Disruption of Plasmodium sporozoite transmission by depletion of sporozoite invasion-associated protein 1

Abstract

Accumulation of infectious Plasmodium sporozoites in Anopheles spp. salivary glands marks the final step of the complex development of the malaria parasite in the insect vector. Sporozoites are formed inside midgut-associated oocysts and actively egress into the mosquito hemocoel. Traversal of the salivary gland acinar cells correlates with the sporozoite's capacity to perform continuous gliding motility. Here, we characterized the cellular role of the Plasmodium berghei sporozoite invasion-associated protein 1 (SIAP-1). Intriguingly, SIAP-1 orthologs are found exclusively in apicomplexan hemoprotozoa, parasites that are transmitted by arthropod vectors, e.g., Plasmodium, Babesia, and Theileria species. By fluorescent tagging with mCherry, we show that SIAP-1 is expressed in oocyst-derived and salivary gland-associated sporozoites, where it accumulates at the apical tip. Targeted disruption of SIAP-1 does not affect sporozoite formation but causes a partial defect in sporozoite egress from oocysts and abolishes sporozoite colonization of mosquito salivary glands. Parasites with the siap-1(-) mutation are blocked in their capacity to perform continuous gliding motility. We propose that arthropod-transmitted apicomplexan parasites specifically express secretory factors, such as SIAP-1, that mediate efficient oocyst exit and migration to the salivary glands.

FIG. 1.

Plasmodium SIAP-1. (A) Schematic diagram of the primary structures of the apicomplexan SIAP-1 proteins. The primary structure of P. berghei SIAP-1 (PB000251.01.0; reannotated) and the orthologs of P. yoelii (PY00455), P. falciparum (PFD0425w), Plasmodium vivax (Pv000815), Babesia bovis (XP_001609197), and Theileria parva (XP_765499) are indicated with white boxes. The predicted cleavable signal peptide is represented in gray. Amino acid sequence identities are indicated as percentages of identical residues compared to the P. berghei sequence. (B) Quantitative RT-PCR analysis of P. berghei SIAP-1 gene expression in midgut oocysts (day 12 postinfection of mosquitoes), salivary gland sporozoites (day 18 postinfection of mosquitoes), early liver stages (6 h postinfection of HepG2 cells) and mixed blood stages. Relative gene expression was normalized to GFP expression level and is shown as the mean (± standard deviation) of the results from two independent experiments.

FIG. 2.

Expression and localization of PbSIAP-1/mCherry in sporozoites. Expression of the mCherry-tagged SIAP-1 (red) was analyzed by confocal fluorescence microscopy of SIAP-1/mCherry P. berghei parasites constitutively expressing GFP (green). (A) Midgut sporozoites. Bar, 20 μm. (B) Salivary gland-associated sporozoites. Bar, 10 μm. (C) Fluorescent imaging of a motile sporozoite. Sequential acquisition of the red fluorescence and differential interference contrast (DIC) confirms SIAP-1 accumulation mainly at the apical tip. Note that the distribution of SIAP-1/mCherry is not modified as the sporozoite glides. Bar, 5 μm. (D) Higher magnification of a fixed salivary gland-associated sporozoite. (E) Confocal microscopy analysis of HepG2 cell cultures 4 h, 24 h, and 72 h after infection with P. berghei parasites expressing GFP (green) and SIAP-1/mCherry (red). Nuclei were stained with Hoechst 33342 (blue). Bars, 10 μm.

FIG. 3.

