The N-terminal domain and glycosomal localization of Leishmania initial acyltransferase LmDAT are important for lipophosphoglycan synthesis.

Ether glycerolipids of Leishmania major are important membrane components as well as building blocks of various virulence factors. In L. major, the first enzyme of the ether glycerolipid biosynthetic pathway, LmDAT, is an unusual, glycosomal dihydroxyacetonephosphate acyltransferase important for parasite's growth and survival during the stationary phase, synthesis of ether lipids, and virulence. The present work extends our knowledge of this important biosynthetic enzyme in parasite biology. Site-directed mutagenesis of LmDAT demonstrated that an active enzyme was critical for normal growth and survival during the stationary phase. Deletion analyses showed that the large N-terminal extension of this initial acyltransferase may be important for its stability or activity. Further, abrogation of the C-terminal glycosomal targeting signal sequence of LmDAT led to extraglycosomal localization, did not impair its enzymatic activity but affected synthesis of the ether glycerolipid-based virulence factor lipophosphoglycan. In addition, expression of this recombinant form of LmDAT in a null mutant of LmDAT did not restore normal growth and survival during the stationary phase. These results emphasize the importance of this enzyme's compartmentalization in the glycosome for the generation of lipophosphoglycan and parasite's biology.

Leishmania dihydroxyacetonephosphate acyltransferase LmDAT is important for ether lipid biosynthesis but not for the integrity of detergent resistant membranes.

Glycerolipid biosynthesis in Leishmania initiates with the acylation of glycerol-3-phosphate by a single glycerol-3-phosphate acyltransferase, LmGAT, or of dihydroxyacetonephosphate by a dihydroxyacetonephosphate acyltransferase, LmDAT. We previously reported that acylation of the precursor dihydroxyacetonephosphate rather than glycerol-3-phosphate is the physiologically relevant pathway for Leishmania parasites. We demonstrated that LmDAT is important for normal growth, survival during the stationary phase, and for virulence. Here, we assessed the role of LmDAT in glycerolipid metabolism and metacyclogenesis. LmDAT was found to be implicated in the biosynthesis of ether glycerolipids, including the ether lipid derived virulence factor lipophosphoglycan and glycosylphosphatidylinositol-anchored proteins. The null mutant produced longer lipophosphoglycan molecules that were not released in the medium, and augmented levels of glycosylphosphatidylinositol-anchored proteins. In addition, the integrity of detergent resistant membranes was not affected by the absence of the LmDAT gene. Further, our genetic analyses strongly suggest that LmDAT was synthetic lethal with the glycerol-3-phosphate acyltransferase encoding gene LmGAT, implying that Leishmania expresses only two acyltransferases that initiate the biosynthesis of its cellular glycerolipids. Last, despite the fact that LmDAT is important for virulence the null mutant still exhibited the typical characteristics of metacyclics.