Graduation Year

2014

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Global Health

Major Professor

John H. Adams, Ph.D.

Committee Member

Dennis E.Kyle, Ph.D.

Committee Member

Julian C. Rayner, Ph.D.

Committee Member

Stanley M. Stevens, Jr, Ph.D.

Keywords

cytoadherence, malaria, MAPK, PfMKP1, phosphatase, piggyBac

Abstract

Plasmodium falciparum is a human intracellular parasite that is the causative agent of a deadly form of malaria. This species alone is responsible for 200 million cases of malaria annually resulting in over 1 million deaths worldwide. The excessive mortality due to P. falciparum infection is due to its ability to cause severe pathogenesis through hyperparasitemia and cytoadherence defined as the ability of infected red blood cells to adhere to host vasculature. Cytoadherence is mediated through the export of parasite proteins to the surface of the infected red blood cell (RBC). Exported proteins have been identified but the pathway for protein export is still being elucidated.

Many protein coding genes in the P. falciparum genome are hypothetical and therefore still need to be studied. Random transposon mutagenesis using the piggyBac transposable element in P. falciparum has given us a library of mutants to use for forward genetic studies. In this work, we describe a novel approach for screening P. falciparum piggyBac mutants to look for differences in cytoadherence. We utilized an image-based approach in order to quantify cytoadherence in P. falciparum NF54 wild-type and thirty-four piggyBac mutants. We found cytoadherence to be affected by the expression of genes with specific gene ontologies including nucleic acid metabolism and post-translational modification. Many of these genes are annotated as hypothetical or putative and this work may result in further revelations of a role for these genes in parasite pathogenesis.

We further characterized a piggyBac mutant that was increased in cytoadherent abilities as an atypical mitogen-activated protein kinase phosphatase (MKP) named PfMKP1. We were able to demonstrate phosphatase activity in a generic substrate-based assay and identify a putative substrate as mitogen-activated protein kinase 1 (Pfmap1). Furthermore, we found Pfmap1 to be differentially phosphorylated and a difference in localization (through immunofluorescence assay) in the PfMKP1 mutant line compared to the wild-type and complemented mutant. This adds to the recent work characterizing this gene as important in cell cycle progression within the erythrocytic cycle and lends to the hypothesis of a functioning mitogen-activated protein kinase (MAPK) pathway in P. falciparum for which it is currently unknown.

Supplementary work focused on the development of tools to further investigate the MAPK pathway and the role for PfMKP1 as an atypical MKP of Pfmap1 in this pathway. A mass spectrometry-based technique was developed to look at Pfmap1 and its potential active state based on its phosphorylation status. This approach needs further development but the methods are described here within. In addition, the tools needed to further characterize the binding interaction between PfMKP1 and Pfmap1 were established. The mass spectrometry screen and immunofluorescence screening of Pfmap1 can further our knowledge of the MAPK pathway in P. falciparum and lead to the identification of external stimuli that can induce growth or stress response in the parasite. Taken together, the elucidation of mechanisms for cytoadherence and signal transduction pathways in the parasite can lead to new drug target identification.

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