Construction And Study On Phenotypes And Transcriptome Of Toxoplasma Gondii RH-ΔTgcsp2 Strain

Toxoplasma gondii,an important zoonotic pathogen,can cause a variety of acute clinical symptoms in immunocompromised humans and animals,such as fever,pneumonia and myocarditis.In addition,the infection can be acquired during pregnancy,causing fetal encephalitis,retinitis and more serious consequences.In order to develop safe and effective vaccines,there have been many studies on virulence-related proteins of T.gondii,such as dense granule proteins(GRAs)and rhoptry proteins(ROPs).Previous studies have indicated that Cold Shock Proteins(CSPs)could regulate processes of transcription and translation in bacteria,plants and animals.During the invasion of Salmonella spp.into host cells,CspC and CspE were identified to be involved in the regulation of gene expression,and play important roles in motility,biofilm formation and virulence of these bacteria.Additionally,CSPs are crucial for plant cell development and animal cell proliferation and migration,but there have been no studies about the function of CSPs in T.gondii.In the present study,the CRISPR/Cas9 technology was used to construct a Tgcsp2 gene knockout strain of T.gondii,and the changes in phenotype and transcriptome were also studied to analyze the effects of Tgcsp2 gene of this parasite.1.The Tgcsp2 knockout T.gondii strain(RH-ΔTgcsp2)was successfully constructed.The resistance marker was inserted into the Tgcsp2 gene of RH tachyzoites by using the CRISPR/Cas9 method.After selection by pyrimethamine,the parasites resistant to pyrimethamine were identified by the diagnostic PCR and real-time PCR.Compared with the wide-type RH strain,the resistance marker was indeed inserted into the RH-ΔTgcsp2 strain.2.Phenotypes of Toxoplasma gondii RH-ΔTgcsp2 strain were studied.Human foreskin fibroblasts(HFF)cells were infected with wide-type RH tachyzoites and RH-ΔTgcsp2 tachyzoites.Compared with the wide-type RH strain,the plaques produced by RH-?Tgcsp2 strain were smaller and fewer.When HFF cells were infected with different doses of the parasites,RH-?Tgcsp2 strain caused less monolayer lysis compared with wide-type RH strain.Indirect immunofluorescent assay(IFA)showed that RH-?Tgcsp2 strain was less able to invade HFF host cells compared to the RH strain,and the intracellular replication and egress of RH-?Tgcsp2 strain were impaired compared with RH strain.Virulence assay indicated that mutation of the Tgcsp2 gene attenuated the virulence of T.gondii during in vivo infection.3.The transcriptome of Toxoplasma gondii RH-ΔTgcsp2 strain was obtained.In order to explore the effect of Tgcsp2 gene deletion on gene expression of T.gondii,in this study,RNAseq technology was used to obtain the transcriptome data of RH-ΔTgcsp2 and RH strain.The results showed that there were 7392 and 7492 genes expressed in RH-ΔTgcsp2 and RH strains,respectively.Compared with RH strain,there were 169 genes specifically expressed in RH-ΔTgcsp2 strain,and 5000 genes were differentially expressed(2582 up-and 2418 downregulated),but 269 genes did not express in RH-ΔTgcsp2 strain.Functional analysis of these differentially expressed genes(DEGs)showed that they were enriched into 3873 GO terms and 72 KEGG pathways.GO analysis indicated that these DEGs were likely to participate in metabolic process,catalytic activity and regulation of biosynthesis,while KEGG analysis showed that they would be mainly related to protein processing,proteasome,DNA replication,biosynthesis and toxoplasmosis.Taken together,the present study indicated that the deletion of Tgcsp2 weakened the invasion,intracellular replication and egress and the virulence to mice of T.gondii.Transcriptome analysis suggested that the differences in phenotypes would be associated with expression of these DEGs.These results provided basic data for studying the function of TgCSP2 of T.gondii,as well we the theoretical foundations for the development of vaccines using attenuated strains.