BALB/c mice were injected with 2 × 106 parasite-infected red blood cells (iRBCs) intraperitoneally to initiate the infection. The mice were anesthetized when the parasitemia reached approximately 5–8%, and mosquitoes were allowed to feed on them for 10 min. The mosquitoes were dissected on days 14 and 18 after feeding to measure the presence and number of oocysts and sporozoites, respectively. On day 14, approximately 40 midguts were individually visualized under a fluorescence microscope, and the numbers of oocysts were counted. On day 18, salivary glands were individually dissected from the remaining 40 mosquitoes and homogenized gently to release sporozoites, which were then counted in a hemocytometer to calculate the average number of sporozoites per mosquito.
Parasite infection and parasitemia
BALB/c mice were intravenously injected with 1 × 104 blood stage parasites. Parasite growth was monitored by daily parasite counting in approximately 5000 erythrocytes until mice died. For this, thin blood films made from mouse blood were stained using Giemsa staining. To screen the ability of parasites to undergo sexual development and sporogony, groups of BALB/c mice were infected with 2 × 106 knockout or wild-type blood stage parasites intraperitoneally and monitored for gametocyte formation by analyzing Giemsa-stained thin blood films under a light microscope.
Transcript profiling
Total RNA was isolated from different parasitic stages, and cDNA was prepared by performing reverse transcription from RNA using random hexamer primers following the protocol mentioned in the kit (Bio-Rad #1708891). qPCR was performed using a gene-specific primer pair (primer details given in Supplementary Table 1). To check target gene expression under heat stress, infected blood at approximately 3% parasitemia was collected from mice and cultured at 37 °C and 41 °C in vitro. The parasites were harvested at 3 h and 6 h after heat stress.
Cloning, expression and purification of HspJ62 protein
The E. coli codon-optimized (Entelechon, Germany) HspJ62 gene (amino acids 2–424 of PBANKA_0938300) was cloned (primer details given in Supplementary Table 1) between the XhoI and BamHI restriction sites of the pGEX-6P1 vector (GE Healthcare). The vector contains a glutathione S-transferase (GST) tag that is located at the N-terminus of the fusion protein. The calculated fusion protein size is 72,530 Da. The HspJ62 gene cloned vector was transformed into E. coli strain BL21-CodonPlus-RIL (Stratagene), and the cells were grown in culture medium containing 100 μg/ml ampicillin at 37 °C. Isopropyl 1-thio-β-d-galactopyranoside (IPTG, BioSynth Inc., USA) was added to a final conc. of 1 mM, was used to induce the culture when its O. The D600 reached between 0.5 and 0.6 and was further incubated at 18 °C for 22 h. The cells were harvested by centrifuging the culture at 10,000 rpm for 10 min at 4 °C and suspended in cell lysis buffer containing 0.025 mg/ml lysozyme (Bio Basic Inc. Canada) and 1 × Protease Inhibitor mixture (Roche, Germany). The culture was sonicated at 4 °C (ice-cold H2O) for 10 min at an amplitude of 35 (Qsonica Sonicator, Microprobe). The lysed sample was centrifuged at 13,000 rpm for 20 mint at 4 °C in an R247 rotor (REMI-CPR30 centrifuge). GST beads (GE Healthcare) were added to the cleared lysate and kept for binding at 4 °C for 8 h. Following binding, beads were loaded on a column and washed with 0.2 mM reduced glutathione-containing buffer. Elution buffer containing 25 mM reduced glutathione was used to elute the protein.
Rat immunization and antibody generation
Four-week-old SD rats (n = 2) were primed with recombinant HspJ62 protein (50 μg) emulsified in complete Freund’s adjuvant. The first boost was given 14 days post priming with protein (30 μg/rat) emulsified in incomplete Freund’s adjuvant. The two subsequent boosts were followed using the same amount of protein as in the first boost at one-week intervals each. Preimmune sera were collected two days before immunization from the same rat. Blood was collected from the retro-orbital sinus one week after the second boost and from cardiac puncture one week after the third boost. The blood was allowed to clot at room temperature for 1 h and centrifuged at full speed (Eppendorf model 5415R), and serum containing antibodies was collected and stored at − 20 °C.
