Positive selection on the Plasmodium falciparum clag2 gene encoding a component of the erythrocyte-binding rhoptry protein complex

A protein complex of high-molecular-mass proteins (PfRhopH) of the human malaria parasite Plasmodium falciparum induces host protective immunity and therefore is a candidate for vaccine development. Clarification of the level of polymorphism and the evolutionary processes is important both for vaccine design and for a better understanding of the evolution of cell invasion in this parasite. In a previous study on 5 genes encoding RhopH1/Clag proteins, positive diversifying selection was detected in clag8 and clag9 but not in the paralogous clag2, clag3.1 and clag3.2. In this study, to extend the analysis of clag polymorphism, we obtained sequences surrounding the most polymorphic regions of clag2, clag8, and clag9 from parasites collected in Thailand. Using sequence data obtained newly in this study and reported previously, we classified clag2 sequences into 5 groups based on the similarity of the deduced amino acid sequences and number of insertions/deletions. By the sliding window method, an excess of nonsynonymous substitutions over synonymous substitutions was detected in the group 1 and group 2 clag2 and clag8 sequences. Population-based analyses also detected a significant departure from the neutral expectation for group 1 clag2 and clag8. Thus, two independent approaches suggest that clag2 is subject to a positive diversifying selection. The previously suggested positive selection on clag8 was also supported by population-based analyses. However, the positive selection on clag9, which was detected by comparing the 5 sequences, was not detected using the additional 34 sequences obtained in this study

Novel hemagglutinating, hemolytic and cytotoxic activities of the intermediate subunit of Entamoeba histolytica lectin

Galactose and N-acetyl-D-galactosamine (Gal/GalNAc) inhibitable lectin of Entamoeba histolytica, a common protozoan parasite, has roles in pathogenicity and induction of protective immunity in mouse models of amoebiasis. The lectin consists of heavy (Hgl), light (Lgl), and intermediate (Igl) subunits. Hgl has lectin activity and Lgl does not, but little is known about the activity of Igl. In this study, we assessed various regions of Igl for hemagglutinating activity using recombinant proteins expressed in Escherichia coli. We identified a weak hemagglutinating activity of the protein. Furthermore, we found novel hemolytic and cytotoxic activities of the lectin, which resided in the carboxy-terminal region of the protein. Antibodies against Igl inhibited the hemolytic activity of Entamoeba histolytica trophozoites. This is the first report showing hemagglutinating, hemolytic and cytotoxic activities of an amoebic molecule, Igl

Distinct effects on the secretion of MTRAP and AMA1 in Plasmodium yoelii following deletion of acylated pleckstrin homology domain-containing protein

Plasmodium, the causative agents of malaria, are obligate intracellular organisms. In humans, pathogenesis is caused by the blood stage parasite, which multiplies within erythrocytes, thus erythrocyte invasion is an essential developmental step. Merozoite form parasites released into the blood stream coordinately secrets a panel of proteins from the microneme secretory organelles for gliding motility, establishment of a tight junction with a target naive erythrocyte, and subsequent internalization. A protein identified in Toxoplasma gondii facilitates microneme fusion with the plasma membrane for exocytosis; namely, acylated pleckstrin homology domain-containing protein (APH). To obtain insight into the differential microneme discharge by malaria parasites, in this study we analyzed the consequences of APH deletion in the rodent malaria model, Plasmodium yoelii, using a DiCre-based inducible knockout method. We found that APH deletion resulted in a reduction in parasite asexual growth and erythrocyte invasion, with some parasites retaining the ability to invade and grow without APH. APH deletion impaired the secretion of microneme proteins, MTRAP and AMA1, and upon contact with erythrocytes the secretion of MTRAP, but not AMA1, was observed. APH-deleted merozoites were able to attach to and deform erythrocytes, consistent with the observed MTRAP secretion. Tight junctions were formed, but echinocytosis after merozoite internalization into erythrocytes was significantly reduced, consistent with the observed absence of AMA1 secretion. Together with our observation that APH largely colocalized with MTRAP, but less with AMA1, we propose that APH is directly involved in MTRAP secretion; whereas any role of APH in AMA1 secretion is indirect in Plasmodium

The Cytoplasmic Region of Plasmodium falciparum SURFIN4.2 Is Required for Transport from Maurer’s Clefts to the Red Blood Cell Surface

