Understanding Anchoring of Plasmodium Falciparum Exported Proteins to the Erythrocyte Cytoskeleton and Investigating Their Function

Bikash Shakya, Purdue University

Abstract

Plasmodium falciparum causes the most severe form of human malaria and is responsible for most malaria-associated deaths. In 2015, about 214 million cases of malaria occurred worldwide, killing 438,000 people, mostly children. The remodeling of parasite-infected red blood cells contributes to the pathogenesis of falciparum malaria. P. falciparum extensively modifies the infected red blood cell (iRBC), resulting in changes in iRBC deformability, shape and surface properties. These alterations suggest that the red blood cell (RBC) cytoskeleton is a major target for modifications during infection. These modifications are attributed to the actions of hundreds of proteins that are exported by the parasite to iRBC. However, the cytoskeletal binding partners of most of these exported proteins are not known. This dissertation summarizes the findings from the studies designed to identify and investigate protein-protein interactions between P. falciparum exported proteins and erythrocyte cytoskeletal proteins. A large-scale screening using the split luciferase assay was performed to identify the cytoskeletal targets of twenty-four parasite-exported proteins, which yielded cytoskeletal binding partners for 15 parasite proteins. In total, 56 interactions were identified between 15 parasite proteins and 14 RBC cytoskeletal protein fragments. Seventeen of the above interactions were confirmed using protein co-precipitation assays. The polyhelical interspersed subtelomeric (PHIST) domain-containing proteins (PFD0090c, PF14_0018, MAL8P1.163 and PFD0095c) interacted with both ankyrin 1 (ANK1) and band 4.1 (4.1R). Out of these four proteins, PF14_0018 and PFD0095c also contained the MESA erythrocyte cytoskeletal (MEC) binding motif. These MEC motif proteins including MESA and PF10_0381 targeted the ANK1 fragment composed of spectrin binding domain. Yeast two hybrid assays and split-luciferase assays were used to investigate the minimum-binding region of MESA and PF14_0018. The MEC motif was sufficient for the interaction of MESA and PF14_0018 with the ANK1 fragment and in absence of the MEC motifs, the interactions were obliterated. Similarly, protein co-purification assays were used to identify the minimum-binding domain of ANK1 repeats targeted by parasite proteins. I found that the parasite proteins that targeted the ANK1 repeats interacted with D2 subdomain of the ANK1 repeats, suggesting that the interaction may disrupt the interaction between ANK1 and band 3 in the iRBC membrane. In order to assess the role of parasite proteins in host cell rigidity, I performed microsphiltration assays by loading erythrocyte ghosts with the parasite proteins and testing their retention rate in a matrix composed of metal beads. Ghost erythrocytes loaded with RESA, PF14_0018 and PFD0090c were retained at a significantly higher rate than control samples, suggesting the possible role of these parasite proteins in host cell rigidity. In summary, the research reported in this thesis identified and characterized protein-protein interactions between P. falciparum exported proteins and erythrocyte cytoskeletal proteins and began to define their roles in altering host cell rigidity.

Degree

Ph.D.

Advisors

LaCount, Purdue University.

Subject Area

Molecular biology|Microbiology|Parasitology

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