Table 1.
Life cycle progression and infectivity of FCKOmCh parasites following outcrossing.
Fig 1.
Crossing FCKOmCh to FCWT parasites allows normal development of FCKOmCh sporozoites.
A) Schematic representation of the predicted parasite genotypes at the polyploid oocyst stage and haploid sporozoite stage after allowing mosquitoes to feed on mice dually infected with FCKOmCh and FCWT-GFP parasites. Three genetic combinations are possible in the polyploid oocyst: self- fertilization yielding oocysts with only the FCKOmCh or the FCWT-GFP allele or outcrossing creating an oocyst with both FCKOmCh and the FCWT-GFP alleles. Following sporogony, the parasite returns to the haploid state yielding four possible genetic combinations of the FCKOmCh and the FCWT-GFP genes: The original FCKOmCh or FCWT-GFP alleles alone, a combination of both the FCKOmCh and FCWT-GFP alleles or parasites carrying neither allele. B) Schematic representation of the predicted parasite genotypes at the polyploid oocyst stage and haploid sporozoite stage after allowing mosquitoes to feed on mice dually infected with FCKOmCh–nek4-wt and FCWT-nek4-ko parasites. The nek4ko mutation prevents self-fertilization of this parasite line, so two genetic combinations are possible in the polyploid oocyst: self- fertilization yielding oocysts with only the FCKOmCh allele or outcrossing creating an oocyst with both FCKOmCh and the FCWT-nek4ko alleles. Following sporogony, the parasite returns to the haploid state yielding four possible genetic combinations of the FCKOmCh and the FCWT-nek4ko genes: The original FCKOmCh or FCWT-nek4ko alleles alone, a combination of both the FCKOmCh and FCWT-nek4ko alleles or parasites carrying neither allele. C) A midgut infected by feeding on an FCKOmCh x FCWT-GFP infected mouse showing the presence of the three oocyst phenotypes. Red oocysts are self-fertilized FCKOmCh parasites, green are self-fertilized FCWT-GFP, and orange oocysts are FCKOmCh x FCWT-GFP recombinants. scale bar: 20μm. (D) Complementation of the FCKO phenotype demonstrated by comparing the mean surface area of oocysts from FCKOmCh self-fertilization with oocysts produced by the FCKOmCh x FCWT-GFP and FCKOmCh nek-4wt x FCWT-nek-4ko crosses. The female specific defect resulting from the nek4-ko genotype prevents development of colourless FCWT-nek-4ko oocysts, so all oocysts produced from FCKOmCh nek-4wt x FCWT-nek-4ko crosses carry the FCKOmCh and are red in colour. ns; not significant, ***—P value <0.0001 (Student t test) (E) Immunofluorescence assay showing the presence of mCherry fluorescence, and hence the presence of the FCKOmCh allele in midgut (day 17 post infection) and salivary gland (day 21 post infection) sporozoites from the FCKOmCh nek-4wt x FCWT-nek-4ko cross and the salivary gland sporozoites from the FCKOmCh x FCWT-GFP cross. scale bar: 10μm F) FC genotype (by PCR) and matching fluorescent phenotype of parasites through the complete life cycle showing the presence of parasites carrying both the FCKOmCh and FCWT genes until the liver stage of the life cycle. Parasites carrying the FCKOmCh fail to complete liver stage development so neither red fluorescence nor the FCKOmCh gene is detectable in the subsequent blood stage infection. Sporozoite and liver stage genotypes were assessed with a nested PCR reaction, yielding a band of different size to that seen in blood stages. scale bar: 10μm.
Fig 2.
Sporozoite development involves significant carryover of oocyst cytoplasm.
A) Schematic representation of the predicted parasite genotypes at the polyploid oocyst stage and haploid sporozoite stage after allowing mosquitoes to feed on mice dually infected with parasites expressing either GFP or tdTomato. The fluorophore is inserted into the same genetic locus in each parasite line, so three genetic combinations are possible in the polyploid oocyst: self- fertilization yielding oocysts with either the GFP or tdTomato allele, whereas outcrossing generates a heterzygous oocyst with both the GFP and tdTomato alleles. Following sporogony, the parasite returns to the haploid state and can carry either the GFP or tdTomato allele, but not both. B) Progeny of the GFP x tdTomato cross through the life cycle. Three oocyst phenotypes are present, GFP only (green) and tdTomato only (red) from self-fertilizations and heterozygous, polyploid parasites expressing both fluorophores (orange). Only two phenotypes, GFP (green) or tdTomato (red) are present in haploid liver and red blood cell stages. C) Sporozoites generated from crossing GFP and tdTomato parasites showed many haploid sporozoites still carrying both fluorphores (arrow heads). Single colour (green or red) sporozoites are presumably offspring from self-fetilizations, while two-colour (orange) sporozoites are from recombinant oocysts.
Fig 3.
Parasites lacking FC have a severe liver stage growth phenotype.
A) Immunofluorescence assay of FCKOmCh and FCWT parasites grown in HepG2 liver cells for 24, 48 and 68 hours post infection (hpi) showing the reduced growth of FCKOmCh parasites. scale bar 20 μm. B) Quantification of liver stage parasite size showing that FCKOmCh parasites are significantly smaller than FCWT parasites after 48 and 68 hours of incubation in HepG2 cells. ***—P value < 0.0001 (Student t-test), data pooled from six independent experiments. C) Quantification of parasite nuclei 68 hours after infection showing that FCKOmCh parasites produced significantly fewer nuclei than FCWT parasites by 68 hpi. P value <0.0001 (χ2). D) Immunofluorescence assay of MSP-1 expression, a marker for mid to late stage parasite development in the liver stage. FCKOmCh parasites have a defect in MSP-1 expression, with only 45% of FCKOmCh parasites producing MSP-1 compared to 79% of FCWT. ***—P value <0.001(Fisher’s exact test) 801 parasites assessed, pooled from three independent experiments with no fewer than 58 parasites measured in any one sample. Error bars show 99% confidence interval.
Table 2.
Production of FCKOmCh merosomes following outcrossing (no. of trials = 3).
Fig 4.
FC deficient parasites cannot complete the liver stage in vivo.
(A) Epi-fluorescent images comparing the expression of mCherry and GFP before and after transmission. Both GFP and mCherry are visible in parasites recovered from the blood of a donor mouse dual infected with FCKOmCh and FCWT-GFP parasites (upper panel). In contrast only GFP expression is observed in a naïve mouse infected with the progeny (P0) of this cross (lower panel), confirming the essential nature of FC during liver stage development. scale bar: 100μm. B) Summary of the genotypes and pyrimethamine resistance phenotype following infection of naïve mice with sporozoites from the FCKOmCh x FCWT-GFP cross. PCR screening detects parasites with both of the FC and GFP alleles in the donor mouse used to infect mosquitoes. The FCKOmCh genotype cannot be detected in the blood of mice infected by parasites found in those mosquitoes, demonstrating its lethality in the liver stage. Pyrimethamine selection for parasites carrying the hDHFR inserted in the FC locus kills all parasites, confirming that FCKOmCh sporozoites cannot infect naïve mice. FCWT-GFP parasites are susceptible to pyrimethamine treatment and are, therefore, not present after drug treatment.