Characterization of Plasmodium berghei Pbg37 as Both a Pre- and Postfertilization Antigen with Transmission-Blocking Potential

Mice, parasites, and mosquitoes.BALB/c mice aged 6 to 8 weeks were bought from the Beijing Animal Institute. The P. berghei ANKA strain was maintained by serial passages as described previously (28). Mouse infection was initiated by intraperitoneal injection of 1 × 10⁷ P. berghei-infected red blood cells (iRBCs). Anopheles stephensi mosquitoes (Hor strain) were maintained in an insectary at 25°C and 50 to 80% humidity. All animal experiments were approved by the animal ethics committee of China Medical University.

Expression and purification of recombinant protein.For the expression of rPbg37, total RNA was extracted from purified gametocytes and RT-PCR was performed to amplify the pbg37 fragment encoding amino acids 26 to 88 with primers pbg37-F and pbg37-R (see Table S1 in the supplemental material). The PCR product was digested with BamHI and NotI and cloned into pET32a(+). Expression of the rPbg37 in Escherichia coli Rosetta-gami B (DE3) cells was induced with 1 mM isopropyl-d-1-thiogalactoside (Sigma) at 20°C for 8 h, and rPbg37 fused with the thioredoxin (Trx) from the expression vector was purified on an Ni-NTA agarose column (Novagen). The Trx-His tag protein expressed from the empty vector pET32a(+) was similarly purified and used as a control for immunization. Purified recombinant proteins were dialyzed extensively in phosphate-buffered saline (PBS; pH 7.4) at 4°C overnight and analyzed on a 10% SDS-PAGE gel. While the rPbg37 fusion protein was used for immunization, the rPbg37 polypeptide was further purified from the Ni-NTA agarose column after digestion with enterokinase (Solarbio). The released rPbg37 polypeptide (∼7 kDa) was concentrated, and its purity was determined by electrophoresis on a Tris-Tricine-SDS-PAGE gel.

Immunization of BALB/c mice.BALB/c mice (n = 6) were immunized with the rPbg37-Trx fusion protein (50 μg each) emulsified in complete Freund's adjuvant (Sigma) via subcutaneous injection. Two booster immunizations with the same rPbg37 fusion protein (25 μg/mouse) were done at a 2-week interval. The same immunization procedure was followed with another group of mice (n = 6) using the Trx control protein. Serum was collected and pooled for each group of mice on the day before each immunization and at 14 days after the final immunization.

ELISA was used to assess the antibody titers in the rPbg37 immunization group as previously described (28, 46). To estimate the endpoint titer of antiserum from the rPbg37-immunized group and the control group, 96-well plates were coated overnight at 4°C with the purified Pbg37 polypeptides at 5 μg/ml. Serum was serially diluted in 1% bovine serum albumin (BSA) in PBS from 1:2,000 to 1:128,000. The endpoint was defined as the highest dilution of the antiserum when the optical density (OD) value at 490 nm was above the average for the control antiserum plus 3 times the standard deviation.

Western blotting.To determine the expression of the Pbg37 protein in different development stages of the parasite, P. berghei schizonts, gametocytes, zygotes, and ookinetes were purified on Nycodenz gradients essentially as previously described (28). Proteins from purified parasites were extracted with 2% SDS containing protease inhibitors. To determine the localization of the Pbg37 protein in the cytosol or membrane fractions, purified gametocytes were separated into membrane and soluble protein fractions using a membrane protein isolation kit (Invent Biotechnologies). Briefly, purified gametocytes were treated with 0.15% saponin to lyse the erythrocytes, and parasites were collected by centrifugation and washed once with PBS. Pellets were lysed in buffer A with protease inhibitors (Roche) in a filter cartridge. After centrifugation at 14,000 rpm for 30 s, the pellets were resuspended and centrifuged at 3,000 rpm for 1 min. The supernatant was collected, centrifuged at 14,000 rpm for 10 min, and kept as the cytoplasmic protein fraction. The pellet (total membrane fraction) was resuspended in buffer B and centrifuged at 10,000 rpm for 20 min. The supernatant was then centrifuged again at 14,000 rpm for 30 min, and the pellet was collected as the membrane protein fraction. Protein concentrations were determined using a bicinchoninic acid assay kit (Thermo Fisher Scientific). Equal amounts of parasite proteins (10 μg) of the four parasite stages and equal amounts (30 μg) of the gametocyte cytosolic and membrane proteins were separated in a 10% SDS-PAGE gel under reducing conditions and transferred to polyvinylidene difluoride membranes (Bio-Rad). The membranes were blocked with 5% nonfat milk in Tris-buffered saline with 0.1% Tween 20 (TBS-T) at room temperature for 2 h and probed with mouse anti-rPbg37 antiserum and control serum against Trx (1:200). After three washes with TBS-T, the membrane was incubated with horseradish peroxidase-conjugated anti-mouse immunoglobulin antibody. Protein bands were visualized by chemiluminescence using a Pierce ECL Western blotting kit.

