Essential role of a Plasmodium berghei heat shock protein (PBANKA_0938300) in gametocyte development

Abstract

The continued existence of Plasmodium parasites in physiologically distinct environments during their transmission in mosquitoes and vertebrate hosts requires effector proteins encoded by parasite genes to provide adaptability. Parasites utilize their robust stress response system involving heat shock proteins for their survival. Molecular chaperones are involved in maintaining protein homeostasis within a cell during stress, protein biogenesis and the formation of protein complexes. Due to their critical role in parasite virulence, they are considered targets for therapeutic interventions. Our results identified a putative P. berghei heat shock protein (HSP) belonging to the HSP40 family (HspJ62), which is abundantly induced upon heat stress and expressed during all parasite stages. To determine the role HspJ62, a gene-disrupted P. berghei transgenic line was developed (ΔHspJ62), which resulted in disruption of gametocyte formation. Such parasites were unable to form subsequent sexual stages because of disrupted gametogenesis, indicating the essential role of HspJ62 in gametocyte formation. Transcriptomic analysis of the transgenic line showed downregulation of a number of genes, most of which were specific to male or female gametocytes. The transcription factor ApiAP2 was also downregulated in ΔHspJ62 parasites. Our findings suggest that the downregulation of ApiAP2 likely disrupts the transcriptional regulation of sexual stage genes, leading to impaired gametogenesis. This finding also highlights the critical role that HspJ62 indirectly plays in the development of P. berghei sexual stages and in facilitating the conversion from the asexual blood stage to the sexual stage. This study characterizes the HspJ62 protein as a fertility factor because parasites lacking it are unable to transmit to mosquitoes. This study adds an important contribution to ongoing research aimed at understanding gametocyte differentiation and formation in parasites. The molecule adds to the list of potential drug targets that can be targeted to inhibit parasite sexual development and consequently parasite transmission.

Figure 1

HspJ62 is induced under heat stress conditions. (A) In silico analysis of the functional domains of HspJ62 shows that the DnaJ domain (34–93 amino acids) and thioredoxine-like domain (188–289 amino acids) are typical of the Type II HSP40 protein family. (B) The result of PCR using cDNA synthesized from RNA isolated from different stages of parasite is shown. PCR analysis shows the presence of gene transcripts throughout the life cycle of parasites. Genomic DNA (gDNA) was used as a positive control, while water was used in place of a template in the negative control; EEF: exoerythrocytic forms 24 and 48 h post infection; Blood: mixed blood stages. Full blots are shown in Supplementary Fig. 5A and B. (C) Bands present in figure A were quantified by densitometry and normalized to Gapdh and plotted as graph. (D) Quantitative PCR using RNA extracted from blood stage parasites cultured at normal 37 °C and higher 41 °C temperatures. The transcript levels were normalized to the Gapdh housekeeping gene, and PbHsp70-1 transcript was included as a positive control. The data are shown as the gene expression at 41 °C compared to 37 °C and represent the mean value of two separate experiments performed in triplicate along with standard error of the mean (SEM) shown as error bars. Statistical analysis was performed using the unpaired T-test; **P < 0.01, *P < 0.1.

Figure 2

HspJ62 is expressed throughout the different stages of the parasite life cycle. (A) Representative cells show the export of protein across the PV membrane into the host cytosol. An anti-HspJ62 polyclonal antibody was used as the primary antibody, followed by an Alexa 594-conjugated secondary antibody. The parasite nuclei were stained with DAPI (blue), and the slides were visualized under a fluorescence microscope. Preimmune rat sera were used as a negative control. (B) Immunofluorescence assays to localize HspJ62 in the different developmental stages of parasites using anti-HspJ62 antibodies. The WT-GFPcon parasites (green) were used for IFA, and an anti-rat Alexa 594-conjugated secondary antibody (red) was used to detect the expression and localization of HspJ62 in all stages of P. berghei. The parasite nuclei were stained with DAPI (blue), and the slides were visualized under a fluorescence microscope. Preimmune rat sera were used as a negative control (Supplementary Fig. 2B).

