Virulence Test Of Toxoplasma Gondii CK1α Negatively Infect Mice And Mechanism Research

Toxoplasma gondii, belonging to the phylum Apicomplexa, is an intracellular protozoan parasite that infects almost all warm-blooded animals and humans, and may cause devastating diseases in the developing fetus and immunocompromised individuals. In recent years, a growing body of research suggests that T. gondii infection may affect behaviors of hosts and may be responsible for certain psychiatric disorders.Protein kinases(PKs) of T. gondii play crucial roles in its proliferation,differentiation, and pathogenesis. Previous studies have demonstrated that calcium-dependent protein kinase 1(CDPK1) is an essential regulator of exocytosis in T. gondii, CDPK3 regulates calcium-dependent egress of the parasite from host cells,and mitogen-activated protein kinase 1(MAPK1) controls parasite growth by inhibiting host interferon(IFN)-γ mediated inducible nitric oxide synthase(iNOS).Rhoptry kinases, the T. gondii-specific PKs, are involved in host manipulation, and co-opt host transcription factors and eventually modulate gene expression. The casein kinase 1 family(CK1) is evolutionary conserved within eukaryotes and regulates a wide variety of vital cellular processes, including Wnt signaling, circadian rhythms,membrane traffiCKing, and cytoskeleton maintenance. Recently, CK1 has also been described in various protozoan organisms, such as Toxoplasma, Plasmodium,Leishmania and Trypanosma. Additionally, the inhibitors of CK1(trisubstituted pyrrole and imidazopyridine) can block the growth of L. major promastigotes and T.brucei bloodstream forms in vitro.Toxoplasma gondii genome can encode two CK1 subtypes like CK1 alpha and CK1 beta, but only CK1 has the enzyme activity. CK1 plays an important role in many physiological activities, such as cell division and apoptosis, DNA repair, P53 regulation, and other cyclical rhythms of the body.As an important model organism and original zoonosis, the role of Toxoplasma gondii CK1α is unclear in process of infect host.CK1α deficient strains(△ck1α) which the laboratory has been constructed is used to infect BALB/c mice. The parent strain GT1 infect BALB/c mice is used as control group to determine the impact of CK1α when t.gondii infect the host.Quantitative PCR, ELISA, transcriptome sequencing and Western blot are used to deduced the specific mechanisms that how CK1α impact the virulence of t.gondii when infecting the host.Our aim is to provide some basis on the prevention of toxoplasma and the development of vaccines and drugs..Research contents:1. Three kinds of T.gondii are used to infect BALB/C mice,to determine the influence of CK1 a delection on T.gondii.2. To determine the specific pathway of CK1 a affect the virulence of T.gondii.Research method:1. BALB/c mice were infected intraperitoneally with 102, 103, or 104 parental GT-1, Δck1α, or complemented tachyzoites,to investigate the effect of CK1α deletion on parasite virulence in mice. ELISA are used to determine the levels of hepatocyte cytoplasmic enzymes released into the serum. quantitative PCR are used to measure the parasite tissue burden. Fluorescence are used to determine the levels of serum cytokines2. Western blot to compare the difference of MAPK、NF-κB and STAT activation between parental GT-1 or Δck1α parasites. transcriptome sequencing to investigate whether the ΔCK1α strain might have global changes in gene expression.Research results:1. The mice that were infected with 104 parental parasites started to show clinical signs, including swollen abdomen and ruffled fur at day 5 post-infection(pi), and died between day 7 and 9 pi, with mean survival time(MST) of 7.7 days. The mice infected with 102 and 103 parental parasites died between days 10 and days 13 pi, with MST of 11.4 days and 10.4 days, respectively. In contrast, the mice that were challenged with 104 Δck1α parasites showed signs of disease at days 4 pi, and all died between days 6 and days 8 pi, with MST of 6.8 days, and all the mice injected with102 and 103 Δck1α parasites died between day 8 and day 12 pi, with MST of 10.5 days and 8.4 days, respectively. Significant difference of MST between parental GT-1 andΔck1α parasites was observed in the two groups with different infection doses. No significant difference was observed in MST between the parental GT-1 and complemented parasites.Research target:2. Severe pathology caused by Δck1α parasites was found in the liver tissue,showing generalized hepatocyte enlargement, cytoplasmic vacuolization, nucleus dissolution and loss of sinusoid architecture at days 7 pi, while the parental GT-1parasites induced little tissue pathology at that time. For AST, the levels of mice infected with Δck1αparasites were elevated 2-5 times at days 3 and 7 pi over the mice inoculated with parental GT-1 parasites. The level of the liver associated enzyme ALT was 10-12 times higher in Δck1α-infected mice compared with the control mice infected with GT-1 parasites. Δck1α tachyzoites showed significantly higher parasite burdens than the parental GT-1 parasites in liver and spleen tissues. CK1α deletion can inhibit the IL-12 and IFN-γ induction triggered by infection with the parental GT-1 in parasites.3. Infection with Δck1α parasites only stimulated an increase in MAPK phospho-p38 in liver tissues, and no significant difference was found in other phosphorylated proteins, including NF-κB p-p65, MAPKs p-ERK and p-JNK. In spleen tissues, no significant difference was found in the phosphorylation levels between the two groups infected with parental GT-1 and ΔCK1α parasites. Compared with the parental GT-1 parasite, infection with Δck1α parasite caused an increase in phosphorylation of STAT3 at Tyr705 and STAT6 at Y604, and a decrease in phosphorylation of STAT1 at Tyr701 and STAT4 at Y705 in both liver and spleen tissues. The transcriptome of the Δck1α and parental GT-1 parasites was compared using RNA-seq,show the up-regulated and down-regulated genes in the Δck1α.Among the up-regulated genes, we found many genes encoding rhoptry proteins(ROPs), which are secreted from rhoptry organelles, and constitute important virulence factors for T. gondii, including ROP5, ROP16, and ROP18.Research conclusion:1. We constructed a T. gondii CK1α deletion mutant(Δck1α), and a complementation mutant with restored CK1α expression, by which the biological role of CK1α during T. gondii infection was identified. Infected mice with ΔCK1α parasite resulted in decreased amounts of IL-12p70 production, severe liver damage, and short survival time relative to the CK1α-positive GT-1 parental strain.2. CK1α negatively regulates virulence of T. gondii in mice by inhibiting production of IL-12 via activation of STAT3.