the control. Additionally, I-TASSER and Discovery Studio software were used to predict the tertiary structures of BoRON4 and its ligands, CH-PKA and CH-complex. These ligands can serve as lead compounds for the development of anti-babesiosis drugs. In conclusion, BoRON4 emerges as a promising candidate antigen for distinguishing water buffalo infected with B. orientalis from their normal counterparts. This study positions BoRON4 as a potential diagnostic antigen for babesiosis in water buffalo, contributing valuable insights to the field of parasitology.

•We produced recombinant ROR5 protein and recombinant SAG1 protein.•Mice immunized with subunit vaccines showed significantly increased survival time.•Humoral and cellular (Th1/Th2) immune responses were induced by rROP5.•T. gondii ROP5 is a promising vaccine candidate against animal toxoplasmosis. Infections with the intracellular protozoan parasite Toxoplasma gondii pose a serious public health problem and are of great economic importance worldwide. The parasite rhoptry protein 5 (ROP5) has been implicated as a major virulence factor that reduces the accumulation of immunity-related GTPases (IRG) in parasitophorous vacuole membrane (PVM), which maintains PVM integrity and evades IFNγ-mediated killing by intracellular parasites. To study the immunoprotective value of ROP5, BALB/c mice were immunized with a recombinant form of the protein administered alone or in combination with another promising vaccine antigen, rSAG1. All mice vaccinated with the recombinant antigens developed a high level of specific antibody responses against soluble tachyzoite antigens (STAg), a statistically significant increase of the splenocyte proliferation response, and significant levels of IFN-γ and IL-2 production. In contrast to rSAG1, which only stimulated the release of IFN-γ and IL-2, rROP5 induced the specific production of IL-10, the Th2-type cytokine, in addition to IFN-γ and IL-2. These results demonstrated that rROP5 could induce significant cellular and humoral (Th1/Th2) immune responses. Moreover, mice immunized with rROP5 displayed a prolonged survival time against a lethal challenge with the T. gondii RH strain. Additionally, vaccination with the mixture of rROP5+rSAG1 resulted in higher levels of T. gondii-specific IgG antibodies and lymphocyte proliferative responses and conferred more efficient protection against T. gondii challenge compared to immunization with rROP5 or rSAG1 alone. Our studies show that recombinant ROP5 antigen may be a promising vaccine candidate against toxoplasmosis. To our knowledge, this is the first report to evaluate the immunoprotective value of ROP5.

Malaria causes a half a million deaths annually. The parasite intraerythrocytic lifecycle in the human bloodstream is the major cause of morbidity and mortality. Apical organelles of merozoite stage parasites are involved in the invasion of erythrocytes. A limited number of apical organellar proteins have been identified and characterized for their roles during erythrocyte invasion or subsequent intraerythrocytic parasite development. To expand the repertoire of identified apical organellar proteins we generated a panel of monoclonal antibodies against Plasmodium falciparum schizont-rich parasites and screened the antibodies using immunofluorescence assays. Out of 164 hybridoma lines, 12 clones produced monoclonal antibodies yielding punctate immunofluorescence staining patterns in individual merozoites in late schizonts, suggesting recognition of merozoite apical organelles. Five of the monoclonal antibodies were used to immuno-affinity purify their target antigens and these antigens were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two known apical organelle protein complexes were identified, the high-molecular mass rhoptry protein complex (PfRhopH1/Clags, PfRhopH2, and PfRhopH3) and the low-molecular mass rhoptry protein complex (rhoptry-associated proteins complex, PfRAP1, and PfRAP2). A novel complex was additionally identified by immunoprecipitation, composed of rhoptry-associated membrane antigen (PfRAMA) and rhoptry neck protein 3 (PfRON3) of P. falciparum . We further identified a region spanning amino acids Q 221 -E 481 within the PfRAMA that may associate with PfRON3 in immature schizonts. Further investigation will be required as to whether PfRAMA and PfRON3 interact directly or indirectly.

