TgMIC6: Virulence Determinant Of Chinese1 Toxoplasma And Its Pathogenesis

Background:Toxoplasma gondii is an obligate intracellular protozoan parasite potentially invading all nucleated cells of warm-blooded vertebrates,infecting approximately one-third of the human population worldwide.In immunocompetent individuals,a generally asymptomatic acute infection is followed by the onset of a quiescent bradyzoite parasite stage,leading to long-term chronic infections.Previous studies have reported that multiple genetic types of T.gondii are distributed worldwide,of which types ?,? and ? are the classical strains in North America and Europe.Furthermore,T.gondii strains are much more genetically diverse in South America;however,most parasites in East Asia fall within one nonclassical lineage:Chinese 1.Among laboratory mice,excluding wild mice or other intermediate hosts,type ?parasites kill the host in the acute phase of the infection,possibly due to hyperinflammation-inducible cytokine storm and uncontrolled parasite dissemination.Type ? and ? strains typically exhibit less virulence and may even be avirulent.Furthermore,our previous studies have demonstrated that the isolates of Chinese I vary in their virulence to mice,of these WH3 exhibit high virulence,while WH6 display low virulence.In T.gondii-infected human/murine macrophages and nonhematopoietic cells,CD40 interacts with CD40L(CD154)expressed on the surface of T cells to trigger the killing of the parasite through an autophagy-dependent pathway.The Toxoplasma micronemal proteins MIC3,and MIC6 act as ligands for EGFR,inducing phosphorylation of this protein in host cells and activating the PI3K/Akt signaling pathway.This event prevents the expression of the autophagy protein LC3 and vacuole-lysosomal fusion.Subsequently,the parasite is prevented from being targeted by the host autophagy machinery.In mammalian cells,Akt phosphorylation also results in the activation of mTORC1,which negatively regulates autophagosome formation,thus inhibiting autophagyObjective:In this study,whole-genome sequencing and classical virulence gene polymorphism analysis were performed on the representative strains WH3 and WH6 of Toxoplasma Chinesel.To investigate the regulatory effect of differentially expressed TgMIC6 on Toxoplasma Chinesel and its mechanismMethods:(1)Complete WH3 and WH6 genomes sequencing,assembly and annotation The WH3 and WH6 tachyzoites were collected and purified,and the DNA was extracted and sent to Annooad Genomics Inc.,for library construction,and sequenced using PacBio Sequel/Sequel ? platform.HQRF(High Quality Region Finder)was used to identify the longest region where Singly-loaded enzyme remained active,and SNR(Signal No ISE Rat IO)was used to filter the low-quality region.After three generations of data assembly and correction,they were spliced into chromosomes;(2)Polymorphism analysis of virulence related genes of WH3 and WH6:The polymorphism analysis of several important virulence factors(ROP18,ROP5,ROP16,GRA15)regulating classic genotypes of Toxoplasma gondii:Type ?,Type ? and Type? were compared and analyzed(3)Verification of WH3 and WH6 transcriptome data and differential expression of MIC6 protein between WH3 and WH6:the differential expression of TgMIC6 in WH3 and WH6 was verified by qPCR and Western-blot;(4)Construction and validation of WH3?mic6 strain:The TgMIC6 knockout strain was further prepared by CRISPR/Cas9 technology;(5)Detection of wild-type WH3(WH3-WT),TgMIC6 knockout WH3(WH3?micc6)and WH6 invasion and proliferation in vitro;(6)Toxicity test of WH3-WT,WH3?and WH6 mice and detection of insect load and inflammatory factors:1000 T.gondii tachyzoites were injected intraperitoneally The death of mice in each group was observed within 20 days,and the formation of intracranial cystic in surviving mice was observed after 40 days.Tissue DNA was extracted from the brain,eyes,liver,spleen and lymph nodes on 1/3/5 day after infection Real-time PCR was used to detect T.gondii 529 gene load.The expression levels of IFN-?,IL-12,TNF and iNOS were detected by ELISA(7)Different effects of WH3-WT,WH3?mic6 and WH6 on the classical autophagy pathway induced by CD40-CD154:CD 154/IFN-? recombinant protein was added in vitro and autophagosomes were observed under electron microscopy.Western-blot analysis:LC3II/I,Tyr1068 p-EGFR,Tyr1148-p-EGFR,Akt,Ser473-p-Akt,mTOR Autophagy flow was observed by mRFP-GFP-LC3 tandem fluorescent protein adenovirusResults:(1)After correction,the WH3 genome size was 63,476,350 bp,Contig N50 was 2,433,820 bp,and the number of assembled Contig was 64.There was no obvious GC-depth segmentation.Using the 19.86g genome sequencing data of Toxoplasma gondii and using k-mer=21 for analysis,the genome size was 70.63MbP,the modified WH6 genome size was 64.53MBP,the heterozygosity rate was 0.78%,and the repeat sequence proportion was 8.37%.At the same time,K-mer=35 was used for preliminary assembly,and the assembled Contign50 was 150bp with a total length of 436,373,055bp.The Scaffold N50 was 150bp with a total length of 440,639,185bp(2)By comparing the CDS sequences of ROP18,ROP5,ROP16 and GRA15,it was found that there was no difference between WH3 and WH6,and the mRNA expression levels of ROP18 and ROP5 were not significantly different between WH3 and WH6 These effector molecules may not be the virulence factors that lead to the difference in the virulence of WH3 and WH6(3)The expression level of TgMIC6 in WH3 was significantly higher than that of WH6 by qPCR and Western blot,and the protein expression level of WH3 was 2.8 times higher than that of WH6.WH3?mic6 parasites showed lower virulence than that of the wild type in infected mice.We through the CRISPR/CAS9 targeted gene knockout technology successful build TgMIC6 defect strains WH3-?mic6.Compared with wild type,WH3-?mic6 virulence in mice and the invasion of cell efficiency,intracellular proliferation and plaques are reduced,serological detection WH3-?mic6 infected mice induced strong IFN-?,IL-12,and TNF alpha secretion.Western blot,electron microscopy,and mRFP-GFP-LC3 tandem fluorescent protein adenovirus were revealed that TgMIC6 antagonizes host cell defenses by regulating the classical CD40-CD154 autophagy pathway.Conclusions:Whole-genome sequencing data of WH3 and WH6 showed that the non-functional ROP5/18 combination and the same copy number of ROP5 did not explain the significant difference in virulence between WH3 and WH6.We found that the expression of TgMIC6 in the WH3 was significantly higher than that of the weak strain WH6.TgMIC6,in addition to its role in Toxoplasma invasion,it can also inhibit autophagy activity after invasion of host cells.In conclusion,we speculated that TgMIC6 might be the effector molecule responsible for the difference in virulence between WH3 and WH6.This molecule antagonizes the host cell defense by regulating the classical CD40-CD154 autophagy pathway,which leads to different proliferation and pathogenicity of the two representative strains of Chinese 1 in the host.