Tryptophan C-mannosylation is critical for Plasmodium falciparum transmission

Abstract

Tryptophan C-mannosylation stabilizes proteins bearing a thrombospondin repeat (TSR) domain in metazoans. Here we show that Plasmodium falciparum expresses a DPY19 tryptophan C-mannosyltransferase in the endoplasmic reticulum and that DPY19-deficiency abolishes C-glycosylation, destabilizes members of the TRAP adhesin family and inhibits transmission to mosquitoes. Imaging P. falciparum gametogenesis in its entirety in four dimensions using lattice light-sheet microscopy reveals defects in ΔDPY19 gametocyte egress and exflagellation. While egress is diminished, ΔDPY19 microgametes still fertilize macrogametes, forming ookinetes, but these are abrogated for mosquito infection. The gametogenesis defects correspond with destabilization of MTRAP, which we show is C-mannosylated in P. falciparum, and the ookinete defect is concordant with defective CTRP secretion on the ΔDPY19 background. Genetic complementation of DPY19 restores ookinete infectivity, sporozoite production and C-mannosylation activity. Therefore, tryptophan C-mannosylation by DPY19 ensures TSR protein quality control at two lifecycle stages for successful transmission of the human malaria parasite.

Conservation, abundance, glycosylation profile, and localization of the TSP protein family in Cryptosporidium parvum

“…The first enrichment strategy made use of the 5G12 antibody, which was developed to recognize peptides bearing the C-mannosyl tryptophan protein modification ( 31 , 32 ). This unusual type of glycosylation, as well as O-linked glucosyl-β(1→3)-fucosylation [βGlc (1→3)αFuc], is commonly associated with TSR domains in metazoans ( 33 ) and apicomplexans like Plasmodium spp ( 34 , 35 ) and T. gondii ( 36 , 37 ). We suspected that these modifications existed in Cryptosporidium spp.…”

“…S2 ). It is likely that this glycan is localized to the classical CXX( S/T) S motif of the TSR domain, as it is in other apicomplexans ( 34 , 35 , 36 , 37 ) and metazoans ( 33 ). These data confirm that C-mannosylation occurs in C. parvum sporozoites and that it is only found on TSR proteins, at least in this stage of the life cycle.…”

“…Similarly, the tryptophan C-mannosylation detected here on Cp TSP1–4,7–8,11 has been observed to play important roles in T. gondii and Plasmodium spp. biology ( 35 , 36 , 51 ). In T. gondii , tryptophan C-mannosylation stabilizes the TSR-containing adhesin MIC2 and is thus important for parasite motility ( 36 ).…”

“…In T. gondii , tryptophan C-mannosylation stabilizes the TSR-containing adhesin MIC2 and is thus important for parasite motility ( 36 ). In P. falciparum and Plasmodium berghei , tryptophan C-mannosylation is essential for transmission to the mosquito through its role in stabilization of the TSR-containing adhesins MTRAP and CTRP, which are required for gamete egress and ookinete motility, respectively ( 35 , 51 ). These findings are commensurate with the general observation that tryptophan C-mannosylation stabilizes proteins with a TSR domain ( 52 , 53 ).…”

Plasmodium falciparum exoerythrocytic forms require the PTEX translocon for development in human hepatocytes

“…Cell traversal was measured using a cell wounding assay 74 . HC-04 hepatocytes (1x10 5 ) were seeded onto the bottom of a 96-well plate using IMDM (Life Technologies, 11360-070).…”