Construction Of Knockout Toxoplasma Gondii Strains Of Five Lipid Metabolizing Enzyme Genes And Their Basic Biological Functions

Toxoplasma gondii is an obligate intracellular protozoan which can infect almost all warm-blooded animals and humans,and cause zoonotic toxoplasmosis.Infection in individuals with impaired immune response can have serious or even fatal consequences.Lipids are crucial for T.gondii,which relies on complex absorption system as well as synthetic machinery to meet their lipid requirements.Many lipid metabolizing enzymes are involved in this process,among which malonyl Co A acyl carrier protein transacylase(Fab D),acyl ACP thiolesterase(TE)are involved in the FASII pathway;cytochrome b5 heme binding protein(CB5)is involved in the FAE pathway;diacylglycerol acyltransferase(DGAT)is involved in triglyceride biosynthesis;Glycerol-3-phosphate dehydrogenase(G3PDH)is involved in the biosynthesis of phospholipids.In this study,CRISPR-Cas9-mediated homologous recombination technique was used to insert a 6×HA tag proteins gene at the end of Fab D,TE,CB5,DGAT,and G3 PDH genes.The cellular localization of these five proteins in tachyzoite stage was observed by indirect immunofluorescence assay(IFA).It was found that Fab D was localized in the apicoplast of T.gondii,CB5 was localized in the endoplasmic reticulum of T.gondii,while TE,DGAT and G3 PDH were not observed by IFA with the relevant localization fluorescence.IFA results were consistent with the results of Western blot experiments in which bands consistent with the expected protein sizes were seen for both Fab D and CB5,but not for TE,DGAT and G3 PDH.In this study,Fab D,TE,DGAT,and G3 PDH knockouts as well as CB5 knockouts and complements in T.gondii RH/Pru strains were successfully constructed using CRISPR-Cas9 technology.The results showed that knockout of TE,DGAT and G3 PDH genes had no effect on T.gondii tachyzoite egress,plaque formation,replication,virulence,bradyzoite differentiation and brain cyst formation.Knockdown of the fabd and CB5 genes had no effect on tachyzoite egress,bradyzoite transformation,and virulence of RH,but both Fab D and CB5 deletions reduced the rate of T.gondii proliferation and formed fewer plaques;When we exogenously supplemented the Fab D deletion strain with fatty acids(C16:0),the ability to form plaques was restored;The ability to form plaques was restored after exogenous supplementation of CB5 deletion strains with fatty acids(C20:1).In the experiment of chronic infection,deletion of CB5 geneattenuated the virulence of Pru strain and reduced the formation of cysts in vivo.Mice inoculated with PruΔCB5 tachyzoites had 100% survival on day 30 post-infection,whereas mice inoculated with Pru wild strains and PruΔCB5C tachyzoites had only 10% survival.The survived mice were then re-inoculated with 100 tachyzoites of RH wild strain and the mice immunized with PruΔCB5 tachyzoites did not die,indicating that PruΔCB5 strain induced good immunoprotective effect on mice.In summary,this study successfully constructed T.gondii Fab D,TE,CB5,DGAT and G3 PDH localization and knockout strains,and CB5 gene complement strain in RH and Pru strains.The basic biological functions of knockout strains were studied,and it was found that knockout of Fab D and CB5 genes respectively reduced the proliferation rate,the plaques formed were smaller and the number was smaller,and attenuated the virulence of Pru strain and reduced the formation of cysts in vivo.These results lay a foundation for further in-depth study on lipid metabolism of T.gondii,and have important implications for the development of effective drugs and vaccines against toxoplasmosis.