Role of Plasmodium berghei ookinete surface and oocyst capsule protein, a novel oocyst capsule-associated protein, in ookinete motility

Nakayama, Kazuhiko; Kimura, Yuta; Kitahara, Yu; Soga, Akira; Haraguchi, Asako; Hakozaki, Jun; Sugiyama, Makoto; Kusakisako, Kodai; Fukumoto, Shinya; Ikadai, Hiromi

doi:10.21203/rs.3.rs-326425/v1

Cited by 3 publications

(7 citation statements)

References 35 publications

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“…P. berghei (ANKA strain) that constitutively expresses green fluorescent protein (GFP) was maintained via serial passage in ICR mice (Charles River Laboratories Japan Inc., Yokohama, Japan) from frozen stock. Ookinete culture of P. berghei was performed as previously described [28,29]. Blood from infected mice was collected via cardiac puncture and placed in 10 volumes of ookinete medium containing RPMI medium 1640 (Thermo Fisher Scientific Inc., Waltham, MA, USA), 25 mM HEPES (Dojindo Laboratories, Kumamoto, Japan), 0.4 mM hypoxanthine (Sigma-Aldrich, St. Louis, MO, USA), 24 mM NaHCO 3 (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), and 12.5 mg L -1 gentamicin reagent solution (Gibco) before incubation for 24 h at 19 °C.…”

Section: Methodsmentioning

confidence: 99%

“…P. berghei ookinetes and P. falciparum gametocytes and zygotes were purified using a MidiMACS separator system (LS Columns, Miltenyi Biotec, Bergisch Gladbach, Germany), as previously described [28,29]. Ookinetes, gametocytes, and zygotes were recovered by passing ookinete medium, incomplete medium comprising complete medium without 10% (v/v) heat-inactivated type A + human plasma, and exflagellation-inducing medium, respectively.…”

Section: Methodsmentioning

confidence: 99%

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Molecular mechanisms of Plasmodium development in male and female Anopheles mosquitoes

Haraguchi

Takano

Hakozaki

et al. 2022

Preprint

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Vector competence influences the ability of Anopheles mosquitoes to transmit Plasmodium parasites. The aim of this study was to determine the competence of male and female Anopheles mosquitoes to support the development of Plasmodium parasites. Male and female A. stephensi (STE2 strain) were infected with in vitro-cultured P. berghei (ANKA strain) ookinetes. We found that the number of oocysts produced was higher in males than in females. RNA-seq analysis of male and female mosquitoes injected with P. berghei ookinetes showed that predominantly genes of unknown function changed in expression levels in response to ookinete infection; however, further studies are required to elucidate their functions. Moreover, male mosquitoes were injected with in vitro-cultured P. falciparum (3D7 strain) gametocytes or zygotes. The development of P. falciparum in males was detected using nested polymerase chain reaction. We found the DNA content was higher on day 15 than on day 0, indicating that P. falciparum developed in the mosquito hemocoel. This study revealed promising new mechanisms underlying the interactions between Plasmodium and mosquitoes.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Molecular mechanisms of Plasmodium development in male and female Anopheles mosquitoes

Haraguchi

Takano

Hakozaki

et al. 2022

Preprint

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“…When parasitemia reached approximately 10% in ve mice per group, the gametocytes per infected red blood cells were enumerated and the gametocyte sex ratios were determined by Giemsa-stained tail blood smears. Male gametocyte ex agellation was quanti ed as previously described [27]. Brie y, 1 µL gametocyte-infected blood was drawn from the tail vein and immediately mixed with 1 µL heparin and 38 µL complete ookinete culture medium (OKM: RPMI 1640 medium (Gibco, Grand Island, NY, USA) and 20% (v/v) heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich Corp., St. Louis, MO, USA)).…”

Section: Phenotypic Analysis Of Pbbrca2-ko Parasitesmentioning

confidence: 99%

“…Ten volumes OKM was added to the blood and the suspension was cultured at 19°C for 24 h. Ookinetes were enumerated and their structure was examined using culture suspension smears. They were puri ed in a MidiMACS Separator System (Miltenyi Biotec, Bergisch-Gladbach, Germany) as previously described [27]. A total of 3 mL culture was passed through the system thrice before removing the column from the magnet.…”

Section: Ookinete Culture and Puri Cationmentioning

confidence: 99%

Plasmodium berghei Brca2 Is Required for Normal Development and Differentiation in Mice and Mosquitoes

