Evidence that free GPI glycolipids are essential for growth of Leishmania mexicana

The cell surface of the parasitic protozoan Leishmania mexicana is coated b y glycosylphosphatidylinositol (GPI)-anchored glycoproteins, a GPI-anchored lipophosphoglycan and a class of free GPI glycolipids, To investigate whet her the anchor or free GPIs are required for parasite growth we cloned the L.mexicana gene for dolichol-phosphate-mannose synthase (DPMS) and attempte d to create DPMS knockout mutants by targeted gene deletion. DPMS catalyzes the formation of dolichol-phosphate mannose, the sugar donor for all manno se additions in the biosynthesis of both the anchor and free GPIs, except f or a alpha 1-3-linked mannose residue that is added exclusively to the free GPIs and lipophosphoglycan anchor precursors. The requirement for dolichol -phosphate-mannose in other glycosylation pathways in L.mexicana is minimal . Deletion of both alleles of the DPMS gene (lmdpms) consistently resulted in amplification of the lmdpms chromosomal locus unless the promastigotes w ere first transfected with an episomal copy of lmdpms, indicating that lmdp ms, and possibly GPI biosynthesis, is essential for parasite growth. As evi dence presented in this and previous studies indicates that neither GPI-anc hored glycoproteins nor lipophosphoglycan are required for growth of cultur ed parasites, it is possible that the abundant and functionally uncharacter ized free GPIs are essential membrane components.