Background: Plasmodium, the causative agent of malaria, exports many proteins to the surface of the infected red blood cell (iRBC) in order to modify it toward a structure more suitable for parasite development and survival. One such exported protein, SURFIN4.2, from the parasite of human malignant malaria, P. falciparum, was identified in the trypsin-cleaved protein fraction from the iRBC surface, and is thereby inferred to be exposed on the iRBC surface. SURFIN4.2 also localize to Maurer’s clefts?parasite-derived membranous structures established in the RBC cytoplasm and tethered to the RBC membrane?and their role in trafficking suggests that they are a pathway for SURFIN4.2 transport to the iRBC surface. It has not been determined the participation of protein domains and motifs within SURFIN4.2 in transport from Maurer’s clefts to the iRBC surface; and herein we examined if the SURFIN4.2 intracellular region containing tryptophan-rich (WR) domain is required for its exposure on the iRBC surface. Results: We generated two transgenic parasite lines which express modified SURFIN4.2, with or without a part of the intracellular region. Both recombinant SURFIN4.2 proteins were exported to Maurer’s clefts. However, only SURFIN4.2 possessing the intracellular region was efficiently cleaved by surface treatment of iRBC with proteinase K. Conclusions: These results indicate that SURFIN4.2 is exposed on the iRBC surface and that the intracellular region containing WR domain plays a role on the transport from Maurer’s clefts to the iRBC membrane

熱帯熱マラリア原虫のSURFIN4.1の赤血球への効率的な輸送にはN末端領域の複数の荷電アミノ酸が重要である

Plasmodium falciparum, an obligate intracellular protozoan parasite which causes the severe form of human malaria, exports numerous proteins to the infected red blood cell that are important for its survival and of severe pathological effect to its host. These proteins and their export mechanisms are candidates for drug and vaccine development, and among them is the Plasmodium SURFIN family of proteins. Previously we showed that the N-terminal region along with the sequence surrounding the transmembrane domain of SURFIN4.1 is essential for its export to Maurer\u27s clefts in the red blood cell cytoplasm. We proposed that this region is recognized by a machinery responsible for protein translocation across the parasitophorous vacuole membrane surrounding the parasite. To understand the export mechanism further, we utilized a fluorescent protein-tagged mini-SURFIN4.1 consisting of the minimum essential components for export. Alanine scanning of all charged amino acids within the N-terminal region revealed that replacement of 3 glutamic acid and 2 lysine residues significantly impairs the export efficiency of this protein across the parasitophorous vacuole membrane. In addition, N-terminally Myc-tagged mini-SURFIN4.1 and mini-SURFIN4.2 with similar architectures were detected with anti-Myc antibody at Maurer\u27s clefts, indicating that elements required for export to Maurer\u27s clefts are conserved between SURFIN4.1 and SURFIN4.2, and that N-terminal sequences of these SURFIN members are not cleaved during export. Our results implicate a conserved nature of SURFIN export to the red blood cell, particularly an important role of multiple glutamic acid and lysine residues in the SURFIN N-terminal region.長崎大学学位論文 学位記番号:博(医歯薬)甲第1171号 学位授与年月日:令和元年6月5日Author: Ben-Yeddy Abel Chitama, Shinya Miyazaki, Xiaotong Zhu, Wataru Kagaya, Kazuhide Yahata, Osamu KanekoCitation: Parasitology International, 71, pp.186-193; 2019Nagasaki University (長崎大学)課程博

cAMP-dependent protein kinase regulates secretion of apical membrane antigen 1 (AMA1) in Plasmodium yoelii

Malaria remains a heavy global burden on human health, and it is important to understand the molecular and cellular biology of the parasite to find targets for drug and vaccine development. The mouse malaria model is an essential tool to characterize the function of identified molecules; however, robust technologies for targeted gene deletions are still poorly developed for the widely used rodent malaria parasite, Plasmodium yoelii. To overcome this problem, we established a DiCre-loxP inducible knockout (iKO) system in P. yoelii, which showed more than 80% excision efficacy of the target locus and more than 90% reduction of locus transcripts 24 h (one cell cycle) after RAP administration. Using this developed system, cAMP-dependent protein kinase (PKAc) was inducibly disrupted and the phenotypes of the resulting PKAc-iKO parasites were analyzed. We found that PKAc-iKO parasites showed severe growth and erythrocyte invasion defects. We also found that disruption of PKAc impaired the secretion of AMA1 in P. yoelii, in contrast to a report showing no role of PKAc in AMA1 secretion in P. falciparum. This discrepancy may be related to the difference in the timing of AMA1 distribution to the merozoite surface, which occurs just after egress for P. falciparum, but after several minutes for P. yoelii. Secretions of PyEBL, Py235, and RON2 were not affected by the disruption of PKAc in P. yoelii. PyRON2 was already secreted to the merozoite surface immediately after merozoite egress, which is inconsistent with the current model that RON2 is injected into the erythrocyte cytosol. Further investigations are required to understand the role of RON2 exposed on the merozoite surface

A novel Plasmodium yoelii pseudokinase, PypPK1, is involved in erythrocyte invasion and exflagellation center formation