Yoshikawa

Kimura

Soga

et al. 2022

Preprint

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Background: Malaria is a major global parasitic infestation caused by Plasmodium spp. Its zygotes undergo meiosis and develop into haploid ookinetes which differentiate into oocysts that undergo sporogony. Homologous recombination (HR) occurs during meiosis and introduces genetic variation. However, the mechanisms of HR in Plasmodium are unclear. In humans, the recombinases Rad51 and Dmc1 are required for HR and are regulated by BRCA2. Most eukaryotes harbor BRCA2 homologs. Nevertheless, they have not been reported for Plasmodium.Methods: A Brca2 candidate was salvaged from a database to identify Brca2 homologs in Plasmodium. To confirm that the candidate protein was Brca2, interaction activity between Plasmodium berghei (Pb) Brca2 and Rad51 was tested by a mammalian two-hybrid assay. To elucidate the functions of PbBrca2, it was knocked out and parasite proliferation and differentiation were assessed in mice and mosquitoes. Transmission electron microscopy was used to identify sporogony.Results: The candidate protein was conserved among Plasmodium species and indicated that they harbored critical BRCA2 domains including BRC repeats, tower, and oligonucleotide/oligosaccharide-binding-fold domains. The P. berghei BRC repeats interacted with PbRad51. Hence, this candidate was considered a Brca2 homolog. PbBrca2 knockout parasites were associated with reduced parasitemia, gametocytemia, female gametocyte ratio, oocyst number, and ookinete development in both mice and mosquitoes. Nevertheless, gametocyte and ookinete morphology was comparable with that of wild type parasites. Transmission electron microscopy disclosed that sporogony never progressed in Brca2-knockout parasites.Conclusions: Brca2 is implicated in nearly all Plasmodium life cycle stages and especially in sporogony. PbBrca2 contributes to homologous recombination during meiosis.

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“…The oocyst has a thick wall, unlike other parasite stages. A few proteins present in the oocyst wall have been reported [10,12,17]. These proteins are oocyst wall components, and knocking out one of these, PbCap380, decreases oocysts in mosquitoes [17].…”

Section: Introductionmentioning

confidence: 99%

Formation of free oocysts in <i>Anopheles</i> mosquitoes injected with <i>Plasmodium</i> ookinetes

Haraguchi

Takano

Hakozaki

et al. 2023

The Journal of Veterinary Medical Science

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Malaria needs new strategies for its control. Plasmodium spp., the causative agent of malaria, is transmitted by mosquitoes. These parasites develop into oocysts and sporozoites in the body of the mosquitoes. A deeper understanding of oocysts that produce the infectious form of the parasite, sporozoites, can facilitate the development of novel countermeasures. However, the isolation of Plasmodium oocysts is challenging as these are formed between midgut epithelial cells and basal lamina after gametocytes enter the mosquito’s body through blood feeding. Further research on oocysts has been impeded by issues related to oocyst isolation. Therefore, in this study, we injected Plasmodium into mosquitoes—an artificial and unique method—and aimed to clarify how oocysts were formed in mosquitoes after Plasmodium injection and whether free oocysts were formed from the mosquito tissue. Plasmodium berghei (ANKA strain) ookinetes cultured in vitro were injected into the thoracic body cavity (hemocoel) of female and male Anopheles stephensi mosquitoes. Oocysts were formed in the body of female and male mosquitoes at 14 days post injection. In addition, oocysts formed as a result of injection developed into sporozoites, which were infectious to mice. These findings suggest that P. berghei can complete its developmental stage in mosquitoes by injection. Some of the oocysts formed were free from mosquito tissue, and it was possible to collect oocysts with minimal contamination of mosquito tissue. These free oocysts can be used for investigating oocyst proteins and metabolism.

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Role of Plasmodium berghei ookinete surface and oocyst capsule protein, a novel oocyst capsule-associated protein, in ookinete motility

Cited by 3 publications

References 35 publications

Molecular mechanisms of Plasmodium development in male and female Anopheles mosquitoes

Molecular mechanisms of Plasmodium development in male and female Anopheles mosquitoes

Plasmodium berghei Brca2 Is Required for Normal Development and Differentiation in Mice and Mosquitoes

Formation of free oocysts in <i>Anopheles</i> mosquitoes injected with <i>Plasmodium</i> ookinetes

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