Levels of circumsporozoite protein in the Plasmodium oocyst determine sporozoite morphology

Abstract

The sporozoite stage of the Plasmodium parasite is formed by budding from a multinucleate oocyst in the mosquito midgut. During their life, sporozoites must infect the salivary glands of the mosquito vector and the liver of the mammalian host; both events depend on the major sporozoite surface protein, the circumsporozoite protein (CS). We previously reported that Plasmodium berghei oocysts in which the CS gene is inactivated do not form sporozoites. Here, we analyzed the ultrastructure of P.berghei oocyst differentiation in the wild type, recombinants that do not produce or produce reduced amounts of CS, and corresponding complemented clones. The results indicate that CS is essential for establishing polarity in the oocyst. The amounts of CS protein correlate with the extent of development of the inner membranes and associated microtubules underneath the oocyst outer membrane, which normally demarcate focal budding sites. This is a first example of a protein controlling both morphogenesis and infectivity of a parasite stage.

Fig. 1. Electron micrographs of oocyst development in wild-type P.berghei. (A) An early oocyst (oo) in A.stephensi midgut epithelium. The oocyst is limited from mosquito tissue (mo) by an electron-dense capsule (arrow), which interfaces with the hemocoel (he). Several nuclei (n) are present. (B) The oocyst plasma membrane then retracts from the capsule and invaginates into the oocyst cytoplasm, subdividing the oocyst into several sporoblasts (spr). Inner membranes emerge immediately beneath the plasma membrane (arrows) and demarcate sites of sporozoite budding. (C) As these areas evaginate (arrows), the oocyst plasma membrane and inner membranes become the trimembranous pellicle of the nascent sporozoites (b), formed anterior pole first. Nuclei (n) are seen entering sporozoite buds (b). Sporozoites that have budded off (sp) are seen in cross-section. (D) Eventually, the oocyst becomes packed with regularly shaped sporozoites (sp). Bars: 5 µm.

Fig. 2. Sporozoite budding in wild-type P.berghei. (A) Budding starts with the focal emergence of the inner membranes (arrows) beneath the oocyst plasma membrane. Bar: 0.5 µm. (B) Focal extension of the inner membranes. Subpellicular microtubules are visible in cross- section immediately below the inner membranes (arrows). Bar: 0.5 µm. (C) Inner membranes and microtubules (arrow) grow, shaping the nascent sporozoite. A rhoptry (Rh) is visible at the apical pole of the bud, and a nucleus (n) is seen entering the bud. Bar: 0.4 µm. (D) Enlarged view of the triple membrane structure of the pellicle. The closed end of the vesicle that gives rise to the two inner membranes is visible (arrow). Bar: 0.3 µm. (E) Cross-section of a sporozoite showing the trimembrane pellicle and the 15 subpellicular microtubules around two-thirds of the section. The arrow shows the single microtubule in the remaining third. Bar: 0.5 µm. (F) Longitudinal section of the sporozoite pellicle shows the plasma membrane, inner membranes and an associated microtubule. Bar: 0.5 µm.

Fig. 3. Immunoelectron micrographs showing CS expression in wild-type P.berghei oocysts. Sections of wild-type P.berghei oocysts at increasing stages of development from (A) to (D) were stained with anti-CS monoclonal antibodies followed by anti-mouse IgG conjugated to 15 nm gold particles. (A) Gold particles are associated with the plasma membrane, which is still closely attached to the capsule (c) in the early oocyst, as well as with vesicles fusing to the plasma membrane (arrows). Bar: 0.8 µm. (B) CS labeling increases at the plasma membrane, while the cytoplasm remains poorly labeled. Bar: 1.5 µm. (C) CS labeling continues to increase as the plasma membrane retracts from the oocyst capsule. Bar: 0.8 µm. (D) At later stages, CS covers the surface of budding sporozoites. Bar: 1.5 µm.

