Exit of Plasmodium sporozoites from oocysts is an active process that involves the circumsporozoite protein

Abstract

Plasmodium sporozoites develop within oocysts residing in the mosquito midgut. Mature sporozoites exit the oocysts, enter the hemolymph, and invade the salivary glands. The circumsporozoite (CS) protein is the major surface protein of salivary gland and oocyst sporozoites. It is also found on the oocyst plasma membrane and on the inner surface of the oocyst capsule. CS protein contains a conserved motif of positively charged amino acids: region II-plus, which has been implicated in the initial stages of sporozoite invasion of hepatocytes. We investigated the function of region II-plus by generating mutant parasites in which the region had been substituted with alanines. Mutant parasites produced normal numbers of sporozoites in the oocysts, but the sporozoites were unable to exit the oocysts. In in vitro as well, there was a profound delay, upon trypsin treatment, in the release of mutant sporozoites from oocysts. We conclude that the exit of sporozoites from oocysts is an active process that involves the region II-plus of CS protein. In addition, the mutant sporozoites were not infective to young rats. These findings provide a new target for developing reagents that interfere with the transmission of malaria.

Figure 1. Gene Targeting at the CS Locus of P. berghei

(A and B) Replacement plasmid pRCS-WT and pRCS-RIImut, wild-type (WT) CS locus and recombinant locus. ORFs are symbolized by boxes. Small black box in the CS ORF indicates the mutation in region II-plus (R290A, K291A, R292A, and K293A). A PstI site is introduced in the mutation site to differentiate CS-RIImut from CS-WT. Thick lines indicate 5′- and 3′-UTRs of DHFR-TS; thin lines, 5′- and 3′UTRs of CS; dashed lines, plasmid vector sequence. B, BamHI; K, KpnI; N, NotI; P, PstI; S, SacI; Xb, XbaI; Xh, XhoI. Recombinant CS-WT or CS-RIImut was generated by double crossover occurring between the CS sequences in the KpnI-SacI fragment of plasmid pRCS-WT or pRCS-RIImut and their homologous sequence in the wild-type CS genomic locus. The CS probe used in the genomic Southern hybridization is symbolized by a thick dash-dot line. Restriction fragments of the wild type and of the expected recombinants are shown below the corresponding locus. Locations of primers used for PCR are indicated in (B). (C) Schematic structure of the CS protein and sequences of the region II-plus of wild-type and mutant CS. (D) Genomic Southern hybridization of the wild-type P. berghei strain (WT), the recombinant lines, control (CS-WT), and mutant (CS-RIImut) parasites upon digestion with XbaI and PstI, using a CS probe. (E) PCR amplification with primers CS1 and PB103 of the expressed CS protein at the 5′ recombinant locus in CS-RIImut and CS-WT. The WT is used as a negative control (lack of recombination events). The amplicons (2.3 kb) were subjected to PstI restriction enzyme digestion (two fragments released, 1.6 and 0.7 kb) to examine the presence of mutation in CS-RIImut, which is absent in CS-WT. (F) PCR amplification of the 3′ recombinant locus using primers PB106 and CS4.

Figure 2. CS-RIImut Oocyst Sporozoites Are Not Released from the Midguts

Represented in each graph are the mean numbers of sporozoites per mosquito at different days PI. Each number is calculated based on an average of 20 mosquitoes. (A) Numbers of oocyst sporozoites per mosquito from CS-WT– and CS-RIImut–infected mosquitoes. In CS-RIImut, the number of sporozoites increases progressively until day 18 PI, whereas in CS-WT a plateau is reached at day 14. (B) Numbers of sporozoites in the hemolymph from CS-WT– and CS-RIImut–infected mosquitoes from the same lot as in (A). CS-WT oocysts release sporozoites into the hemolymph from day 12 to day 18 PI. The peak is around day 18 PI. In contrast, the hemolymph from CS-RIImut contains very few sporozoites. (C) In another feeding experiment, the number of hemolymph sporozoites from CS-WT and CS-RIImut infected mosquitoes were calculated from day 14 to day 28 PI. CS-WT releases sporozoites into the hemolymph up to day 28 PI, whereas CS-RIImut does not.

Figure 3. Ultrastructure and CS Localization of CS-WT and CS-RIImut

(A) Immunofluorescence labeling of oocyst sporozoites from CS-WT and CS-RIImut at day 18 PI. Sporozoites were stained with anti-CS antibody and detected by FITC-conjugated anti-IgG antibodies, without prior permeabilization. (B) Transmission electron micrographs showing a CS-RIImut oocyst at day 14 PI. The oocyst is surrounded by a capsule and the mosquito basal lamina, and contains sections of sporozoites with normal morphology. Fully developed sporozoites are found within the CS-RIImut oocyst. Sporozoites have homogenous size and morphology. Scale bar represents 1 μm. (C) Longitudinal section of the CS-RIImut sporozoite pellicle shows the plasma membrane, inner membrane complex, and an associated microtubule. Scale bar represents 0.5 μm. (D) Cross-section of a CS-RIImut sporozoite showing the trimembrane pellicle and subpellicular microtubules. Scale bar represents 0.5 μm. (E) Immuno-electron micrographs showing CS localization in the CS-RIImut oocyst. CS protein is predominantly found on the surface of sporozoites and the residual body, and on the inner surface of the capsule. Scale bar represents 1 μm. (F) Enlarged picture of the CS-RIImut oocyst capsule. CS protein is detected on the inner surface of the capsule. Scale bar represents 1 μm. (G) Western blot analysis of extracts from CS-WT (WT) and CS-RIImut (RII) oocyst sporozoites. Numbers of sporozoites loaded are indicated on the top of each lane. Sporozoites were collected from oocysts in mosquito midguts at days 14 and 18 PI. C, capsule; IMC, inner membrane complex; Mo, mosquito tissue; MT, microtubule; Oo, oocyst; PM, plasma membrane; Rb, residual body; SPZ, sporozoite.

Figure 4. In Vitro Oocyst Sporozoite Release Assay

Release of oocyst sporozoites at 25 °C in vitro in the presence of trypsin (50 μg/ml) at day 14 PI. Y-axis represents released sporozoites as a percentage of total oocyst sporozoites. X-axis indicates the time point when the samples are collected. In the absence of the trypsin, very few sporozoites are released from the midguts. In the presence of the trypsin, oocyst sporozoites from both CS-WT (WT) and CS-RIImut (RII) are released in a time-dependent manner. Compared with CS-WT, CS-RIImut sporozoites are released more slowly.

Figure 5. Region II-plus is Important for Sporozoite Adhesion to HepG2 Cells

Midgut sporozoites of CS-WT and CS-RIImut (100,000 each) were added to confluent HepG2 cells. Adhesion is shown as the mean number of bound sporozoites in one microscopic field (400× magnification). Results are from three independent experiments.