Targeted deletion of the P. berghei SIAP-1 gene. (A) Replacement strategy for targeted gene disruption of PbSIAP-1. The WT SIAP-1 locus (WT) is targeted with a KpnI (K)/SpeI (S)-linearized replacement plasmid (pΔSIAP-1) containing the 5′ and 3′ UTR of PbSIAP-1 and the positive selection marker TgDHFR-TS. After double-crossover homologous recombination, the SIAP-1 ORF is substituted by the selection marker, resulting in the mutant siap-1(−) allele. Replacement- and WT-specific test primer combinations and expected fragments are shown as lines. (B) Replacement-specific PCR analysis. Confirmation of the predicted gene targeting is done with primer combinations that amplify only a signal in the recombinant locus (test). The absence of a WT-specific signal in the clonal siap-1(−) parasite population confirms the purity of the mutant parasite line. (C) Depletion of SIAP-1 transcripts in siap-1(−) parasites. cDNA from WT and siap-1(−) sporozoites were used as a template for SIAP-1-specific PCRs (top). Amplification of the circumsporozoite protein (CSP) transcripts was used as a positive control (bottom).

FIG. 4.

siap-1(−) sporozoites are impaired in egress from oocysts. (A) Representative immunofluorescence pictures of infected A. stephensi midguts at day 14 after infection. (B) Quantification of oocyst-associated sporozoites per infected mosquito for siap-1(−) (gray circles) and WT (white circles) parasites. The bars represent the mean values. (C) Representative merged bright field and immunofluorescence pictures of infected A. stephensi salivary glands at day 17 after infection. (D) Quantification of salivary gland-associated sporozoites per infected mosquito for siap-1(−) (gray circles) and WT (white circles) parasites. Note the logarithmic scale. Bars, 200 μm.

FIG. 5.

siap-1(−) sporozoites fail to colonize the mosquito salivary glands. (A) Replacement strategy for targeted gene disruption of PbSIAP-1 and insertion of a GFP expression cassette. The WT SIAP-1 locus is targeted with a SacII/KpnI-linearized replacement plasmid (siap1rep-GFP construct) containing the 5′ and 3′ UTR of PbSIAP-1, the positive selection marker TgDHFR-TS, and the GFP coding sequence under the control of the CSP promoter region. After double crossover homologous recombination, the SIAP-1 ORF is substituted by the selection marker and the GFP cassette, resulting in the mutant siap-1(−)/GFP allele. (B) Fluorescence imaging of whole mosquitoes infected with WT or siap-1(−)/GFP parasites, day 17 and day 22 after the infectious blood meal. Note the presence of green fluorescent WT parasites in the mosquito midgut (M), hemocoel (H, best visualized in the wings with arrows), and salivary glands (S, arrowheads). siap-1(−)/GFP parasites were only detected in the midgut (M) and the hemocoel (H, arrows), but not in salivary glands of infected mosquitoes. (C) Fluorescence microscopy of a dissected mosquito midgut 21 days after infection with siap1(−)/GFP parasites. Note the presence of oocysts and the release of high numbers of sporozoites. Bar, 200 μm. (D) Fluorescence microscopy of a dissected mosquito wing 21 days after infection with siap-1(−)/GFP parasites, demonstrating the presence of sporozoites in the hemocoel. Bar, 50 μm.

FIG. 6.

Quantitative assessment of siap-1(−) sporozoite release into the mosquito hemocoel. Shown are quantifications of hemocoel sporozoites per infected mosquito for siap-1(−) (gray circles) and WT (white circles) parasites. The bars represent the mean values.

FIG. 7.

Defective gliding locomotion in siap-1(−) sporozoites. (A) Representative immunofluorescence pictures of sporozoites isolated from Anopheles midguts or hemocoels. Sporozoites were deposited onto coated coverslips, and trails were visualized with the anti-circumsporozoite protein (CSP) antibody. Note that siap-1(−) hemocoel sporozoites lack productive motility and are indistinguishable from midgut-associated sporozoites. (B) Quantification of the WT and siap1(−) motility patterns. Shown is the percentage of nonmotile, i.e., detached (white) and attached (light gray), sporozoites and sporozoites that display nonproductive (dark gray) and productive (black) motility. In contrast to WT sporozoites that mature upon egress to the mosquito hemocoel, siap-1(−) parasites remain nongliding.