The long-term goals of the proposed work are to understand the molecular mechanisms involved in transcriptional regulation in the malaria-causing parasite Plasmodium falciparum. During the malaria infection, the red blood cell stage of Plasmodium development is responsible for all of the clinical manifestations of this disease. This developmental stage is characterized by a cyclical 48-hour growth and replication phase that must be exquisitely regulated at the transcriptional level to maximize developmental efficiency and fidelity. The mechanisms used by the parasite to achieve this high level of transcriptional control remain unclear, although there is mounting evidence that transcription is strictly regulated in this organism. Our hypothesis is that a recently identified set of putative transcriptional regulators - the Apicomplexan AP2 (ApiAP2) proteins - are a major family of key transcriptional regulators during parasite development. The role of ApiAP2 proteins as transcriptional regulators will be analyzed using biochemistry, molecular biology and functional genomics tools. The first approach will determine the DNA recognition sequences for ApiAP2 proteins in vitro using two complementary methods. One method is a PCR-based selective enrichment of ligands by exponential enrichment (SELEX) using recombinant ApiAP2 proteins and a random library of putative DNA binding sites. Alternatively, a protein binding microarray (PBM) will be used to hybridize ApiAP2 proteins to a comprehensive collection of double stranded oligonucleotides. The second approach will be to establish the in vivo role of ApiAP2 proteins by 1) modulating transcriptional levels of these putative regulators and 2) generating knockout parasite lines. The effects of these genetic modifications will be assayed globally using DNA microarrays to detect changes in gene expression. Next, the binding of ApiAP2 proteins to DNA will be measured in vivo by chromatin immunoprecipitation with DNA microarray detection (ChIP-chip). These results will define the gene sets directly regulated by the ApiAP2 proteins and will be compared to the in vitro binding results in Aim 2 as well as to prior gene expression data to establish a model for stage-specific transcriptional regulation during parasite development. Finally, we plan to establish and experimentally verify a peptide- based signaling motif that will allow us to predict the active nuclear localization of Plasmodial proteins. The ApiAP2 proteins are the first large family of putative transcription factor domains identified in the genome of P. falciparum and hold great promise for antimalarial intervention since there are no mammalian counterparts to these proteins.

Public Health Relevance

Malaria is a major worldwide disease caused by the Plasmodium parasite with over half a billion cases annually. With the sharp rise in drug-resistant parasites, the challenge is to identify and characterize novel drug targets for anti-malarial strategies. Our research plan will investigate the mechanisms underlying the regulation of gene expression during parasite development as a potential avenue for new therapeutic interventions.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Pathogenic Eukaryotes Study Section (PTHE)
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Joy, Deirdre A
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Pennsylvania State University
Schools of Arts and Sciences
University Park
United States
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Santos, Joana Mendonca; Josling, Gabrielle; Ross, Philipp et al. (2017) Red Blood Cell Invasion by the Malaria Parasite Is Coordinated by the PfAP2-I Transcription Factor. Cell Host Microbe 21:731-741.e10
Josling, Gabrielle A; Llinás, Manuel (2015) Sexual development in Plasmodium parasites: knowing when it's time to commit. Nat Rev Microbiol 13:573-87
Sinha, Abhinav; Hughes, Katie R; Modrzynska, Katarzyna K et al. (2014) A cascade of DNA-binding proteins for sexual commitment and development in Plasmodium. Nature 507:253-257
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Kafsack, Björn F C; Rovira-Graells, Núria; Clark, Taane G et al. (2014) A transcriptional switch underlies commitment to sexual development in malaria parasites. Nature 507:248-52
Carter, Lucy M; Kafsack, Björn F C; Llinás, Manuel et al. (2013) Stress and sex in malaria parasites: Why does commitment vary? Evol Med Public Health 2013:135-47
Radke, Joshua B; Lucas, Olivier; De Silva, Erandi K et al. (2013) ApiAP2 transcription factor restricts development of the Toxoplasma tissue cyst. Proc Natl Acad Sci U S A 110:6871-6
Pino, Paco; Sebastian, Sarah; Kim, Eunbin Arin et al. (2012) A tetracycline-repressible transactivator system to study essential genes in malaria parasites. Cell Host Microbe 12:824-34
Kafsack, Björn F C; Painter, Heather J; Llinás, Manuel (2012) New Agilent platform DNA microarrays for transcriptome analysis of Plasmodium falciparum and Plasmodium berghei for the malaria research community. Malar J 11:187

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