ELISA
The recombinant HspJ62 protein was coated in each well of a 96-well plate, incubated at 37 °C for 3 h and then stored at 4 °C. The next day, the wells were washed three times with 1X PBST and three times with 1 × PBS and then blocked with 3% bovine serum albumin (BSA) for 1 h at room temperature. Serum collected from rats was added to the plate at increasing dilutions (100 µl/well) and incubated at 37 °C for 2 h. The plate was washed thrice with 1X PBST and 1X PBS. After washing, 100 µl HRP-conjugated goat anti-rat IgG antibodies (Invitrogen) diluted 1:4000 was added to the plate and further incubated at 37 °C for 1 h. The plate was washed again with PBST, followed by the addition of 50 µl of TMB substrate (Sigma). The color was allowed to develop for 10 min. The reaction was stopped by adding 50 µl of 2 mM H2SO4, to each well. The plate was read immediately in a plate reader at 490 nm.
Immunofluorescence assay (IFA)
The parasites were collected at different stages of their life cycle. Erythrocytic stage parasites were collected from mouse blood; ookinetes were collected from in vitro culture; oocysts and sporozoites were collected from the mosquito gut and salivary gland, respectively; and liver stage parasites were collected from in vitro infected HepG2 cells. Slides of each stage were prepared, fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% saponin (Sigma) for 10 min. Samples were blocked using 3% BSA, followed by incubation in anti-HspJ62 antibody (1:5,00 dilution) at room temperature for 2 h. The slides with samples were washed and incubated with Alexa Fluor-conjugated goat anti-rat IgG (1:1000 dilution) for 1 h. After three washes, parasite nuclei were stained with 4′,6′-diamidino-2-phenylindole (DAPI, Invitrogen, USA). Slides were mounted with ProLong Gold antifade reagent (Invitrogen USA), and images were captured with a fluorescence microscope (Zeiss Axio-Imager M2).
Pull-down assay
The infected RBCs were collected from mice, and RBCs were lysed using 0.2% saponin. The free parasites obtained were washed thrice with 1X PBS and lysed in IP lysis buffer (150 mM NaCl, 250 mM Tris, 1 mM EDTA, 5% glycerol, 1% NP-40, pH 7.4), followed by centrifugation at 12,000×g to obtain a clear lysate. The lysate protein content was quantified using a Pierce BCA protein assay kit. The beads coated with recombinant HspJ62 protein were added to parasite lysate and incubated at 4 °C under mild agitation for 12 h (10 μg of recombinant HspJ62 protein on beads was incubated with 100 μg of total parasite proteins). The beads were washed three times in IP buffer, and the bound proteins were eluted from the beads using elution buffer (25 mM reduced glutathione, 150 mm NaCl, 100 mm Tris, 0.5 mm EDTA, 10% glycerol, pH 8.5). Eluted proteins were treated with the enzyme trypsin in solution. An AB SCIEX MALDI-TOF/TOF-4800 mass spectrometer coupled to nano-LC 1000 was employed to analyze peptides generated upon trypsin treatment. The raw data obtained during nano LC–MS/MS analysis were processed and searched with the P. berghei PlasmoDB database.
In-solution trypsin digestion
The proteins in the samples were treated before digestion with trypsin in solution. The samples were vacuum dried and volume compacted to nearly 100 µL in 50 mM ammonium bicarbonate (Sigma) buffer at pH 7.8, reduced using 10 mM DTT (final concentration) and alkylated with 40 mM iodoacetamide (Sigma Aldrich, USA) for 1 h in the dark at RT. The samples were then treated with sequencing grade trypsin (Promega) at a ratio of 1:50 (w/w) trypsin:protein and placed in a water bath at 37 °C for 18 h for complete digestion. Subsequent to digestion, the peptides were acidified in 0.1% formic acid.
