Glycosylation of Plasmodium falciparum TRAP is essential for infection of mosquito salivary glands.

The human malaria parasite Plasmodium falciparum ( Pf ) expresses ten thrombospondin type 1 repeat (TSR) domain-bearing proteins at different stages throughout its life cycle. TSRs can be modified by two types of glycosylation: O-fucosylation at conserved serine (S) or threonine (T) residues and C-mannosylation at conserved tryptophan (W) residues. Pf TRAP, which is expressed in mosquito-stage sporozoites, has one TSR domain that is O-fucosylated at Thr 256 and C-mannosylated at Trp 250 . We employed site-directed mutagenesis by CRISPR/Cas9 gene editing to generate two Pf TRAP glyco-null mutant parasites, Pf TRAP_T256A and Pf TRAP_W250F. The fitness of these mutant parasites across the life cycle was compared to the parental NF54 wild type as well as a Pf TRAP knockout line. The glyco-null parasites exhibited major fitness defects comparable to knockout: sporozoites were unable to productively colonize the salivary glands and were highly impaired in gliding motility and the ability to invade cultured human hepatocytes. Protein analysis revealed significantly reduced Pf TRAP abundance in the glyco-null mutants despite normal transcript levels. These findings demonstrate that glycosylation of Pf TRAP's TSR is critical for its proper expression and function, and underscores the importance of TSR glycosylation in the mosquito stage of the life cycle.