Transposon mutagenesis identifies genes essential for
            <i>Plasmodium falciparum</i>
            gametocytogenesis

Ikadai, Hiromi; Shaw-Saliba, Kathryn; Kanzok, Stefan M.; McLean, Kyle Jarrod; Tanaka, Takeshi; Cao, Jun; Williamson, Kim C.; Jacobs-­Lorena, Marcelo

doi:10.1073/pnas.1217712110

Cited by 72 publications

(79 citation statements)

References 64 publications

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“…It appears that a key step in Plasmodium gametocytogenesis is accomplished by transcriptional repression via an AP2 family transcription factor that blocks asexual replication and promotes conversion to the sexual stage (40). In fact, the P. falciparum ortholog of the principal AP2 gene identified in our study has been associated with transcriptional regulation of gametocytogenesis (41). Similarly, processes governing hypnozoite dormancy and activation that underlie P. vivax relapse may be under epigenetic regulation.…”

Section: Discussionmentioning

confidence: 80%

Selective sweep suggests transcriptional regulation may underlie Plasmodium vivax resilience to malaria control measures in Cambodia

Parobek

Lin

Saunders

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Cambodia, in which both Plasmodium vivax and Plasmodium falciparum are endemic, has been the focus of numerous malaria-control interventions, resulting in a marked decline in overall malaria incidence. Despite this decline, the number of P. vivax cases has actually increased. To understand better the factors underlying this resilience, we compared the genetic responses of the two species to recent selective pressures. We sequenced and studied the genomes of 70 P. vivax and 80 P. falciparum isolates collected between 2009 and 2013. We found that although P. falciparum has undergone population fracturing, the coendemic P. vivax population has grown undisrupted, resulting in a larger effective population size, no discernable population structure, and frequent multiclonal infections. Signatures of selection suggest recent, species-specific evolutionary differences. Particularly, in contrast to P. falciparum, P. vivax transcription factors, chromatin modifiers, and histone deacetylases have undergone strong directional selection, including a particularly strong selective sweep at an AP2 transcription factor. Together, our findings point to different population-level adaptive mechanisms used by P. vivax and P. falciparum parasites. Although population substructuring in P. falciparum has resulted in clonal outgrowths of resistant parasites, P. vivax may use a nuanced transcriptional regulatory approach to population maintenance, enabling it to preserve a larger, more diverse population better suited to facing selective threats. We conclude that transcriptional control may underlie P. vivax's resilience to malaria control measures. Novel strategies to target such processes are likely required to eradicate P. vivax and achieve malaria elimination.D uring the last decade, western Cambodia has been the focus of numerous and multimodal interventions to control the spread of artemisinin-resistant Plasmodium falciparum (1, 2). Such interventions, including increased vector control, increased surveillance, and improved access to quality artemisinin-combination therapy (ACT), would be expected to curtail coendemic Plasmodium vivax as well. However, even as P. falciparum infections in Cambodia decreased by 81% between 2009 and 2013, P. vivax cases have increased, making it the predominant species in the Mekong region (3-6). This scenario, repeated in Brazil and other areas of coendemicity, has led to growing awareness that P. vivax, although infecting the same populations and transmitted by the same mosquito vectors, will likely be the more challenging species to eradicate (6-9). In this study, we use population genomics to gain insight into the evolutionary factors underlying P. vivax's resilience to malaria control measures.Population genetic studies have previously hinted at the resilience of P. vivax populations in comparison with P. falciparum. Studies of microsatellites and highly variable antigens of sympatric P. vivax and P. falciparum populations in Southeast Asia and the Southwest Pacific have consistently shown P. viv...

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Section: Discussionmentioning

confidence: 80%

Selective sweep suggests transcriptional regulation may underlie Plasmodium vivax resilience to malaria control measures in Cambodia

Parobek

Lin

Saunders

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…The ortholog of this gene is resistant to genetic disruption in P. berghei (9), and its role in parasite development has not been investigated previously, although its putative function in gametocytogenesis was implicated in a previous study of P. falciparum parasites (24,25). Here, we successfully disrupted the Pyap2-g3 gene and showed that disruption of this gene significantly reduced the numbers of male and female gametocytes (Fig.…”

Section: Resultsmentioning

confidence: 89%

“…Our results also demonstrate that PY17X_1417400 (Pyap2-g3) is a gene that plays an important role in gametocyte development. Interestingly, the insertion of a transposon into the coding sequence of the P. falciparum ortholog gene (PF3D7_1317200) resulted in the absence of gametocytes, suggesting that it might play a role in gametocytogenesis; however, the role of the ortholog of this gene in gametocytogenesis could not be conclusively determined because the parasite line containing a transposon insertion in the PF3D7_1317200 gene also had a missense mutation in the Pfap2-g gene (24,25). Our data clearly showed that disruption of Pyap2-g3 significantly reduced the number of gametocytes, leading to the absence of sporozoites even though a small number of day 8 oocysts were observed (Fig.…”