Far Western blot analysis
The predicted functional domain of the ApiAP2 protein (amino acids 708–1485 of PBANKA_1453700) was cloned into the pET28a vector, purified recombinant ApiAP2 protein was used as prey, and purified HspJ62 protein was used as bait using a modified protocol published earlier45. ApiAP2 protein was run on SDS-PAGE gels and transferred to nitrocellulose membranes. The denatured protein was renatured on the membrane in buffer (10% glycerol, 100 mM NaCl, 100 mM Tris pH 7.5, 0.001 EDTA, 1% Tween-2-, 6 M guanidine–HCl, 2% milk powder, 0.001 DTT) by gradually reducing the guanidine–HCl concentration from 6 to 0.1 M while rocking at room temperature. The membrane was washed three times with AC buffer [100 mM NaCl, 20 mM Tris (pH 7.6), 0.5 mM EDTA, 10% glycerol, 0.1% Tween-20, 2% skim milk powder and 1 mM DTT]. After blocking with 5% skimmed milk, the membrane was incubated with purified bait (recombinant HspJ62) protein overnight at 4 °C. The membrane was washed, added to buffer containing anti-HspJ62 antibody and incubated at RT for 2 h. The membrane was washed once again and incubated with secondary antibody (HRP-labeled anti-rat IgG) for one hour at 37 °C. The antibody concentrations used were 1:1000 anti-HspJ62 antibody and 1:5000 secondary antibody (HRP-labeled anti-rat). The membrane was developed by DAB using a standard western blot protocol.
RNA isolation, library preparation, sequencing
Infected blood was collected from mice and passed though Plasmodipure (Euro Proxima, Netherlands, cat# 8011) to remove WBCs. The purified RBCs were lysed in 0.2% saponin, and the free parasite pellet was washed three times with PBS to obtain a pellet devoid of any RBC contamination. The parasite RNA was isolated using Trizol. RNA extraction and Illumina mRNA sequencing were performed in duplicate. For this, total RNA was isolated and quantified using a NanoQuant-M200 spectrophotometer (Tecan, UK). The RNA integrity number (RIN) was employed to check the quality of RNA, and it was more than 7. The mRNA was purified, subjected to cDNA synthesis and fragmented, and for adapter ligation, a single ‘A’ base was added at the end. The purified products were enhanced with PCR to make the final cDNA library. The quality of the cDNA library was analyzed on a Bioanalyzer, and deep sequencing of validated cDNA libraries was achieved by the Illumina HiSeq 1000 platform using manufacturer guidelines.
Data processing
The Fast QC quality control tool (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used to check the quality of raw reads, which were then cleaned by using TrimGalore software (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore). Subsequently, TopHat2 software was used to map transcriptome data with the reference genome (PlasmoDB Release 33). Nonexpressed contigs (with FPKM = 0) were excluded from this transcriptomic analysis. Raw read count data were used to identify differentially expressed genes using edgeR46.
Differential expression
Gene expression differences in all the different experimental groups were analyzed employing Cuffdiff (version 2.2.1). Specific groups were matched with linear contrasts, and the P-values were optimized by multiple testing (i.e., false discovery rate). A P value (FDR) below 0.005 and a minimal log FC of 1.5 were considered differentially expressed47.
Gene ontology enrichment
Differentially expressed gene (DEG) functional enrichment of gene ontology (GO) analyses was performed using the REVIGO tool in conjunction with the GO annotation available from PlasmoDB48 The analysis was based on a gene set of interest compared to all annotated genes using the “weight” algorithm with Fisher’s exact test (both implemented in REVIGO). A P-value of below 0.05 was considered significant.
Validation of NGS data by real time PCR
NGS data was validated by RT-PCR. A random set of 20 differentially expressed genes was selected. 18S rRNA and GAPDH were used as housekeeping gene controls. The primers (primer details given in Supplementary Table 1) against each of these genes were designed using Primer3 software and synthesized. cDNA was made from independent biological replicates of WT and HspJ62-KO parasite lines of blood stage RNA. All reactions were performed on an Eppendorf RealPlex detection system using SYBR Green PCR master mix. The fold change in each selected gene was determined using the 2 −ΔΔCt method.
Contribution to the field statement
This work has provided insights into the critical role of a heat shock protein and the processes underlying gametocyte commitment and development. HspJ62, a putative chaperone molecule, is essential for the development of P. berghei sexual stages. The transgenic mutant P. berghei parasite line (ΔHspJ62) was unable to make gametocytes. This study characterizes HspJ62 protein as a fertility factor, as parasites lacking it are unable to transmit to mosquitoes. Our study adds an important contribution to ongoing research aimed at understanding gametocyte differentiation and formation in parasites.