Fig. 4. Electron micrographs showing the development of CSko oocysts. Inner membranes and microtubules rapidly delineate large areas of the oocyst plasma membrane. (A) Extended stretches of inner membranes (arrowheads) are visible beneath the plasma membrane at early stages of plasma membrane retraction from the oocyst capsule (arrow). n, nucleus; mo, mosquito midgut tissue. Bar: 5 µm. (B) Continuous lining by inner membranes of an extensive area of the plasma membrane that has not retracted from the capsule (c). Bar: 2.5 µm. (C) The arrow shows a longitudinal section of a subpellicular microtubule. c, oocyst capsule. Bar: 0.5 µm. (D) Arrows show subpellicular microtubules in cross-section. Bar: 0.5 µm.

Fig. 5. Electron micrographs of CSko oocysts showing aberrant budding in the subcapsular space (A–C) and in the sporoblast cytoplasm (D–G). (A) Parallel stacking of bud-like structures. Bar: 3 µm. (B) An abnormal bud extends parallel to the sporoblast periphery instead of away into the subcapsular space. Bar: 2.5 µm. (C) Inner membranes and microtubules (arrows) underneath the oocyst plasma membrane and the bud outer membrane. Bar: 0.5 µm. (D–G) Cytoplasmic membranes lined with inner membranes (arrows), either totally (D and G) or partially (E and F). Rh, rhoptry; n, nucleus. Bars: 2.5 µm.

Fig. 6. Genetic complementation of the CSko clone by reintroduction of the CS gene into the disrupted CS locus. (A) Gene targeting at the CS knockout locus (CS disruptant) using the insertion plasmid pCSComp linearized at the unique KpnI site (K) prior to transfection. The complemented CS locus expected to result from plasmid integration is shown below. The predicted sizes (in kb) of restriction fragments generated upon digestion of parasite genomic DNA with EcoRV (E5) in the CS knockout or the complemented CS locus are shown. Open boxes, CS coding sequences; black boxes, pyrimethamine resistance P.berghei DHFR-TS selection cassette; gray boxes, human DHFR (hDHFR) selection cassette; thin lines, 5′- and 3′-UTRs of CS; thick lines, pUC19 vector sequences; double-headed arrows, internal probes from CS, P.berghei DHFR-TS or hDHFR coding sequences used in Southern hybridization. (B) Southern blot hybridization of EcoRV-digested genomic DNA of wild-type P.berghei (WT), CSko [CS(-)1], complemented CS (CSComp1) and blood stages resulting from a sporozoite-induced infection by CSComp1 sporozoites (CSComp1-SI). The same blot was probed successively with CS, hDHFR and PbDHFR-TS probes. Endog., endogenous PbDHFR-TS gene; exog., exogenous PbDHFR-TS copies originating from the CS knockout locus (7.3 kb) and from the complemented CS locus (12.1 kb); *, cross-reaction of the PbDHFR-TS probe with sequences in pCSComp. (C) Western blot analysis of midgut sporozoite extracts from wild-type (lane 1) and complemented (CSComp1, lane 2) parasites. Sporozoites were collected from oocysts in mosquito midguts dissected at day 15 post-infection. Crude extracts from ∼5 × 10³ sporozoites from each population resolved by SDS–PAGE and transferred to a membrane were probed with anti-TRAP polyclonal antibodies (α-TRAP) and anti-CS monoclonal antibodies (α-CS). (D) Indirect immunofluorescence assay of CSComp1 sporozoites collected from the salivary glands of infected mosquitos dissected at day 18 post-infection. Sporozoites were permeabilized and stained using FITC-conjugated, anti-CS monoclonal antibodies.

