Construction And Biological Function Of Four FAS? Synthase Gene Deletion Strains Of Toxoplasma Gondii

Toxoplasma gondii(T.gondii)is an intracellular Apicomplexa protozoan,with a wide range of hosts,and it is distributed all around the world,seriously endangering human health and the development of animal husbandry.Parasitophorous vacuole membrane(PVM)is formed in the process of T.gondii invading host cells,following the recruiting of mitochondria and endoplasmic reticulum in host cells,to maintain its own growth,promote its movement and provide necessary nutrition for the development of newborn tachyzoites by using nutrients in host cells as energy substrates.Fatty acids are the main components of parasitophorous vacuole(PV)and organelles of T.gondii,and the metabolism of fatty acids plays a key role in the survival and replication of T.gondii.However,at present,the research on the metabolic process of fatty aci ds in T.gondii is still very limited,and the functional analysis of the key genes in the process of fatty acid biosynthesis is still not comprehensive.In this study,CRISPR-Cas9 gene editing technology was used to explore the biological function of four genes[beta-hydroxyacyl-acyl carrier protein dehydratase(FabZ),3-oxoacyl-acyl-carrier protein synthase I/II,putative(FabB/F),3-oxoacyl-[acyl-carrier-protein]reductase(ODSCI)and 3-oxoacyl-[acyl-carrier-protein]reductase(ODSCII)]in type II fatty acid synthesis(FAS?)pathway of T.gondii.FAS? pathway is a unique and very important pathway of T.gondii,while little is known about the role of FAS? related genes in the growth and development of T.gondii.Therefore,we used CRISPR-Cas9 gene editing technique to construct knockout strains targeting the four genes(FabZ,FabB/F,ODSCI and ODSCII)on two strains of T.gondii(Type I RH strain and Type II PRU strain).Through immunofluorescence experiment and q PCR analysis,we found that,compared with wide type(WT)strain,the apicoplast of RH?FabZ and RH?FabB/F strains was partially lost,and the expression of apicoplast genome was significantly decreased,which indicated that FabZ and FabB/F genes played an important role in the integrity of apicoplast and maintenance of T.gondii genome.The results of plaque assay and replication test showed that the growth ability of RH?FabZ and RH?FabB/F strains was significantly inhibited in vitro,while similar changes were not detected in RH?ODSCI and RH?ODSCII strains.Compared with RH?ODSCI and RH?ODSCII,the virulence of RH?FabZ and RH?FabB/F in mice was lower and the survival rate was higher.Compared with PRU?ODSCI and PRU?ODSCII,the PRU?FabZ and PRU?FabB/F significantly reduced the number of brain cysts in mice.These findings suggest that FabZ and FabB/F play important roles in the growth,development and pathogenicity of T.gondii,while ODSCI and ODSCII do not contribute to these characteristics.According to the phenotypic differences between WT strain and the RH?FabZ and RH?FabB/F strains,we speculated that deletion of FabZ and FabB/F should also have influence on gene expression profiles,so we analyzed the transcriptomes of RH WT,RH?FabZ and RH?FabB/F.Compared with the WT strain,1994 differentially expressed genes were identified in RH?FabB/F strain;of which,1156 genes were up-regulated and 838 genes were down-regulated;while 1846 differentially expressed genes were identified in FabZ deletion strain,among them,1210 genes were up-regulated and 636 down-regulated.Further analysis of differentially expressed genes showed that eight genes were up-regulated simultaneously in RH?FabZ and RH?FabB/F,and these genes were involved in the FAS? pathway;9 genes were down-regulated at the same time,and they are mainly AP2 domain transcription factors.It was found that they were mainly involved in lipid metabolism,cell biosynthesis,enzyme activity,amino sugar and nucleotide sugar metabolism and secondary metabolites through GO and KEGG analysis of differentially expressed genes,indicating that when FabZ and FabB/F in T.gondii were knocked out,T.gondii provided itself with energy and nutrients to maintain its own growth and development through the metabolism and synthesis process involved by these differentially expressed genes.Significantly lower content of myristic acid and palmitic acid in RH?FabB/F strain was detected when compared with WT strain through analysis of fatty acid content by GC-MS and isotope labeling techniques.The isotopic maps of C14:0 and C 16:0 showed that there were a large number of M8-M14/16 isotope isomers(corresponding to ¹³C labeled 8-14 or16 carbon)in the WT strain,while that of the majority of RH?FabB/F strain were located in the M0 component(without¹³C labeled).This suggested that FabB/F plays an important role in the flow of ¹³C into medium-chain fatty acids.In summary,the present study evaluated the effects of FabZ,FabB/F,ODSCI and ODSCII genes involved in FAS? pathway on the development and pathogenicity of T.gondii RH strain and PRU strain by using CRISPR-Cas9 gene editing technology,transcriptome sequencing and metabolic techniques,and the results indicated that FabZ and FabB/F play an important role in the development and pathogenicity of T.gondii.The differentially expressed genes are mainly involved in lipid metabolism,cell biosynthesis,enzyme activity,amino sugar and nucleotide sugar metabolism and secondary metabolites,indicating that the phenomenon of slow growth and reduced virulence of the deleted strains is due to the up-regulation or down-regulation of genes to support its continued growth.¹³C-glucose isotope labeling experiment showed that the deletion of FabB/F led to the inhibition of fatty acid synthesis of T.gondii,which laid a foundation for further elucidation of the mechanism of fatty acid metabolism in Toxoplasma gondii.