IFA.The localization of Pbg37 in parasites was detected by IFA (47). Briefly, on day 4 postinfection, gametocytes were collected in PBS and kept on ice to avoid activation. To produce gametes, gametocyte-infected blood was taken from a mouse and immediately mixed with ookinete culture medium for 15 min at 25°C. Then, cultured parasites were washed twice with PBS. Both activated (gametes) and nonactivated gametocytes and other parasite stages (schizonts, zygote, retort, and ookinete) were fixed with 4% paraformaldehyde and 0.0075% glutaraldehyde in PBS for 30 min at room temperature (47). Parasites were washed once with 0.15% glycine in PBS for 10 min. The fixed cells were either permeabilized with 0.1% Triton X-100 in PBS for 10 min and then blocked with 3% BSA–PBS for 60 min or blocked without permeabilization. Following another PBS wash, mouse anti-rPbg37 antiserum and control serum (diluted to 1:200 in 3% BSA–PBS) were added and the mixture was incubated at 37°C for 1 h. After washing the slides with PBS, fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin antibody (Invitrogen) diluted to 1:500 in 3% BSA–PBS was added and the mixture was incubated for 1 h. To distinguish extracellular female gametes from intraerythrocytic female gametocytes, a monoclonal antibody against Pbs21 was used to show the surface staining of female gametes and ookinetes. Parasite nuclei were counterstained with 10 μl DAPI (4′,6-diamidino-2-phenylindole; Sigma). To observe the localization of Pbg37 under native conditions, unfixed parasites were collected in PBS or RPMI 1640 culture medium and IFA was performed as described above with anti-rPbg37 antiserum. IFA slides were mounted with ProLong Gold antifade reagent (Invitrogen) and observed under an Olympus BX53 microscope.

Generation of transgenic parasites.A double-crossover homologous recombination strategy was used to generate the Δpbg37 lines (48). The pbg37 5′ and 3′ flanking regions were amplified using primer pairs 5UTR-F–5UTR-R and 3UTR-F–3UTR-R, respectively (Table S1) and cloned into the vector to flank the human dhfr expression cassette. Ten micrograms of the plasmid was linearized by HindIII and XhoI digestion and electroporated into purified P. berghei schizonts using a Nucleofector system. After transfection, the parasites were mixed with 50 μl of complete culture medium and the mixture was inoculated into mice. Twenty-four hours later, parasites were selected with pyrimethamine (70 μg/ml) via the drinking water for mice. Two independent transfection experiments were performed to obtain different parasite clones. Parasite genomic DNA was extracted from infected blood to confirm pbg37 gene deletion using integration-specific diagnostic PCR with primer pairs P1-P2, P1-P3, and P4-P5 (Table S1).

