Screening And Identification Of Host Proteins That Interact With Toxoplasma Gondii Micronemal Proteins

As an important zoonotic pathogen at worldwide, Toxoplasma gondii is an obligate intracellular protozoan that belongs to the phylum apicomplexa parasite. This parasite can infect with all kinds of warm-blood animals including human. The broad host range of T. gondii is due to the successful mechanism of host infection, which including invasion and modulation of host cells. During parasite infection, host cell adhesion and modulation require the secretion of proteins from micronemes, a unique organelles in apicomplexan parasites that are specialized in protein secretion. Although most micronemal proteins(MICs) contain several adhesive domains, the specific host proteins that interact with MICs are still unknown. Thus, identification of host proteins that specific interact with MICs will help us to better understand the mechanism of T. gondii invasion and host modulation, and will also support the molecular basis of the critical role of host proteins in pathogenic process.In this study, we sought to identify host proteins interacting with MICs by yeast-two-hybrid screens. Using micronemal protein AMA1, MIC2 and MIC3 as bait to screen a library expressing mouse proteins, we found two host proteins interacting with integrin A-like domain of MIC2 and eight host proteins interacting with full-length MIC3. Two of MIC3 screened host proteins, spermatogenesis-associated protein 3(Spata3) and dickkopf-related protein 2(Dkk2), were further confirmed to interact with MIC3 both in vitro and in vivo by additional protein-protein interaction tests. Finally, we tested the effect of Dkk2-mediated host Wnt/β-catenin signaling pathway by Toxoplasma gondii and MIC3 protein.(1) Screening of host proteins that interacting with Toxoplasma gondii micronemalproteinsTotal RNA was extracted from purified tachyzoites of Toxoplasma gondii RH strain. The coding sequence of ectodomain of AMA1, ectodomain of MIC2 and full-length MIC3 were PCR amplified from tachyzoties c DNA using specific primers and cloned into p GBKT7 vector to make the bait plasmids for yeast-two-hybrid screening. After auto-activation and toxicity test, we found that MIC2 ectodomain bait plasmid possesses auto-activation activity for yeast-two-hybrid system. Thus, the ectodomain of MIC2 was truncated to two domains, integrin A-like domain(A/I) and thrombospondin repeats domain(TSR) and the two domains were constructed into bait plasmids. We detected robust auto-activation activity for TSR domain, but not for A/I domain, which was used for host protein screening. We screened a library expressing mouse proteins and found two host proteins that showed positive interactions with MIC2-A/I, including LAMTOR1 and RNase H2 b, and eight host proteins showed positive interactions with MIC3, they are Svil, Phf7, Dkk2, Tmem100, Spata3 iso2, Spata3 iso3 and two uncharacterized protein LOC210940, LOC72128, but there is no any host proteins showed positive interactions with AMA1.(2) Confirmation of interaction between MIC3 and positive hitsThe full-length coding sequence of MIC3 screened host protein Phf7, Dkk2, Tmem100 and Spata3 iso3, and the prey plasmid inserted fragment of Svil and two uncharacterized proteins were PCR amplified from mouse c DNA using specific primers. Firstly, the point-to-point yeast-two-hybrid was performed to exclude the fasle positive in yeast cells. We found that interaction between MIC3 and host protein Spata3, Dkk2 and two uncharacterized proteins are truly positive interaction in yeast system. Secondly, we confirmed that MIC3 interacts with Spata3 and Dkk2 using in vitro GST Pull-down. Finally, interaction between MIC3 and host protein Spata3 and Dkk2 were also confirmed using Co-IP and Bi Fc. Taken together, micronemal protein MIC3 can be interacted with host protein Spata3 and Dkk2 in vitro and in vivo.(3) Identification of interacted domain of MIC3 that interacting with screened hostproteinsFull-length MIC3 was truncated to two domains, N-terminal chitin binding-like domain(CBL) and an C-terminal tandem repeat of epidermal growth factor-like domains(EGF). Coding sequence of the two domains were cloned into p GADT7 vector, and mated with MIC3 screened host proteins cloned into p GBKT7 vector. The result showed that none of screened host protein interacted with the CBL domain of MIC3. Same as full-length MIC3, the EGF domain mating with host protein Spata3, Dkk2 and two uncharacterized proteins were showed positive interaction. These indicate that the EGF domain of MIC3 is the key domain in mediating MIC3-host protein interactions.(4) Effect of Toxoplasma gondii on host Wnt/β-catenin signaling pathwayAs a MIC3 screened host protein, Dkk2 plays an important role in modulation of host Wnt//β-catenin signaling pathway. Thus, we focus on the effect of Toxoplasma gondii on host Wnt/β-catenin signaling pathway. Using Wnt mediated-TOP-Flash luciferase reporter gene system, we found that T. gondii infection was unable to activate or inhibit the signaling transduction. Similarly, MIC3 protein also did not alter Wnt signaling transduction. We also found that T. gondii infection and MIC3 transfection could not effect on the expression level of β-catenin using Western blot analysis. Taken together, these results suggest that although Tg MIC3 can directly interact with host protein Dkk2, neither Toxoplasma gondii infection nor Tg MIC3 affect the host Wnt/β-catenin signaling pathway.These results of the present study that interaction between Toxoplasma gondii micronemal proteins and host proteins are not only greatly help us to better understand the mechanism of host invasion and infection, but also provide the theoretical basis for discovery of anti-Toxoplama new drug and vaccine.