Eimeria tenella (E. tenella) is a highly pathogenic and prevalent species of Eimeria that infects chickens, and it causes a considerable disease burden worldwide. The secreted proteins and surface antigens of E. tenella at the sporozoite stage play an essential role in the host–parasite interaction, which involves attachment and invasion, and these interactions are considered vaccine candidates based on the strategy of cutting off the invasion pathway to interrupt infection. We selected two highly expressed surface antigens (SAGs; Et-SAG13 and Et-SAG) and two highly expressed secreted antigens (rhoptry kinases Eten5-A, Et-ROPK-Eten5-A and dense granule 12, Et-GRA12) at the sporozoite stage. Et-ROPK-Eten5-A and Et-GRA12 were two unexplored proteins. Et-ROPK-Eten5-A was an E. tenella-specific rhoptry (ROP) protein and distributed in the apical pole of sporozoites and merozoites. Et-GRA12 was scattered in granular form at the sporozoite stage. To evaluate the potential of rEt-ROPK-Eten5-A, rEt-GRA12, rEt-SAG13 and rEt-SAG proteins as a coccidiosis vaccine, the protective efficacy was examined based on survival rate, lesion score, body weight gain, relative body weight gain and oocyst output. The survival rate was significantly improved in rEt-ROPK-Eten5-A (100%) and rEt-GRA12 (100%) immune chickens compared to the challenged control group (40%). The average body weight gains of rEt-ROPK-Eten5-A, rEt-GRA12, rEt-SAG13 and rEt-SAG immunized chickens were significantly higher than those of unimmunized chickens. The mean lesion score and oocyst output of the rEt-ROPK-Eten5-A immunized chickens were significantly reduced compared to unimmunized challenged chickens. These results suggest that the rEt-ROPK-Eten5-A protein effectively triggered protection against E. tenella in chickens and provides a useful foundation for future work developing anticoccidial vaccines.

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that affects humans and various vertebrate livestock and causes serious economic losses. To develop an effective vaccine against T. gondii infection, we constructed a DNA vaccine encoding the T. gondii rhoptry protein 17 (TgROP17) and evaluated its immune protective efficacy against acute T. gondii infection in mice. The DNA vaccine (p3×Flag-CMV-14-ROP17) was intramuscularly injected to BALB/c mice and the immune responses of the vaccinated mice were determined. Compared to control mice treated with empty vector or PBS, mice immunized with the ROP17 vaccine showed a relatively high level of specific anti-T. gondii antibodies, and a mixed IgG1/IgG2a response with predominance of IgG2a production. The immunized mice also displayed a specific lymphocyte proliferative response, a Th1-type cellular immune response with production of IFN-γ and interleukin-2, and increased number of CD8+ T cells. Immunization with the ROP17 DNA significantly prolonged the survival time (15.6 ± 5.4 days, P < 0.05) of mice after challenge infection with the virulent T. gondii RH strain (Type I), compared with the control groups which died within 8 days. Therefore, our data suggest that DNA vaccination with TgROP17 triggers significant humoral and cellular responses and induces effective protection in mice against acute T. gondii infection, indicating that TgROP17 is a promising vaccine candidate against acute toxoplasmosis. Toxoplasma gondii est un Apicomplexa parasite intracellulaire obligatoire, qui affecte l’homme et divers animaux domestiques et provoque de graves pertes économiques. Pour développer un vaccin efficace contre l’infection par T. gondii, nous avons construit un vaccin à ADN codant pour la protéine 17 des rhoptries de T. gondii (TRAP17) et avons évalué son efficacité protectrice immunitaire contre une infection aiguë par T. gondii chez la souris. Le vaccin à ADN (p3×Flag-CMV-14-ROP17) a été injecté par voie intramusculaire à des souris BALB/c et les réponses immunitaires des souris vaccinées ont été déterminées. Par comparaison avec des souris témoins traitées avec le vecteur vide ou du PBS, les souris immunisées avec le vaccin contre la ROP17 ont montré un niveau relativement élevé d’anticorps spécifiques anti-T. gondii et une réponse IgG1/IgG2a mixte avec prédominance de la production d’IgG2a. Les souris immunisées ont également montré une réponse proliférative lymphocytaire spécifique, une réponse immunitaire cellulaire de type Th1 avec production d’IFN-γ et d’interleukine-2, et une augmentation du nombre de cellules T CD8+. L’immunisation avec l’ADN ROP17 a prolongé de façon significative le temps de survie (15.6 ± 5.4 jours, P < 0.05) des souris après infection d’épreuve avec la souche virulente de T. gondii RH (type I), par rapport aux groupes de contrôle qui sont morts dans les 8 jours. Par conséquent, nos données suggèrent que la vaccination par ADN avec TgROP17 déclenche des réponses humorale et cellulaire importantes et induit une protection efficace chez la souris contre une infection aiguë par T. gondii, indiquant que TgROP17 est un candidat vaccin prometteur contre la toxoplasmose aiguë.

