Identification And Functionality Of Toxoplasma Gondii Stearoyl-coenzyme A Desaturase

Toxoplasma gondii is an obligative intracellular protozoan parasite, infecting karyocytes of almost all the warm-blooded animals and human. In the life cycle of T. gondii, indispensable catabolic and anabolic reactions are performed in the parasite through its relative perfect metabolic system, although many nutritive materials are scavenged from the host cells. The paraites is capable to de novo synthesize saturated fatty acid; however, it is still unclear whether unsaturasted fatty acids can be synthesized in T. gondii.Stearoyl-coenzyme A desaturase （SCD） is the key enzyme in unsaturated fatty acids synthesis. By protein alignment search with Plasmodium falciparum SCD in ToxoDB, a putative fatty acyl-CoA desaturase （TGGT1₂38950） was retrieved, with the similarity of 54%. The bioinformatics analysis revealed a typical A 9 desaturase domain in the protein. The 3129 bp full coding sequence of this gene was obtained by RT-PCR with T. gondii RH strain RNA, and was 100% identical with TGGT1₂38950. The truncated prokaryotic protein of this gene was expressed and the mice polyclonal antibodies were prepared. The expression of this gene in T. gondii tachyzoites was confirmed by western blot, with a 120 kDa band of expected molecular weight. The parasite line of endogenous HA-tagged gene was constructed. The results of IFA revealed an endoplasmic reticulum （ER） localization of the endogenouse protein, which was consistent with fatty acid desaturases in other organism. Therefore, the TGGT1₂38950 was designated as T. gondii SCD.Sterculic acid is a cyclopropene fatty acid, which is an inhibitor of SCD. In vitro experiments showed that sterculic acid or its analogue esters all inhibited the proliferation and egress of intracellular T. gondii to some extent. With the lowest EC50 value againt T. gondii by sterculic acid, it was also toxic to host cells. In contrast, test compound INT21 exhibited anti-T. gondii activity, and no cytotoxic effects were observered in host cells. The in vivo experiments showed that the brain parasite load was significantly reduced in mice infected with Pru strain after intraperitoneal injection with INT21. However, INT21 treatment did not protect mice infected with RH strain.For further study of the role of TgSCD playing in the parasite growth, based on CRISPR/Cas9 gene editing technology, SCD conditional knockout mutants in T. gondii TATi strain were obtained. The in vitro growth and phathogenicity in mice of mutants were did not change whether TgSCD was functional or not, suggesting SCD might not be dispensable for tachyzoites growth and proliferation. SCD overexpressed line was constructed for further observation the SCD function. The portion of oleic acid increased in SCD overexpressed parasites, compared with RH parental strain, indicating T. gondii indeed was competent for unsaturated fatty acid synthesis. The SCD overexpressed tachyzoites propogated slower than the parental strain, with decreased invision capability and weaker pathogenicity in mice. The detection of TgIF2a phosphorylation and expression change of several associated gene demonstrated ER stress was triggered in SCD overexpressed parasites, which were more apt to autophagy and apoptosis.To sum up, the existance of SCD in T. gondii was demonstrated and it functions as fatty acid desaturase, involving in the process of parasite autophagy. However, the deletion of TgSCD did not significantly affect tachyzoites growth. The present work was a preliminary research on the biological function of TgSCD, which provided scientific basis for the further study of its functional mechanism.