Proteomic Identification and Biochemical Characterization of Modifications Produced on Plasmodium cynomolgi and Plasmodium knowlesi Infected Erythrocyte Membranes Público

Abstract

Plasmodium parasites modify their host red blood cell (RBC) antigenically and
structurally, enabling parasite protein export, nutrient import, and RBC adherence. While knobs
and Maurer's clefts are observed in P. falciparum-infected erythrocytes, RBCs infected with
other Plasmodium species display alterations such as caveolae and caveola-vesicle complexes
(CVCs). Information on the antigenic makeup and function of these ultrastructures is scarce. This
dissertation utilizes genomics, proteomics and biochemical tools as well as the simian malaria
species P. knowlesi and P. cynomolgi to characterize and gain functional insights into these
structures.

This dissertation first focuses on investigations of glyceraldehyde-3-phosphate
dehydrogenase, an enzyme involved in glycolysis for ATP production. We identified
Plasmodium-specific amino acid substitutions within the functional domains of this protein that
may prove to be of importance, thus providing insights for antimalarial drug-targeting studies.
Secondly, we identified the gene encoding a protein known to be part of the CVCs of P.
vivax, and believed to have an ortholog in the related parasite, P. cynomolgi.
Immunoprecipitation of detergent extracts followed by trypsin peptide cleavage and mass
spectrometric analysis identified this protein as an 81 kDa member of the Plasmodium helical
interspersed sub-telomeric (PHIST) superfamily, termed PcyPHIST-81, with homologs in five
other Plasmodium species. All PHIST-81 homologs have a Plasmodium export motif, conserved
tryptophans, and four consecutive alpha helices in their amino acid sequences. Immunoelectron
tomography studies on P. cynomolgi-infected RBCs using rabbit antisera to recombinant P. vivax
PHIST-81 revealed that PcyPHIST-81 localizes to the tubular extensions of CVCs.
Finally, we utilized a global approach to identify proteins that are localized to the
membranes of P. cynomolgi and P. knowlesi-infected erythrocytes. Mass spectrometric analysis
of parasite-infected RBC membrane ghosts identified 109 P. cynomolgi proteins and 129 P.
knowlesi proteins, which included members of the PHIST and Pk-fam-c protein families as well
as hypothetical proteins.

Table of Contents

Table of Contents

CHAPTER ONE: Malaria and the red blood cell membrane:

1
Ultrastructural modifications, simian models and novel membrane proteins
Malaria









2
The Plasmodium life cycle







7
Clinical
symptoms
of
malaria

10
Plasmodium vivax, P. knowlesi and P.
cynomolgi
11
The
red
blood
cell
membrane

12
Plasmodium and the red blood cell membrane




13
Plasmodium falciparum-infected RBC ultrastructures and proteins

15
RBC ultrastructures and proteins in other Plasmodium
species
19
Proteins associated with caveolae and caveola-vesicle complexes

22
Algorithms to identify putative Plasmodium exported/secreted proteins

23
Monoclonal antibodies raised against P. vivax schizonts that localize specific
proteins
to
parasitized
RBC
ultrastructures
28
Plasmodium helical interspersed subtelomeric (PHIST) family of proteins
29
Overview of Plasmodium-infected red blood cell studies



30
References









31

CHAPTER TWO: Phylogenetic and structural information on

43
glyceraldehyde-3-phosphate dehydrogenase in Plasmodium provides functional
insights
Abstract









44
Introduction
45
Materials
and
Methods 48
Results









50
Discussion









58
References









59

About this Dissertation

Rights statement

• Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.

School	Laney Graduate School
Department	Biological and Biomedical Sciences
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Parasitology Biology, Molecular
Palavra-chave	PHIST Malaria Proteomics Cynomolgi Vivax Plasmodium
Committee Chair / Thesis Advisor	Galinski, Mary R, Emory University
Committee Members	Antia, Rustom, Emory University Moreno, Alberto, Emory University Lammie, Patrick J, Emory University Udhayakumar, Venkatachalam, Centers for Disease Control Mead, Jan R, Emory University

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