Experimental infection of mice by parasites
All the animal experiments were conducted in accordance with the guide for animal experimentation at Ehime University, Graduate School of Medicine. Eight-week-old female BALB/c mice (CLEA Japan Inc., Tokyo, Japan), pretreated with 0.2 mL of 0.6 mg/mL phenylhydrazine, were injected intraperitoneally with P. yoelii 17XNL parasites. To obtain only gametocytes, asexual-stage parasites were eliminated by intraperitoneal injection of 0.25 mL sulfadiazine (2 mg/mL) 24 h before blood collection.
Collection of parasites
For blood-stage parasites, infected blood (at 10–20% parasitaemia) was mixed with 5 volumes of ice-cold incomplete medium (ICM; RPMI1640 medium, pH 7.4, Invitrogen) and then passed through a sterile CF11 filter (Whatman, England) to remove white blood cells. Cells were pelleted at 1,200 × g for 5 min and washed with ice-cold ICM before antigen preparation. For in vitro ookinete culture, infected blood was immediately mixed with 5 volumes of SA buffer (10 mM Tris, 150 mM NaCl, 10 mM glucose, pH 7.3, warmed to 37°C) to prevent gamete exflagellation and then passed through a sterile CF11 filter. After washing with ICM (warmed to 37°C), the parasites were resuspended with 20 volumes of ookinete culture medium (OCM; 24°C), composed of RPMI1640 medium containing 20% heat-inactivated fetal calf serum, 0.367 mM hypoxanthine, 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 5 U/mL heparin (pH 8.3). The parasites were then transferred into culture flasks and incubated at 24°C. At 0, 1, 4, 16, and 24 h after incubation, equal volumes of cultured ookinetes were collected and washed with ice-cold ICM before antigen preparation.
Preparation of parasite antigens
Parasite samples were purified by density gradient centrifugation using Percoll (GE Healthcare, USA). Briefly, parasite pellets were gently mixed with 4 volumes of ICM, layered onto 50% Percoll, and centrifuged at 410 × g for 20 min at room temperature. Purified parasites were collected from the interface, washed twice, and resuspended in 2 volumes of ICM. The purity was checked microscopically using Giemsa-stained blood smears, and the parasite number was determined by counting under a haemocytometer before spotting onto glass slides for the immunofluorescent assay. To prepare the antigens for western blotting, the purified parasites were treated for 10 min at 4°C with 0.15% saponin in ICM containing protease inhibitors (Roche, Germany). The samples were washed twice with 0.1 M phosphate-buffered saline (PBS) containing protease inhibitors (PBS-PI) and were stored as pellets at -80°C until use. For the collection of sporozoites, the salivary glands were dissected from infected mosquitoes at day 15 post-feeding and then ground in ice-cold ICM to release sporozoites.
The genomic sequences used in this study were obtained from PlasmoDB, the Plasmodium Genomics Resource . The putative signal peptide and CPW-WPC domains were also investigated. Multiple sequence alignments of CPW-WPC family members were performed with ClustalW (MegAlign; Lasergene®).
RT-PCR of pycpw-wpc-1 and CPW-WPC family member transcripts
Total RNA was isolated from in vitro cultured ookinetes using the RNeasy Micro Kit (Qiagen). To obtain cDNA, total RNA was subjected to reverse transcription and subsequent PCR using the PrimeScript reagent kit (TAKARA, Japan). The transcriptional profiles of pycpw-wpc-1, py00599, py03515, py04297, and pyhsp70 were determined by conventional PCR at 28 cycles using gene-specific primers (see Additional file 1). cDNA concentrations were normalized across samples to pyhsp70 expression. PCR products were analysed using ImageQuant LAS 4000 (GE Healthcare), and band intensities were measured using Adobe Photoshop (Adobe System Inc., USA).
Expression of recombinant PyCPW-WPC-1 (rPyCPW-WPC-1)
The target gene was amplified from P. yoelii 17XNL zygote-enriched cDNA by PCR using the Phusion High-Fidelity DNA Polymerase (FINNZYMES, Finland). A gene-specific primer set (forward, 5′-CTCGAGAAAACGTTTGTCTTTTCTGGAGACG-3′ and –reverse, 5′-GGATCCTTAAATAATTGATCCAGTTATTGAATCC-3′) was designed to amplify the target sequence (1,539 base pairs), excluding the N-terminal signal sequence. The PCR fragment was cloned into the pEU-E01-GST-TEV-MCS-N2 vector (CellFree Sciences Co. Ltd., Matsuyama, Japan), as previously described . Plasmids were then utilized for protein production in a wheat germ cell-free protein expression system using the bilayer translation reaction method [22, 23]. GST-fused rPyCPW-WPC-1 was bound to a glutathione Sepharose 4B column, followed by cleavage with a tobacco etch virus protease to elute the rPyCPW-WPC-1 protein from the column. The yield was analyzed by Bradford’s assay and conventional SDS-PAGE with Coomassie brilliant blue staining.