Section: Figmentioning

confidence: 99%

Systematic CRISPR-Cas9-Mediated Modifications of Plasmodium yoelii ApiAP2 Genes Reveal Functional Insights into Parasite Development

Zhang

Cui

et al. 2017

mBio

114

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Malaria parasites have a complex life cycle with multiple developmental stages in mosquito and vertebrate hosts, and different developmental stages express unique sets of genes. Unexpectedly, many transcription factors (TFs) commonly found in eukaryotic organisms are absent in malaria parasites; instead, a family of genes encoding proteins similar to the plant Apetala2 (ApiAP2) transcription factors is expanded in the parasites. Several malaria ApiAP2 genes have been shown to play a critical role in parasite development; however, the functions of the majority of the ApiAP2 genes remain to be elucidated. In particular, no study on the Plasmodium yoelii ApiAP2 (PyApiAP2) gene family has been reported so far. This study systematically investigated the functional roles of PyApiAP2 genes in parasite development. Twenty-four of the 26 PyApiAP2 genes were selected for disruption, and 12 were successfully knocked out using the clustered regularly interspaced short palindromic repeat-CRISPR-associated protein 9 (CRISPR-Cas9) method. The effects of gene knockout (KO) on parasite development in mouse and mosquito stages were evaluated. Ten of 12 successfully disrupted genes, including two genes that have not been functionally characterized in any Plasmodium species previously, were shown to be critical for P. yoelii development of sexual and mosquito stages. Additionally, seven of the genes were labeled for protein expression analysis, revealing important information supporting their functions. This study represents the first systematic functional characterization of the P. yoelii ApiAP2 gene family and discovers important insights on the roles of the ApiAP2 genes in parasite development.IMPORTANCE Malaria is a parasitic disease that infects hundreds of millions of people, leading to an estimated 0.35 million deaths in 2015. A better understanding of the mechanism of gene expression regulation during parasite development may provide important clues for disease control and prevention. In this study, systematic gene disruption experiments were performed to study the functional roles of members of the Plasmodium yoelii ApiAP2 (PyApiAP2) gene family in parasite development. Genes that are critical for the development of male and female gametocytes, oocysts, and sporozoites were characterized. The protein expression profiles for seven of the PyApiAP2 gene products were also analyzed, revealing important information on their functions. This study provides expression and functional information for many PyApiAP2 genes, which can be explored for disease management.

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“…One of the best examples of mutagenesis using piggyBac is in vivo mutagenesis in mice for cancer gene discovery. Traditionally, murine leukemia virus and murine mammary tumor virus were used (80,107,135) for oncogene discovery in hematopoietic cells (71,72) and mammary tissues (73), respectively. However, such experiments were not possible in a large fraction of solid tumors due to the limited accessibility of these retroviruses.…”

Section: Piggybac-mediated Genetic Screeningmentioning

confidence: 99%

“…By scaling up the experimental scale, one can generate a library of mutant parasites. A small library consisting of 189 parasites with piggyBac insertion was recently generated and, from this library, 29 parasites that were deficient in gametocytogenesis were successfully isolated (80).…”

Section: Piggybac-mediated Genetic Screeningmentioning

confidence: 99%

piggyBacTransposon

Yusa

2015

Microbiol Spectr

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The piggyBac transposon was originally isolated from the cabbage looper moth, Trichoplusia ni, in the 1980s. Despite its early discovery and dissimilarity to the other DNA transposon families, the piggyBac transposon was not recognized as a member of a large transposon superfamily for a long time. Initially, the piggyBac transposon was thought to be a rare transposon. This view, however, has now been completely revised as a number of fully sequenced genomes have revealed the presence of piggyBac-like repetitive elements. The isolation of active copies of the piggyBac-like elements from several distinct species further supported this revision. This includes the first isolation of an active mammalian DNA transposon identified in the bat genome. To date, the piggyBac transposon has been deeply characterized and it represents a number of unique characteristics. In general, all members of the piggyBac superfamily use TTAA as their integration target sites. In addition, the piggyBac transposon shows precise excision, i.e., restoring the sequence to its preintegration state, and can transpose in a variety of organisms such as yeasts, malaria parasites, insects, mammals, and even in plants. Biochemical analysis of the chemical steps of transposition revealed that piggyBac does not require DNA synthesis during the actual transposition event. The broad host range has attracted researchers from many different fields, and the piggyBac transposon is currently the most widely used transposon system for genetic manipulations.

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Transposon mutagenesis identifies genes essential for Plasmodium falciparum gametocytogenesis

Cited by 72 publications

References 64 publications

Selective sweep suggests transcriptional regulation may underlie Plasmodium vivax resilience to malaria control measures in Cambodia

Selective sweep suggests transcriptional regulation may underlie Plasmodium vivax resilience to malaria control measures in Cambodia

Systematic CRISPR-Cas9-Mediated Modifications of Plasmodium yoelii ApiAP2 Genes Reveal Functional Insights into Parasite Development

piggyBacTransposon

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