Figure 3

Generation and validation of the P. berghei HspJ62 knockout strain (ΔHspJ62). (A) The image shows the schematic summary of the HspJ62 gene knockout strategy by double homologous recombination using linearized vector-pBC-GFP-hDHFR-HSPJ62KO. The replacement cassette consisted of GFP and the hDHFR gene flanked by the 5’UTR (702 bp) and 3’UTR (817 bp) of the HspJ62 gene. (B) The expected product size of 1.2 Kb from diagnostic PCRs was observed. The external primer DP1 anneals to the chromosome, whereas DP2 is specific to the cassette. This primer pair shows specific band size 1.2 Kb only in the KO population. Full blots are shown in Supplementary Fig. 6A. (C) The two internal primers SP1 and SP2 anneal to the HspJ62 genomic locus. The PCR shows a specific band size of 842 bp only in the wild type and not in the knockout parasites. Full blots are shown in Supplementary Fig. 6B. (D) Southern blot analysis of ScaI-digested genomic DNA, obtained from WT and ΔHspJ62-I & II knockout parasite lines, using the 5’UTR of the HspJ62 gene as a probe resulted in the expected 5 Kb band corresponding to the WT gene and 8.3 Kb band corresponding to ΔHspJ62 parasite line. (E) Immunoblot analysis shows the absence of HspJ62 protein (62 kDa) expression in the ΔHspJ62-I & II parasite line compared with the WT parasite line. Pb-aldolase protein was used as a loading control. Full blots are shown in Supplementary Fig. 5F–H. WT wild type parasite lysate, ∆HspJ62-I and ∆HspJ62-II- are the parasite lysates from the two HspJ62 knockout parasite lines.

Figure 4

HspJ62 knockout parasites are unable to develop sexual stages. (A) The blood stage growth curves of the ΔHspJ62 parasite line in comparison with the wild-type strain are shown. Parasitemia was monitored daily from Day 1 until the mice died by Giemsa staining on slides, following intravenous injection of an equal number of WT or ΔHspJ62 parasites. (B) The survival of WT or ΔHspJ62 parasite-infected mice was counted and plotted. All data points are from one of the two representative experiments, showing the average daily parasitemia ± SD of groups of 10 mice each infected with WT or one of the two independent clones of ΔHspJ62 parasite (A, B). Gametocyte formation was monitored in wild-type and ΔHspJ62 parasites following intravenous injection of an equal number of WT or ΔHspJ62 parasites. (C) Gametocytemia was determined per 1000 red blood cells in Giemsa-stained blood smears. The graph shows the percent gametocytes formed in the clonal ΔHspJ62 parasite line in comparison with the wild-type strain; the error bars represent the mean and SD from three independent biological experiments conducted in triplicate. ***P < 0.0002, using one way ANOVA-Kruskal–Wallis test. (D) The effect of chloroquine (10 mg/ml) on WT and two independent clones of ΔHspJ62 parasite lines. (E) Exflagellation center formation was examined 5 min after activation of iRBCs collected from mice infected with WT-GFP or ΔHspJ62 parasites at day 4 post infection. The number of exflagellation centers was calculated from the number of actively moving gametes interacting with neighboring RBCs in 1000 RBCs. (F) The zygote/ookinete conversion rate is the percentage of female sexual stage parasites developing into zygotes or ookinetes. The zygote/ookinete conversion rate is generally calculated as the number of zygotes/ookinetes divided by the total number of female gametocytes. Since no male/female gametocytes were observed in ΔHspJ62 parasites, we looked for the formation of ookinetes per 10,000 RBCs. The number of ookinetes formed in WT and two independent clones of ΔHspJ62 parasites was calculated and plotted. (G) Oocyst formation and numbers were observed and counted in the mosquito midgut 14 days after an infected blood meal. Statistical analysis was performed using one-way ANOVA ****P = 0.0001. The image is representative of one of the three replicates performed.

Figure 5

Transcriptomic Analysis of the ΔHspJ62 parasitic line. (A) Volcano plot of DEGs between the ΔHspJ62 and WT groups with log2 (fold change) as the x-axis and log10 (p value) as the y-axis. The volcano plot was made according to the gene expression level. The red dots indicate significant differentially expressed genes, and black dots indicate nonsignificant differentially expressed genes. (B) The ''Interactive graph'' view of REVIGO; bubble color indicates the user-provided p-value, and bubble size indicates the frequency of the GO term in the underlying GOA database. Highly similar GO terms are linked by edges in the graph, where the line width indicates the degree of similarity. Node initial placement was determined by a '’force-directed'’ layout algorithm that aims to keep the more similar nodes closer together. (C) Pathway enrichment analysis-The significant pathway for differentially expressed genes between ΔHspJ62 KO and control groups. Benjamin value is represented in the logarithm. (D) Heat Map. FG female gamete, MG Male gamete, ES erythrocytic stages, WT wild type, KO HspJ62 knockout.

Figure 6

Transcriptomic Analysis of the ΔHspJ62 parasite line. (A) For validation of NGS data, qPCR was performed on 20 randomly selected up/downregulated genes from independent biological replicates of the experiment. GAPDH was taken as housekeeping genes. Blue and brown bars represent the fold changes (log10 scale) in P. berghei genes observed by qPCR and NGS, respectively. (B) The transcriptional regulatory motif of eight bases, i.e. TCTANAAA was identified upstream of the first ATG codon in gametocyte-specific genes.