Improved transfection and new selectable markers for the rodent malaria parasite Plasmodium yoelii

Jongco, Artemio M.; Ting, Li Min; Thathy, Vandana; Mota, Maria M.; Kim, Kami

doi:10.1016/j.molbiopara.2006.01.001

Cited by 61 publications

(57 citation statements)

References 18 publications

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“…Transgenic Py17XNL parasites with a C-terminal 4xmyc tag attached to PY01024 (PlasmoDB Accession No. PY01024) were created by means of standard single-crossover reverse genetics approaches (21). Transgenic Py17XNL parasites with a C-terminal triple HA tag attached to PY00899 (PlasmoDB Accession No.…”

Section: Methodsmentioning

confidence: 99%

Total and Putative Surface Proteomics of Malaria Parasite Salivary Gland Sporozoites

Lindner

Swearingen

Harupa

et al. 2013

Molecular & Cellular Proteomics

178

274

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Malaria infections of mammals are initiated by the transmission of Plasmodium salivary gland sporozoites during an Anopheles mosquito vector bite. Sporozoites make their way through the skin and eventually to the liver, where they infect hepatocytes. Blocking this initial stage of infection is a promising malaria vaccine strategy. Therefore, comprehensively elucidating the protein composition of sporozoites will be invaluable in identifying novel targets for blocking infection. Previous efforts to identify the proteins expressed in Plasmodium mosquito stages were hampered by the technical difficulty of separating the parasite from its vector; without effective purifications, the large majority of proteins identified were of vector origin. Here we describe the proteomic profiling of highly purified salivary gland sporozoites from two Plasmodium species: human-infective Plasmodium falciparum and rodent-infective Plasmodium yoelii. The combination of improved sample purification and high mass accuracy mass spectrometry has facilitated the most complete proteome coverage to date for a pre-erythrocytic stage of the parasite. A total of 1991 P. falciparum sporozoite proteins and 1876 P. yoelii sporozoite proteins were identified, with >86% identified with high sequence coverage. The proteomic data were used to confirm the presence of components of three features critical for sporozoite infection of the mammalian host: the sporozoite motility and invasion apparatus (glideosome), sporozoite signaling pathways, and the contents of the apical secretory organelles. Furthermore, chemical labeling and identification of proteins on live sporozoites revealed previously uncharacterized complexity of the putative sporozoite surface-exposed proteome. Taken together, the data constitute the most comprehensive analysis to date of the protein expression of salivary gland sporozoites and reveal novel potential surface-exposed proteins that might be valuable targets for antibody blockage of infection. Molecular & Cellular

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Section: Methodsmentioning

confidence: 99%

Total and Putative Surface Proteomics of Malaria Parasite Salivary Gland Sporozoites

Lindner

Swearingen

Harupa

et al. 2013

Molecular & Cellular Proteomics

178

274

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“…Starting at 24 h postinfection, the mice were treated i.p. with 0.8 mg kg of body weight Ϫ1 pyrimethamine (Sigma-Aldrich) daily until parasites were no longer detectable (42). Once drug-resistant parasites appeared, the parasites were passaged into a new mouse by intravenous (i.v.)…”

Section: Methodsmentioning

confidence: 99%

Host Erythrocyte Environment Influences the Localization of Exported Protein 2, an Essential Component of the Plasmodium Translocon

et al. 2015

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Malaria parasites replicating inside red blood cells (RBCs) export a large subset of proteins into the erythrocyte cytoplasm to facilitate parasite growth and survival. PTEX, the parasite-encoded translocon, mediates protein transport across the parasitophorous vacuolar membrane (PVM) in Plasmodium falciparum-infected erythrocytes. Proteins exported into the erythrocyte cytoplasm have been localized to membranous structures, such as Maurer's clefts, small vesicles, and a tubovesicular network. Comparable studies of protein trafficking in Plasmodium vivax-infected reticulocytes are limited. With Plasmodium yoelii-infected reticulocytes, we identified exported protein 2 (Exp2) in a proteomic screen of proteins putatively transported across the PVM. Immunofluorescence studies showed that P. yoelii Exp2 (PyExp2) was primarily localized to the PVM. Unexpectedly, PyExp2 was also associated with distinct, membrane-bound vesicles in the reticulocyte cytoplasm. This is in contrast to P. falciparum in mature RBCs, where P. falciparum Exp2 (PfExp2) is exclusively localized to the PVM. Two P. yoelii-exported proteins, PY04481 (encoded by a pyst-a gene) and PY06203 (PypAg-1), partially colocalized with these PyExp2-positive vesicles. Further analysis revealed that with P. yoelii, Plasmodium berghei, and P. falciparum, cytoplasmic Exp2-positive vesicles were primarily observed in CD71؉ reticulocytes versus mature RBCs. In transgenic P. yoelii 17X parasites, the association of hemagglutinintagged PyExp2 with the PVM and cytoplasmic vesicles was retained, but the pyexp2 gene was refractory to deletion. These data suggest that the localization of Exp2 in mouse and human RBCs can be influenced by the host cell environment. Exp2 may function at multiple points in the pathway by which parasites traffic proteins into and through the reticulocyte cytoplasm.