Fig. 7. Generation of PINA270 and PINA450 clones of P.berghei that produce low or wild-type amounts of CS, respectively. (A) Schematic representation of the wild-type (WT) CS locus and insertion plasmids pPINA270 and pPINA450, which differ only in the length of the CS 3′-UTR, shown enlarged above the WT CS locus. The lollipop symbols in the CS 3′-UTR show the 3′ ends of CS transcripts located between nucleotides 88 and 337 following the CS stop codon. The WT CS gene was targeted with plasmids pPINA270 and pPINA450, whose targeting sequences contain the distal part of the WT CS coding region and 270 or 450 bp of its 3′-UTR, respectively. Plasmids were linearized at a unique AflII site (A) located at 250 bp from the 5′ end of the targeting sequence. Shaded boxes, P.berghei DHFR-TS pyrimethamine resistance cassette; thick lines, pBSKS vector sequences; H, HincII. (B) Structures of the WT CS genomic locus and the CS recombinant loci generated by homologous integration of plasmids pPINA270 and pPINA450. The predicted sizes (in kb) of restriction fragments generated upon digestion with BamHI–EcoRV, BamHI–EcoRI or AflII are shown. A, AflII; B, BamHI; E1, EcoRI; E5, EcoRV. (C) Southern hybridization of WT P.berghei (lane 1) and the PINA270 clone (lane 2), upon digestion with BamHI–EcoRV (lanes 1 and 2) or BamHI–EcoRI (lanes 3 and 4) and using a CS probe. (D) Western blot analysis of midgut sporozoite extracts from the WT and PINA270 (P/270) populations. Sporozoites were harvested from oocysts in mosquito midguts at days 13 and 15 post-infection. Crude extracts separated by SDS–PAGE and transferred to a membrane were probed with anti-CS (α-CS) or anti-TRAP (α-TRAP) antibodies. (E) Southern hybridization of WT P.berghei (lane 1) and the PINA450 clone (lane 2), upon digestion with AflII (lanes 1 and 2) or BamHI–EcoRI (lanes 3 and 4) and using a CS probe. (F) PCR amplification of the expressed CS copy in PINA270 (lane 1) and PINA450 (lane 2) parasites using primers O1CS and T7, which anneal upstream from the region of homology and to the vector sequence, respectively. (G) Western blot analysis of midgut sporozoite extracts from WT, PINA270 (P/270) and PINA450 (P/450) parasites. Sporozoites were harvested from oocysts in mosquito midguts at day 15 post-infection, and analyzed as in (D).

Fig. 8. Transmission electron micrographs showing sections of wild-type and PINA270 oocysts. (A) Focal emergence and extension of inner membranes (arrow) underneath the plasma membrane at a sporozoite bud site in a wild-type oocyst. Bar: 3 µm. (B) In PINA270 oocysts, inner membranes (arrows) delineate areas of the plasma membrane that are larger than in wild-type oocysts. Bar: 3 µm. (C) Sporozoite buds elongate perpendicular to the sporoblast periphery in wild-type oocysts. Bar: 1.5 µm. (D) In PINA270 oocysts, budding often occurs parallel to the sporoblast periphery. Bar: 1.5 µm. (E) Cross- sections of fully formed sporozoites, which are of homogenous sizes and shapes. Bar: 1.5 µm. (F) Longitudinal and cross-sections of sporozoites, which are of variable sizes and shapes. Note the presence of two nuclei surrounded by a single nuclear envelope in one of the sporozoites (arrow) and the ‘corrugated’ appearance of the sporozoite pellicles. Bar: 1.5 µm. (G and H) Scanning electron micrographs of free wild-type (G) and PINA270 (H) midgut sporozoites isolated from infected mosquitos at day 14 post-infection. Note the corrugated surface of the PINA270 sporozoite. Bars: 2 µm.

Fig. 9. Immunofluorescence labeling of wild-type and PINA270 sporozoites collected from the hemolymph of infected mosquitos at day 15 post-infection. Sporozoites were permeabilized and stained with FITC-conjugated, anti-CS monoclonal antibodies. Note the variations in size and shape of PINA270 sporozoites as compared with wild-type sporozoites.