Phenotype analysis of the Δpbg37 parasites.To study the effect of the pbg37 deletion on parasite development, we compared the phenotypes between two pbg37 deletion clones (KO1 and KO2) and the WT parasites. For each group, five mice were used; each was injected intraperitoneally with 1 × 10⁶ iRBCs of the respective parasite clones. Parasitemia and gametocytemia were monitored daily by microscopic examination of Giemsa-stained thin blood films. In each mouse, a total of 100 mature gametocytes were differentiated into male and female gametocytes on the basis of the morphological characteristics to determine the gametocyte sex ratio. Female gametocytes are characterized by a relatively small nucleus with a nucleolus and concentrated pigment. In male gametocytes, the nucleus is larger without a distinctive nucleolus, and the pigment is more diffuse. In Giemsa-stained blood films, male gametocytes are pink cells, whereas female gametocytes are more violet or bluish (17). The proportion of mature gametocytes in the total gametocyte population was also estimated with at least 100 gametocytes. The gametocyte density and the proportion of mature male gametocytes were used to calculate the volume of infected blood to be used for exflagellation and ookinete formation experiments. Exflagellation of male gametocytes and formation of ookinetes were monitored using in vitro cultures (48, 49). Briefly, infected blood containing an equal number of mature male gametocytes was mixed with the ookinete culture medium and incubated at 25°C for 15 min to induce gamete formation. The number of exflagellation centers in each ×100 magnification field was counted under a phase-contrast microscope. A total of 20 fields were counted per mouse. To observe ookinete formation, blood samples were incubated in ookinete culture medium (RPMI 1640, 50 mg/liter penicillin, 50 mg/liter streptomycin, 100 mg/liter neomycin, 25% [vol/vol] heat-inactivated fetal calf serum, 6 U/ml heparin, pH 8.3) at 19°C for 24 h, and ookinetes were monitored by IFA using the monoclonal antibody against Pbs21.

To determine whether pbg37 KO affected the sex ratio of the gametocytes, total RNA was extracted from 1 × 10⁶ gametocytes of the WT, KO1, and KO2 parasites with the TRIzol reagent (Invitrogen). cDNA was synthesized from 1 μg of each RNA sample using a TaKaRa RNA PCR kit. The expression levels of the male-specific pbs230p and the female-specific pbs47 genes were quantified by real-time RT-PCR, with the expression of the hsp70 gene used as the reference. Real-time RT-PCR mixtures consisted of 2 μl cDNA, 5 μl SYBR green fast master mix, 0.5 μl each of the forward and reverse primers (Table S1), and 2 μl diethyl pyrocarbonate-treated water. Analysis was performed using an Applied Biosystems 7500 machine with the following cycling conditions: denaturation at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. Three replicates were used for each mouse. The relative quantitation of gene expression was done following an established protocol (50).

The infectivity of the parasites to mosquitoes was evaluated in a direct mosquito feeding experiment. On day 3 p.i., when asexual parasitemia had reached 5 to 7%, 4-day-old starved A. stephensi mosquitoes (50 per mouse) were allowed to feed on the infected mice for 1 h. Fully engorged mosquitoes were kept for 12 days, and approximately 30 mosquitoes of each group were dissected. Midguts were stained with 0.5% mercurochrome for 10 min to determine the oocyst number per positive midgut and the mosquito infection rate (the number of infected mosquitoes/the number of total mosquitoes) (51).

Quantification of TB activity.The TB potential of Pbg37 was assessed using an in vitro ookinete formation assay and a direct mosquito feeding assay. For the ookinete formation assay, mice were infected with an intraperitoneal injection of 1 × 10⁶ WT P. berghei iRBCs after treatment with phenylhydrazine. At 3 days p.i., anti-rPbg37 serum or the control serum was diluted at 1:5, 1:10, and 1:50 with the ookinete culture medium and mixed with 10 μl of infected mouse blood in a total volume of 50 μl. The exflagellation of male gametocytes was quantified as described above (28). Samples were examined at 15 min, 2 h, 12 h, and 24 h to count the numbers of macrogametes, zygotes, retorts, and ookinetes on the basis of their morphological features after IFA with the monoclonal antibody against Pbs21 and DAPI staining of the nucleus.

For mosquito feeding experiments, mice were immunized with rPbg37 or the control Trx-His tag protein as described above. On day 14 after the final immunization, mice were infected with 1 × 10⁶ parasites. At 3 days after infection, starved female A. stephensi mosquitoes were allowed to feed on the mice (∼50 mosquitoes/mouse). The oocyst density and mosquito infection rate were determined as described above (28).

Statistical analyses.Statistical analyses were carried out using SPSS software, version 17.0 (SPSS Inc., USA). Student's t test was used for statistical comparison between groups of asexual parasitemia, gametocytes, sex ratio, exflagellation, and ookinete numbers. The Mann-Whitney U test was employed to analyze oocyst density (oocyst number per midgut), while Fisher's exact test was used to analyze infection prevalence. All data are presented as the mean ± standard error of the mean (SEM). A P value of <0.05 was considered statistically significant.