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“…Prior to transfection, the targeting plasmid was linearized using an SpeI site created in the pyhmgb2 open reading frame. Transfections were performed as described previously (20,21). Cloning was performed after limiting dilution and infection of 20 BALB/c mice.…”

Section: Methodsmentioning

confidence: 99%

High Mobility Group Protein HMGB2 Is a Critical Regulator of Plasmodium Oocyst Development

Gissot

Ting

Daly

et al. 2008

Journal of Biological Chemistry

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The sexual cycle of Plasmodium is required for transmission of malaria from mosquitoes to mammals, but how parasites induce the expression of genes required for the sexual stages is not known. We disrupted the Plasmodium yoelii gene encoding high mobility group nuclear factor hmgb2, which encodes a DNA-binding protein potentially implicated in transcriptional regulation of malaria gene expression. We investigated its function in vivo in the vertebrate and invertebrate hosts. ⌬pyhmgb2 parasites develop into gametocytes but have drastic impairment of oocyst formation. A global transcriptome analysis of the ⌬pyhmgb2 parasites identified ϳ30 genes whose expression is down-regulated in the ⌬pyhmgb2 parasites. These genes are conserved in all malaria species, and more than 90% of these genes show a peak of mRNA expression at the gametocyte stage. Surprisingly, the transcripts coding for the Plasmodium berghei orthologues of those genes are stored and translated in the ookinete stage. Therefore, sexual stage protein expression appears to be both transcriptionally and translationally regulated with Plasmodium HMGB2 acting as an important regulator of malaria sexual stage gene expression.Plasmodium species, the causative agents of malaria, are the most deadly members of the apicomplexan phylum. As with other members of the Apicomplexa, Plasmodium has a multifaceted life cycle encompassing an asexual phase within the intermediate host and a sexual cycle in the mosquito. Gametocytogenesis and gametogenesis consist of an ensemble of processes leading to the differentiation of erythrocytic forms of the parasite into male or female gametocytes. These complex and coordinated steps ensure the transmission of the parasite to the invertebrate host and are required for transmission of malaria to mammalian hosts. Gametocytes form in mammalian host, are taken up by the mosquito during a blood meal, transform into gametes in the mosquito midgut, and then fuse to form a zygote. Zygotes differentiate into ookinetes that penetrate the midgut wall and differentiate into oocysts, in which sporozoites will develop.Starting from the well controlled vertebrate milieu, the parasite must adapt to the mosquito environment where temperature fluctuates and nutrients are limited. Gametocytogenesis requires growth arrest and a differentiation step that will prepare the parasite for these changes. Sexual differentiation is accompanied by a tightly controlled gene expression program during which ϳ200 -300 mRNAs are specifically expressed or predominantly expressed in sexual forms of the parasite (1-4). Transcriptional control of gene expression is implicated as a crucial step during this process with sex-specific transcript expression in gametocytes controlled by the sequences in the 5Ј-flanking regions (5).For some sexual stage genes post-translational processes mediate gene expression. The genes coding for P25 and P28 ookinete proteins (PY00522 and PY00523, respectively) are transcribed at the gametocyte stage, but their translation is delayed unti...

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Improved transfection and new selectable markers for the rodent malaria parasite Plasmodium yoelii

Cited by 61 publications

References 18 publications

Total and Putative Surface Proteomics of Malaria Parasite Salivary Gland Sporozoites

Total and Putative Surface Proteomics of Malaria Parasite Salivary Gland Sporozoites

Host Erythrocyte Environment Influences the Localization of Exported Protein 2, an Essential Component of the Plasmodium Translocon

High Mobility Group Protein HMGB2 Is a Critical Regulator of Plasmodium Oocyst Development

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