Toxoplasma gondii is an opportunistic pathogen infecting humans and a variety of vertebrate animals. Secretory dense-granule proteins (GRAs) play diverse roles in the mediation of host−parasite interactions and facilitate parasitism, but many of them still remain to be identified. Here, we used two proximity-based protein labeling techniques to identify novel GRA proteins. Taking GRA1 as bait, transgenic strains expressing GRA1-BirA* or GRA1-APEX were constructed to biotinylate GRAs. Using these methods, a total of 46 proteins were identified, 20 of which were known GRA proteins. Among these 46, 17 were identified by both strategies, and 14 out of the 17 were known GRAs. The other three were all confirmed to localize to dense granules. Nonetheless a significant portion of the proteins were only identified by either APEX or BirA*, indicating that there are differences between these methods. Of the 26 novel GRAs, 5 were validated as bona f ide GRAs by localization studies. The majority of these novel GRAs are only present in coccidian parasites and are likely dispensable for parasite growth in vitro; they may play roles during animal infections. The identification of novel GRAs laid the foundation for further studies investigating the mechanisms underlying parasite−host interactions.
Toxoplasma gondii, as a zoonotic protozoan parasite, develops sophisticated strategies to manipulate hosts for efficient intracellular survival. After successful invasion, T. gondii injects many effector proteins into host cells for various purposes. TgROP16 (T. gondii rhoptry protein 16), which is secreted from rhoptries into host cells, can activate the host STAT (signal transducer and activator of transcription) signaling pathway through phosphorylation of STAT3 and STAT6. However, whether there are other host proteins modulated by TgROP16 is currently unknown. In this study, yeast two-hybrid (Y2H) screen was used to look for additional host proteins interacting with TgROP16. Yeast cells expressing a mouse cDNA library cloned into the prey vector were used to mate with yeasts expressing ROP16 without signal peptide. Two mouse proteins, Dnaja1 (DnaJ heat shock protein family member A1) and Gabra4 (gamma-aminobutyric acid A receptor, subunit alpha 4) were identified to interact with ROP16 from this screen. Further analysis suggested that the Predomain of ROP16 played key roles in mediating interactions with these host proteins, whereas the contribution from the Kinase domain was minor. The interactions between Dnaja1 and different parts of ROP16 were also estimated in vivo by co-immunoprecipitation. The results showed that the Predomain of ROP16 was the major region to interact with Dnaja1, which is consistent with the Y2H results. Based on the gene ontology analysis, Dnaja1 is predicted to participate in stress response while Gabra4 is involved in the system development process. The discovery of new host proteins that interact with ROP16 of T. gondii will help us to further investigate the functions of this effector proteins during T. gondii infection.
Several rhoptry proteins (ROPs) have been confirmed to be critical virulence factors of Toxoplasma gondii strains from North America and Europe. The two active kinases ROP17 and ROP18, and pseudokinase ROP5 were thought to be the key determinants of parasites' virulence in laboratory mice. Given the genetic diversity of Toxoplasma strains from different geographical regions, the virulence determinants in other strains, particularly the ones that are phylogenetically distant to the North American and European strains, are yet to be elucidated. In this study, we sought to examine the contribution of three known virulence factors to the virulence of a type I strain (T.gHB1) isolated from Central China. We deleted ROP17 and ROP18 individually, as well as in combination with GRA7 by the CRISPR-Cas9 system in this local isolate. Subsequent virulence tests in mice indicated that deletion of GRA7, ROP17, or ROP18 in T.gHB1showed similar attenuation in mice as the type I RH strain lacking the corresponding proteins. However, in contrast to the reported double knockouts in RH, double deletions of GRA7 plus ROP17 or GRA7 plus ROP18 in T.gHB1 did not show significant further virulence attenuation compared to the ROP17 or ROP18 single knockouts. These results indicated that GRA7, ROP18 and ROP17 may play different roles in virulence determination in genetically diverse strains of Toxoplasma.
Background
Toxoplasma gondii infects almost all warm-blooded animals, and cats play a crucial role in the epidemiology of T. gondii as the definitive host. Despite sporadic reports on the seroprevalence of T. gondii in domestic cats, systematic surveys are lacking and some regions remain in China uninvestigated.
Methods
A total of 1,521 serum samples were collected from 10 regions of China and analyzed by antibodies against T. gondii by ELISA with the purpose of identifying risk factors of T. gondii infection in cats across China and obtaining seroprevalence data from some previously uninvestigated areas.
Results
Antibodies to T. gondii were detected in 62 of 1,478 (4.2%) urban pet cats and in 9 of 43 (20.9%) stray cats. Among the regions examined, the prevalence was 13% in Sichuan, 12.8% in Chongqing, 6.4% in Hunan, 2.5% in Hubei and 0.9% in Guangdong. Additionally, this is the first report on the seroprevalence of T. gondii in urban pet cats from Qinghai (6.2%), Anhui (3.1%), Jiangxi (2.5%), Shaanxi (2.4%) and Ningxia (1.6%). The age and lifestyle (stray or pet) of cats were identified as the risk factors for seropositivity by multivariate analysis of the data.
Conclusions
Our findings improve our understanding of seroprevalence and risk factors of T. gondii infection in cats across China, and provide useful information for the formulating of preventive and control measures against this widespread zoonotic parasite.
Toxoplasma gondii
is an intracellular pathogen that threatens human and animal health. This unicellular parasite is active in many biological processes, such as egress and invasion.
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