Production of anti-PyCPW-WPC-1 antibodies
Eight-week-old female BALB/c mice were immunized by intraperitoneal injection of rPyCPW-WPC-1 in Freund’s adjuvant (Wako, Japan) 3 times (30 μg in 0.2 mL each) at 3-week-intervals. Immune sera were obtained two weeks after the last immunization. The reactivity and specificity of anti-rPyCPW-WPC-1 antisera were determined by ELISA and western blotting.
SDS-PAGE and western blotting
Parasite pellets were extracted with 1% Triton X-100 in PBS-PI. To solubilize the parasite antigens, the mixture was incubated on ice for 1 h. After centrifugation, the supernatant was recovered into a new microcentrifuge tube and mixed with loading buffer containing 2-β-mercaptoethanol at 4% of the final volume. Antigens were then loaded onto 12.5% polyacrylamide gels (ATTO Bioscience & Biotechnology, Japan) at 0.5–1 × 105 parasites/lane and run in an electrophoresis buffer under a constant current. The separated proteins were then transferred onto PVDF membranes (Millipore) using a semidry transfer system. After transfer, membranes were incubated with 5% skim milk in PBS containing 0.1% Tween20 (PBST) at room temperature for 2 h, followed by incubation with mouse anti-PyCPW-WPC-1 antiserum (dilution, 1:100) for 1 h. After washing with PBST, the membranes were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (dilution, 1:20,000; Invitrogen) for 30 min. Unbound antibodies were washed out with PBST, and membranes were incubated with a chemiluminescent-HRP substrate (Immobilon Western, Millipore), wrapped in a cassette, and exposed to X-ray film (Fujifilm Corporation, Tokyo, Japan). Exposed films were developed using a CEPROS SV machine (Fujifilm Corporation).
Indirect immunofluorescent assay (IFA)
Antigen slides were fixed with ice-cold acetone for 5 min. Nonspecific binding was then blocked with 5% skim milk in PBS at 37°C for 30 min before incubation with antibodies (mouse anti-PyCPW-WPC-1 at a dilution of 1:100 and rabbit anti-Pys25 at a dilution of 1:10,000) at 37°C for 1 h. The slides were then washed in PBS for 5 min and subsequently incubated with Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 546 goat anti-rabbit IgG (Invitrogen) diluted in PBS containing 1 μg/mL 4′6-diamidino-2-phenylindole (DAPI) at 37°C for 30 min. After washing out the unbound antibodies, the coverslips were mounted with Prolong Gold antifade reagent (Invitrogen), and the slides were observed under a confocal microscope (LSM710; Carl Zeiss, Germany). The resulting images were analysed using the Zen software (Carl Zeiss, Germany).
Targeted gene disruption of pycpw-wpc-1
Pycpw-wpc-1 was disrupted by double-crossover homologous recombination in P. yoelii 17XNL. Two basic plasmids, pPbDT3UB12 and pHDEF1-mh-R12, were used for the construction of transfection plasmids, as previously described . In brief, target sequences were amplified by PCR using KOD Plus DNA polymerase (Toyobo, Japan) with primer sets targeting the 809-base pair region upstream (-forward, 5′-CTCGAGTATGCATAATTGTGAATAGTTATTGG-3′ and –reverse, 5′-GGATCCGACCCCTATAATAATAAAAGGTCTGTC-3′) and 755-base pair region downstream (-forward, 5′-AGATCTACCAGATGATTATAATGGACCTTGTCC-3′ and –reverse, 5′-CTCGAGCGGATGTATTGAGAAGCTTTACATGTG-3′) of pycpw-wpc-1. PCR products were subsequently digested and inserted into pPbDT3UB12. Using Gateway Technology (Invitrogen), the modified pPbDT3UB12 plasmid was then subjected to a BP recombination reaction with the donor vector pDONR221 to give the entry plasmid. This entry plasmid was then subjected to an LR recombination reaction with pHDEF-1-mh-R12 to yield the final construct, which was linearized with XhoI before use. Enriched schizonts were transfected with 20 μg of linearized construct by electroporation using Nucleofector (Lonza) with a human T cell solution and the U-33 program. Transfected parasites were delivered into 8-week-old BALB/c female mice by intravenous injection. Twenty-four hours later, the mice were treated with pyrimethamine (70 μg/mL) via their drinking water. Infected blood was collected to examine the integration of the disruption cassette by PCR using the KOD FX DNA polymerase with a unique set of primers. Successfully transfected parasites were cloned using the limiting dilution technique, and cloned parasites were cultured for ookinetes. The expression of PyCPW-WPC-1 was measured by western blotting and IFA using mouse anti-PyCPW-WPC-1 antibodies.
Mosquito feeding assay
More than 50 female Anopheles stephensi mosquitoes were allowed to fed on mice infected with wild-type- or gene-disrupted parasites, and only mosquitoes that showed complete engulfment of the blood were selected for an additional two weeks of incubation at 24°C. For infectivity analysis, mosquito midguts were dissected on day 10 post-feeding, and oocyst numbers were counted under a microscope. To examine sporozoite-formation ability, sporozoites were collected from midguts on day 15 post-feeding and counted.