Characterization of a second sterol‐esterifying enzyme in T oxoplasma highlights the importance of cholesterol storage pathways for the parasite

Summary Lipid bodies are eukaryotic structures for temporary storage of neutral lipids such as acylglycerols and steryl esters. Fatty acyl‐ CoA and cholesterol are two substrates for cholesteryl ester ( CE ) synthesis via the ACAT reaction. The intracellular parasite T oxoplasma gondii is incapable of sterol synthesis and unremittingly scavenges cholesterol from mammalian host cells. We previously demonstrated that the parasite expresses a cholesteryl ester‐synthesizing enzyme, TgACAT 1. In this article, we identified and characterized a second ACAT ‐like enzyme, TgACAT 2, which shares 56% identity with TgACAT 1. Both enzymes are endoplasmic reticulum‐associated and contribute to CE formation for storage in lipid bodies. While TgACAT 1 preferentially utilizes palmitoyl‐ CoA , TgACAT 2 has broader fatty acid specificity and produces more CE . Genetic ablation of each individual ACAT results in parasite growth impairment whereas dual ablation of ACAT1 and ACAT2 is not tolerated by T oxoplasma . Δ ACAT 1 and Δ ACAT 2 parasites have reduced CE levels, fewer lipid bodies, and accumulate free cholesterol, which causes injurious membrane effects. Mutant parasites are particularly vulnerable to ACAT inhibitors. This study underlines the important physiological role of ACAT enzymes to store cholesterol in a sterol‐auxotrophic organism such as T oxoplasma , and furthermore opens up possibilities of exploiting TgACAT as targets for the development of antitoxoplasmosis drugs.