Toxoplasma rhoptry protein 16 (ROP16) is crucial in the host-pathogen interaction by acting as a virulent factor during invasion. To improve understanding of the molecular function underlying the effect of ROP16 on host cells, the present study analyzed the transcriptional profile of genes in the ROP16-transfected SH-SY5Y human neuroblastoma cell line. The transcriptional profile of the SH-SY5Y human neuroblastoma cell line overexpressing ROP16 were determined by microarray analysis in order to determine the host neural cell response to the virulent factor. Functional analysis was performed using the Protein Analysis Through Evolutionary Relationships classification system. The ToppGene Suite was used to select candidate genes from the differentially expressed genes. A protein-protein interaction network was constructed using Cytoscape software according to the interaction associations determined using the Search Tool for the Retrieval of Interacting Genes/Proteins. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis of the selected genes confirmed the results of the microarray. The results showed that 383 genes were differentially expressed in response to ROP16 transfection, of which 138 genes were upregulated and 245 genes were downregulated. Functional analysis indicated that the differentially expressed genes (DEGs) were involved in several biological processes, including developmental process, biological regulation and apoptotic process. A total of 15 candidate genes from the DEGs were screened using the ToppGene Suite. No significant differences in expression were observed between the RT-qPCR data and the microarray data. Transfection with ROP16 resulted in alterations of several biological processes, including nervous system development, apoptosis and transcriptional regulation. Several genes, including CXCL12, BAI1, ZIC2, RBMX, RASSF6, MAGE-A6 and HOX, were identified as significant DEGs. Taken together, these results may contribute to understanding the mechanisms underlying the functions of ROP16 and provide scope for further investigation of the pathogenesis of Toxoplasma gondii.

In the apicomplexans, endocytosed cargos (e.g., hemoglobin) are trafficked to a specialized organelle for digestion. This follows a unique endocytotic process at the micropore/cytostome in these parasites. However, the mechanism underlying endocytic trafficking remains elusive, due to the repurposing of classical endocytic proteins for the biogenesis of apical organelles. To resolve this issue, we have exploited the genetic tractability of the model apicomplexan Toxoplasma gondii , which ingests host cytosolic materials (e.g., green fluorescent protein[GFP]). We determined an association between protein prenylation and endocytic trafficking, and using an alkyne-labeled click chemistry approach, the prenylated proteome was characterized. Genome editing, using clustered regularly interspaced short palindromic repaet/CRISPR-associated nuclease 9 (CRISPR/Cas9), was efficiently utilized to generate genetically modified lines for the functional screening of 23 prenylated candidates. This identified four of these proteins that regulate the trafficking of endocytosed GFP vesicles. Among these proteins, Rab1B and YKT6.1 are highly conserved but are non-classical endocytic proteins in eukaryotes. Confocal imaging analysis showed that Rab1B and Ras are substantially localized to both the trans-Golgi network and the endosome-like compartments in the parasite. Conditional knockdown of Rab1B caused a rapid defect in secretory trafficking to the rhoptry bulb, suggesting a trafficking intersection role for the key regulator Rab1B. Further experiments confirmed a critical role for protein prenylation in regulating the stability/activity of these proteins (i.e., Rab1B and YKT6.1) in the parasite. Our findings define the molecular basis of endocytic trafficking and reveal a potential intersection function of Rab1B on membrane trafficking in T. gondii . This might extend to other related protists, including the malarial parasites. The protozoan Toxoplasma gondii establishes a permissive niche, in host cells, that allows parasites to acquire large molecules such as proteins. Numerous studies have demonstrated that the parasite repurposes the classical endocytic components for secretory sorting to the apical organelles, leaving the question of endocytic transport to the lysosome-like compartment unclear. Recent studies indicated that endocytic trafficking is likely to associate with protein prenylation in malarial parasites. This information promoted us to examine this association in the model apicomplexan T. gondii and to identify the key components of the prenylated proteome that are involved. By exploiting the genetic tractability of T. gondii and a host GFP acquisition assay, we reveal four non-classical endocytic proteins that regulate the transport of endocytosed cargos (e.g., GFP) in T. gondii . Thus, we extend the principle that protein prenylation regulates endocytic trafficking and elucidate the process of non-classical endocytosis in T. gondii and potentially in other related protists.