A DOC2 protein identified by mutational profiling is essential for apicomplexan parasite exocytosis

Abstract

Exocytosis is essential to the lytic cycle of apicomplexan parasites and required for the pathogenesis of toxoplasmosis and malaria. DOC2 proteins recruit the membrane fusion machinery required for exocytosis in a Ca(2+)-dependent fashion. Here, the phenotype of a Toxoplasma gondii conditional mutant impaired in host cell invasion and egress was pinpointed to a defect in secretion of the micronemes, an apicomplexan-specific organelle that contains adhesion proteins. Whole-genome sequencing identified the etiological point mutation in TgDOC2.1. A conditional allele of the orthologous gene engineered into Plasmodium falciparum was also defective in microneme secretion. However, the major effect was on invasion, suggesting that microneme secretion is dispensable for Plasmodium egress.

Fig. 1

Mutant F-P2 has a microneme secretion defect. (A) Red-green invasion assays were performed on the 2F-1-YFP2 wild-type and F-P2 mutant parasite lines. Parasites were phenotypically induced for 24 hrs at the restrictive temperature (40°C). Averages of four independent experiments + SD are shown. (B) Conoid extrusion of Ca²⁺-ionophore (A23187) induced or vehicle control treated parasites was determined for parasites grown at 35°C or 40°C. Averages of three independent experiments + SD are shown. (C) Incidence of various motility modes determined by video microscopy over 1 min for wild-type (parent 2F-1-YFP2) and F-P2 parasites at conditions as indicated. Averages of four independent experiments + SEM are shown. (D) Microneme secretion of F-P2 parasites measured by western blot detection of Mic2 protein released in the supernatant upon various stimuli and vehicle control (DMSO). “const.” represents uninduced, constitutive secretion over a 60 min period. Gra1 serves as loading control. (E) IFA of Mic2 and IMC3 (marking the peripheral cytoskeleton) of wild-type and F-P2 at 40°C with or without ionophore stimulation shows micronemes are intact in F-P2. Phase images show vacuolar membrane is intact in F-P2 at 40°C. Asterisk marks egressing parasite.

Fig. 2

Results of paired-end Illumina re-sequencing of parent and F-P2 genomes. (A) Sequencing and alignment statistics. Genomic DNA of F-P2 and its parent were sequenced to >30-fold coverage on an Illumina GA2 instrument. Sequence reads were aligned to the closely related GT1 strain reference genome using the MOSAIK read mapper (9). Coverage was sufficient to call sequence differences between F-P2 and RH in >95% of the GT1 reference. Using the FreeBayes variation calling program (28) and appropriate filtering, 982 single nucleotide polymorphisms (SNPs) are shared between the two samples and 33 are unique to F-P2. (B) Read coverage across the chromosomes of parent and F-P2. Fold coverage is averaged over 100 kb windows. The chromosomal localization of the 33 called SNPs between parent and F-P2 are shown, differentiated by confirmed and false SNP calls. 31 of the latter were positively confirmed by Sanger sequencing. 77% were mutation of an A/T base, corroborating the proclivity of ENU toward AT:GC and AT:TA substitutions when compared to the rate of 45% of the SNPs shared between the RH and GT1 strains.

Fig. 3

Genetic complementation of F-P2 with cosmid PSBMG64 restores the wild-type phenotype. (A) Plaque assays of parent line (2F-1-YFP2), F-P2 and complemented mutant as indicated. (B) Microneme secretion of complemented F-P2 parasites; see legend Fig. 1D. (C) Incidence of various motility modes determined by video microscopy over 1 min at conditions as indicated. Averages of four independent experiments shown + SEM. (D) Plaque assays representing F-P2 complementation with plasmids expressing cDNA encoding the wild-type TgDOC2.1 allele (top) or the F-P2 mutant allele (bottom).

Fig. 4

DOC2.1 has a conserved role in microneme secretion. (A) Schematic of DOC2.1 orthologs in Apicomplexa and ciliate Paramecium tetraurelia. Four conserved blocks are color coded in light blue; C2 domains in dark blue; coiled-coil regions in yellow. The red line marks the conserved phenylalanine mutated to serine in F-P2. (B) Lysates from D10-PfDOC2.1-DD parasites cultured +/− Shld1 probed with antibody to hemagglutinin (HA) (PfDOC2.1; just over 250 kDa, arrow), or antibody to PfLDH (loading control). Normalized PfDOC2.1 ratio (−/+Shld1): 0.43 +/− 0.13. (C) Replication curves of D10-PfDOC2.1-DD parasites maintained +/− Shld1 (mean ± range, N=2). (D) D10-PfDOC2.1-DD parasites were maintained without Shld1 from early ring stage to segmented schizont stage then incubated an additional 8–12 hours +/− Shld1, resulting newly invaded rings were counted by FACS (mean ± SD, N=3 [for D10-CDPK4-DD in experiment 3, N=1], three separate experiments, * p<0.001, t-test). (E) Immunoblots evaluating the release of indicated parasite invasion ligands. Synchronized ring-stage cells were incubated +/− Shld1. Supernatants of ruptured schizonts lysates (saponin) were collected. The asterisk marks 44+/−14% decrease in PfEBA-175 release from D10-PfDOC2.1-DD parasites. (F) Mean ratio of PfEBA-175 released into supernatant (N=4 biological replicates; +95% CI, *p <0.01, t-test).