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, 8 (1), 561

Protein O-fucosylation in Plasmodium Falciparum Ensures Efficient Infection of Mosquito and Vertebrate Hosts

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Protein O-fucosylation in Plasmodium Falciparum Ensures Efficient Infection of Mosquito and Vertebrate Hosts

Sash Lopaticki et al. Nat Commun.

Abstract

O-glycosylation of the Plasmodium sporozoite surface proteins CSP and TRAP was recently identified, but the role of this modification in the parasite life cycle and its relevance to vaccine design remain unclear. Here, we identify the Plasmodium protein O-fucosyltransferase (POFUT2) responsible for O-glycosylating CSP and TRAP. Genetic disruption of POFUT2 in Plasmodium falciparum results in ookinetes that are attenuated for colonizing the mosquito midgut, an essential step in malaria transmission. Some POFUT2-deficient parasites mature into salivary gland sporozoites although they are impaired for gliding motility, cell traversal, hepatocyte invasion, and production of exoerythrocytic forms in humanized chimeric liver mice. These defects can be attributed to destabilization and incorrect trafficking of proteins bearing thrombospondin repeats (TSRs). Therefore, POFUT2 plays a similar role in malaria parasites to that in metazoans: it ensures the trafficking of Plasmodium TSR proteins as part of a non-canonical glycosylation-dependent endoplasmic reticulum protein quality control mechanism.The role of O-glycosylation in the malaria life cycle is largely unknown. Here, the authors identify a Plasmodium protein O-fucosyltransferase and show that it is important for normal trafficking of a subset of surface proteins, particularly CSP and TRAP, and efficient infection of mosquito and vertebrate hosts.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
O-Fucosylation of TSR domains by POFUT2 in P. falciparum. a O-Fucosylation of TSR domains by GDP-fucose, as catalyzed by POFUT2, (illustration generated using 4HQO). b Deconvoluted intact ESI mass spectrum of recombinant P. falciparum TRAP TSR domain treated with GDP-fucose in the absence (blue) and presence (red) of P. vivax POFUT2. c Deconvoluted intact ESI mass spectrum of recombinant P. falciparum CSP TSR domain treated with GDP-fucose in the absence (blue) and presence (red) of P. vivax POFUT2. d Multiple sequence alignment of all TSR domain sequences from P. falciparum revealing the proteins that are likely to be O-fucosylated (red) and in what parasite stage they are expressed
Fig. 2
Fig. 2
POFUT2 is important for P. falciparum transmission to Anopheles stephensi mosquitoes. a Parasite load in mosquito midguts 27 h post-bloodmeal, measured by qRT-PCR of transcripts for Pf18S (total parasites), Pfs25 (gametes, zygotes, ookinetes), and PfCTRP (ookinetes) relative to Anopheles stephensi ribosomal protein, rps7 (AsrpS7). No significant differences were observed relative to NF54 for Pf18S (P = 0.4973), Pfs25 (P = 0.3513), and PfCTRP (P = 0.2547). b Oocyst counts per mosquito midgut 7 days post-bloodmeal. Data are mean ± 95% confidence interval from three independent experiments. c Salivary gland sporozoite count per mosquito 17 days post-bloodmeal. d Salivary gland sporozoite count divided by oocyst count (P = 0.9945). Data is the mean ± S.E.M. from three independent experiments. P-values are for both mutant clones compared to NF54, calculated using the Kruskal–Wallis one-way ANOVA
Fig. 3
Fig. 3
POFUT2 facilitates P. falciparum liver infection. a Percentage of traversed (FITC-dextran positive) human HC-04 hepatocytes by salivary gland sporozoites. b Percentage of HC-04 cells with intracellular parasites 24 h after addition of sporozoites to cells. Data is the mean ± S.E.M. from three (a) and two (b) independent experiments. c Parasite liver load measured by qPCR showing the fitness of ΔPOFUT2 versus parental NF54 sporozoites following coinfection of three humanized chimeric liver mice. Each symbol corresponds to the same coinfected mouse. Data are mean ± S.E.M. d Percent of sporozoites that are non-motile in a two-dimensional gliding motility assay. e Number of circles per trail produced by gliding sporozoites (non-motile parasites removed). Data in d, e is mean ± S.E.M. or 95% confidence interval, respectively, from two independent experiments. P-values are for both mutant clones compared to NF54, calculated using the Kruskal–Wallis one-way ANOVA, except panel c, which compared one mutant clone to NF54 in each of three mice using the paired t-test
Fig. 4
Fig. 4
POFUT2 plays a role in TSR protein trafficking in P. falciparum. a Immunofluorescence microscopy of NF54 and ΔPOFUT2 salivary gland sporozoites showing the localization of CSP (red), TRAP (green), and dsDNA (blue). Purple arrow, TRAP at the sporozoite membrane; white arrow, TRAP internal to parasite. Scale 5 μm. b Total sporozoite pixel intensity for PfTRAP and PfCSP. c Pixel intensity for PfTRAP and PfCSP at the sporozoite membrane only. Data in b, c is the mean ± S.E.M. from two independent experiments. In panel b, a subtle increase in total PfCSP pixels was observed in ΔPOFUT2 D3 relative to NF54 (P = 0.0360) but no difference was observed at the sporozoite membrane (P = 0.1083) in panel c. P-values are for one mutant clone compared to NF54, calculated using the Mann–Whitney test
Fig. 5
Fig. 5
POFUT2 stabilizes TSR proteins in P. falciparum. a Western blot analysis of 30,000 salivary gland sporozoites per lane using antibodies to PfTRAP and PfCSP. Anti-PfPLP1 was used as a loading control. The same blot was probed consecutively with each antibody. b Densitometry of PfTRAP and PfCSP levels in sporozoites measured by immunoblotting and standardized to the PfPLP1 loading control. Data are mean ± S.E.M. and pooled from three independent immunoblots. c Abundance of PfTRAP and PfCSP mRNA transcripts in salivary gland sporozoites relative to Pf18S, measured by qRT-PCR. No differences were observed for either ΔPOFUT2 clone compared to NF54 for PfTRAP (P = 0.5003) and PfCSP (P = 0.3104) mRNA. Data is the mean ± S.E.M. of four independent experiments. P-values are for both mutant clones compared to NF54, calculated using the Kruskal–Wallis one-way ANOVA

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