Malaria parasites proliferate by repeated invasion of and multiplication within erythrocytes in the vertebrate host. Sexually committed intraerythrocytic parasites undergo sexual stage differentiation to become gametocytes. After ingestion by the mosquito, male and female gametocytes egress from erythrocytes and fertilize within the mosquito midgut. A complex signaling pathway likely responds to environmental events to trigger gametogenesis and regulate fertilization; however, such knowledge remains limited for malaria parasites. Several pseudokinases are highly transcribed at the gametocyte stage and are possible multi-functional regulators controlling critical steps of the life cycle. Here we characterized one pseudokinase, termed PypPK1, in Plasmodium yoelii that is highly expressed in schizonts and male gametocytes. Immunofluorescence assays for parasites expressing Myc-tagged PypPK1 confirmed that PypPK1 protein is expressed in schizonts and sexual stage parasites. Transgenic ΔpPK1 parasites, in which the PypPK1 gene locus was deleted by the CRISPR/Cas9 method, showed significant growth defect and reduced virulence in mice. In the blood stage, ΔpPK1 parasites were able to egress from erythrocytes similar to wild type parasites; however, erythrocyte invasion efficacy was significantly reduced. During sexual stage development, no clear changes were seen in male and female gametocytemias as well as gametocyte egress from erythrocytes; but, the number of exflagellation centers and oocysts were significantly reduced in ΔpPK1 parasites. Taken together, PypPK1 has an important role for both erythrocyte invasion and exflagellation center formation

Validation of Plasmodium vivax centromere and promoter activities using Plasmodium yoelii

Plasmodium vivax is the leading cause of malaria outside Africa and represents a significant health and economic burden on affected countries. A major obstacle for P. vivax eradication is the dormant hypnozoite liver stage that causes relapse infections and the limited antimalarial drugs that clear this stage. Advances in studying the hypnozoite and other unique biological aspects of this parasite are hampered by the lack of a continuous in vitro laboratory culture system and poor availability of molecular tools for genetic manipulation. In this study, we aim to develop molecular tools that can be used for genetic manipulation of P. vivax. A putative P. vivax centromere sequence (PvCEN) was cloned and episomal centromere based plasmids expressing a GFP marker were constructed. Centromere activity was evaluated using a rodent malaria parasite Plasmodium yoelii. A plasmid carrying PvCEN was stably maintained in asexual-stage parasites in the absence of drug pressure, and approximately 45% of the parasites retained the plasmid four weeks later. The same retention rate was observed in parasites possessing a native P. yoelii centromere (PyCEN)-based control plasmid. The segregation efficiency of the plasmid per nuclear division was > 99% in PvCEN parasites, compared to ?90% in a control parasite harboring a plasmid without a centromere. In addition, we observed a clear GFP signal in both oocysts and salivary gland sporozoites isolated from mosquitoes. In blood-stage parasites after liver stage development, GFP positivity in PvCEN parasites was comparable to control PyCEN parasites. Thus, PvCEN plasmids were maintained throughout the parasite life cycle. We also validated several P. vivax promoter activities and showed that hsp70 promoter (?1 kb) was active throughout the parasite life cycle. This is the first data for the functional characterization of a P. vivax centromere that can be used in future P. vivax biological research

Gliding motility of Plasmodium merozoites.

Plasmodium malaria parasites are obligate intracellular protozoans that use a unique form of locomotion, termed gliding motility, to move through host tissues and invade cells. The process is substrate dependent and powered by an actomyosin motor that drives the posterior translocation of extracellular adhesins which, in turn, propel the parasite forward. Gliding motility is essential for tissue translocation in the sporozoite and ookinete stages; however, the short-lived erythrocyte-invading merozoite stage has never been observed to undergo gliding movement. Here we show Plasmodium merozoites possess the ability to undergo gliding motility in vitro and that this mechanism is likely an important precursor step for successful parasite invasion. We demonstrate that two human infective species, Plasmodium falciparum and Plasmodium knowlesi, have distinct merozoite motility profiles which may reflect distinct invasion strategies. Additionally, we develop and validate a higher throughput assay to evaluate the effects of genetic and pharmacological perturbations on both the molecular motor and the complex signaling cascade that regulates motility in merozoites. The discovery of merozoite motility provides a model to study the glideosome and adds a dimension for work aiming to develop treatments targeting the blood stage invasion pathways

Gliding motility of Plasmodium merozoites

Plasmodium malaria parasites are obligate intracellular protozoans that use a unique form of locomotion, termed gliding motility, to move through host tissues and invade cells. The process is substrate dependent and powered by an actomyosin motor that drives the posterior translocation of extracellular adhesins which, in turn, propel the parasite forward. Gliding motility is essential for tissue translocation in the sporozoite and ookinete stages; however, the short-lived erythrocyte-invading merozoite stage has never been observed to undergo gliding movement. Here we show Plasmodium merozoites possess the ability to undergo gliding motility in vitro and that this mechanism is likely an important precursor step for successful parasite invasion. We demonstrate that two human infective species, Plasmodium falciparum and Plasmodium knowlesi, have distinct merozoite motility profiles which may reflect distinct invasion strategies. Additionally, we develop and validate a higher throughput assay to evaluate the effects of genetic and pharmacological perturbations on both the molecular motor and the complex signaling cascade that regulates motility in merozoites. The discovery of merozoite motility provides a model to study the glideosome and adds a dimension for work aiming to develop treatments targeting the blood stage invasion pathways