Mutant/mutation
The mutant lacks expression of POFut2 and expresses GFP (under a constitutive promoter).

Protein (function)
Glycosylation is one of the most important post-translational modifications (PTMs) in many proteins. The attachment of carbohydrate residues serves a range of diverse functions, such as the generation of antigenic variation, subcellular localization, and proper folding of protein domains. A large and diverse group of proteins subjected to glycosylation belongs to the TSR superfamily. TSR domains, which play essential roles in cell adhesion and motility, contain six conserved cysteines forming three disulfide bonds. The Plasmodium proteome has several TSR domain-containing proteins. These proteins include circumsporozoite and TRAP-related protein (CTRP), thrombospondin-related anonymous protein (TRAP), and circumsporozoite protein (CSP) in the ookinete and sporozoite stages.3−5 The TSR domains of these essential
proteins are O-fucosylated by POFut2 on the canonical serine/threonine residue of the CXX(S/T)C motif. Both O-linked and C-linked glycosylation events play an important role in the folding and export of TSR domain-containing proteins.
POFut2 has been characterized with conflicting results. Initially,  Plasmodium  falciparum POFut2 was found to be important for both host and vector infections.  While it was found to be nonessential for blood stage infection, it was indicated that a lack of PfPOFut2 affects ookinete implantation, resulting in fewer oocysts. The obtained sporozoites also had impaired liver infectivity in chimeric mice containing humanized livers. However, some studies also reported a nonessential role of POFut2 in P. berghei (see RMgm-4678). Here, we disrupted the POFut2 gene in Plasmodium berghei and demonstrated that POFut2 KO parasites develop normally in blood and mosquito stages but show reduced infectivity in mice. We found that the reduced infectivity of POFut2 KO sporozoites was due to a diminished level of TRAP that affected the parasite gliding motility and hepatocyte infectivity.

Phenotype
Salivary gland sporozoites were injected into C57BL/6 mice. A delay of 1 day in the prepatent period was observed in KO-infected mice compared to the wild type (WT)-infected mice. To analyze the parasite burden in the liver, infected mouse livers were harvested at 40 hpi, and parasite biomass was quantified by measuring 18S rRNA copy number using real-time PCR. A significant decrease in 18S rRNA copy number in KO parasites was found. In in vitro cultures liver stages (EEFs) were observed with normal morphology at 40 hpi, which, upon quantification, revealed a decrease in EEF numbers, while EEF areas remained similar to those of WT EEFs. All POFut2 KO EEFs matured into hepatic merozoites normally. A two-step IFA-based invasion assay was used to assess sporozoite invasion. The invasion of POFut2 KO sporozoites was approximately half compared to WT parasites. POFut2 KO sporozoites showed reduced gliding motility.

Additional information
To check the fucosylation status of TRAP and CSP in POFUT2 KO lines, Western blotting was performed with sporozoite secreted proteins using ulex europaeus agglutinin I (UEA I). UEA I specific signal was found in WT sporozoites but not in the POFUT2 KO parasites. The UEA I identified signals were specific to TRAP and CSP, which was confirmed by reprobing the blot with anti-TRAP and anti-CSP antibodies.
Since O-fucose is added to the TSR domains of sporozoite surface proteins CSP and TRAP,6  the levels of these two proteins was checked by IFA and immunoblotting. The levels of TRAP but not CSP were reduced in POFut2 KO sporozoites. The decreased levels of TRAP were at the protein level and not at the transcript level. The Plasmodium TRAP-TSR domain was found to be always modified with C-mannosylation but not always with O-fucosylation, possibly due to the loss during the experiment and hence not detected.

Other mutants