Characterization of functions in parasite growth and virulence of four Toxoplasma gondii genes involved in lipid synthesis by CRISPR-Cas9 system.

ABSTRACT

Fatty acid uptake is extremely important for the survival and growth of the intracellular parasite Toxoplasma gondii. In this study, CRISPR-Cas9 gene editing technology was used to investigate the role of four lipid synthesis enzymes, namely, glycerol-3-phosphate dehydrogenase (G3PDH), malonyl CoA-acyl carrier protein transacylase (FabD), acyl-ACP thiolesterase (TE), and diacylglycerol acyltransferase (DGAT), in the virulence and infectivity of Type I RH and Type II Prugniaud (Pru) strains of T. gondii. Immunofluorescence analysis of the tachyzoite stage showed that FabD protein was located in the apicoplast; however, the expression level of the other three proteins was undetectable. Compared with wild-type (WT) strains, the growth of RHΔG3PDH, RHΔTE, and RHΔDGAT in vitro and their virulence in vivo were not significantly different. However, RHΔFabD exhibited a significantly reduced growth rate, compared with the WT strain. The deletion of FabD attenuated the virulence of Type II Pru strain and reduced the formation of cysts in vivo. These data improved our understanding of the role of lipid synthesis enzymes in the pathogenesis of T. gondii.