title: Isolation of invasive Plasmodium yoelii merozoites with a long half-life to evaluate invasion dynamics and potential invasion inhibitors.
abstract: Malaria symptoms and pathogenesis are caused by blood stage parasite burdens of Plasmodium spp., for which invasion of red blood cells (RBCs) by merozoites is essential. Successful targeting by either drugs or vaccines directed against the whole merozoite or its antigens during its transient extracellular status would contribute to malaria control by impeding RBC invasion. To understand merozoite invasion biology and mechanisms, it is desired to obtain merozoites that retain their invasion activity in vitro. Accordingly, methods have been developed to isolate invasive Plasmodium knowlesi and Plasmodium falciparum merozoites. Rodent malaria parasite models offer ease in laboratory maintenance and experimental genetic modifications; however, no methods have been reported regarding isolation of high numbers of invasive rodent malaria merozoites. In this study, Plasmodium yoelii-infected RBCs were obtained from infected mice, and mature schizont-infected RBCs enriched via Histodenz™ density gradients. Merozoites retaining invasion activity were then isolated by passing the preparations through a filter membrane. RBC-invaded parasites developed to mature stages in vitro in a synchronous manner. Isolated merozoites were evaluated for retention of invasion activity following storage at different temperatures prior to incubation with uninfected mouse RBCs. Isolated merozoites retained their invasion activity 4h after isolation at 10 or 15 °C, whereas their invasion activity reduced to 0-10% within 30 min when incubated on ice or at 37 °C prior to RBC invasion assay. Images of merozoites at successive steps during RBC invasion were captured by light and transmission electron microscopy. Synthetic peptides derived from the amino acid sequence of the P. yoelii invasion protein RON2 efficiently inhibited RBC invasion. The developed method to isolate and keep invasive P. yoelii merozoites for up to 4h is a powerful tool to study the RBC invasion biology of this parasite. This method provides an important platform to evaluate the mode of action of drugs and vaccine candidates targeting the RBC invasion steps using rodent malaria model.
authors.affiliation: Department of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Sakamoto, Nagasaki 852-8523, Japan; Graduate School of Biomedical Sciences, Nagasaki University, Sakamoto, Nagasaki 852-8523, Japan. Electronic address: joemutungi@gmail.com.
authors.forename: Joe Kimanthi
authors.initials: JK
authors.lastname: Mutungi
authors.affiliation: Department of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Sakamoto, Nagasaki 852-8523, Japan; Graduate School of Biomedical Sciences, Nagasaki University, Sakamoto, Nagasaki 852-8523, Japan. Electronic address: kyahata@nagasaki-u.ac.jp.
authors.forename: Kazuhide
authors.initials: K
authors.lastname: Yahata
authors.affiliation: Central Laboratory, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Sakamoto, Nagasaki 852-8523, Japan. Electronic address: miako@nagasaki-u.ac.jp.
authors.forename: Miako
authors.initials: M
authors.lastname: Sakaguchi
authors.affiliation: Department of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Sakamoto, Nagasaki 852-8523, Japan; Graduate School of Biomedical Sciences, Nagasaki University, Sakamoto, Nagasaki 852-8523, Japan. Electronic address: okaneko@nagasaki-u.ac.jp.
authors.forename: Osamu
authors.initials: O
authors.lastname: Kaneko
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000962:Antimalarials / Q000494:pharmacology*; D004195:Disease Models, Animal; D004912:Erythrocytes / Q000469:parasitology; D005260:Female; D008288:Malaria / Q000469:parasitology*; D052940:Merozoites / Q000187:drug effects / Q000254:growth & development; D051379:Mice; D008813:Mice, Inbred ICR; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology; D010967:Plasmodium yoelii / Q000187:drug effects* / Q000302:isolation & purification* / Q000472:pathogenicity; D015800:Protozoan Proteins / Q000737:chemistry / Q000494:pharmacology; D013170:Spores
chemical_list: D000962:Antimalarials; D010455:Peptides; D015800:Protozoan Proteins
keywords: Inhibitors; Invasion; Malaria; Merozoite; Plasmodium yoelii