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.
Highlights
Plasmodium belongs to phylum protozoa and causes one of the most devastating diseases, is responsible for millions of clinical cases, and affects approximately 1.2 billion lives worldwide[1]
Considering the regulatory role of HSP40 family proteins, we investigated the role of Plasmodium HspJ62 in the parasite using two different approaches: gene knockout and protein–protein interaction methodologies
A remarkable increase in the expression intensity of HspJ62 was observed even when compared with HSP70 protein
Summary
HspJ62 is induced under heat stress condition. We initiated the characterization of HspJ62 by in silico analysis. Parasite aldolase protein detected by anti-aldolase antibodies was used as a loading control (Fig. 3E) These results confirmed the gene deletion of HspJ62 in two clones, which were designated ΔHspJ62-I and ΔHspJ62-II and were used for further phenotypic characterization. Microscopic images displayed a complete absence of gametocytes in the ΔHspJ62 parasites compared with the wild type (Fig. 4C) These results indicate that the HspJ62 protein is essential for gametocyte formation and is the likely reason for rapid asexual growth of knockout strains, as parasites that otherwise would have committed for sexual growth are diverted towards asexual growth. To confirm the defect in gametocyte formation due to HspJ62 gene deletion, groups of mice were infected with ΔHspJ62 and wild-type parasites. We hypothesize that the ApiAP2 transcription factor binds to this conserved sequence and regulates the expression of these 49 gametocyte-specific genes
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