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Date of Award

Summer 8-15-2017

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Microbiology & Microbial Pathogenesis)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Malaria is an immense global health problem which is primarily caused by Plasmodium falciparum and Plasmodium vivax parasites. These parasites have a complex life cycle, with stages in the human host and mosquito vector, and traverse diverse host and vector cells. Cell traversal involves biological processes such as parasite motility, breaching of cell membranes, and immune evasion which are mediated by various Plasmodium proteins that are only now beginning to be identified. Genetic studies have implicated Plasmodium CelTOS (cell-traversal protein for ookinetes and sporozoites) in mediating the traversal of sporozoites through the mammalian liver sinusoidal layer and ookinetes through the mosquito midgut epithelium. Additionally, CelTOS is a promising malaria vaccine candidate, as immunization of mice with CelTOS elicits a protective humoral and cell-mediated immune response. An immune response to CelTOS in humans is also correlated with protection from malaria. Even though CelTOS has critical roles in Plasmodium biology and is a vaccine target, the molecular function and mechanism of CelTOS had been unknown, primarily due to its lack of sequence similarity to proteins of known function.

In our studies, we investigated the biophysical function of CelTOS to gain insight into its role during cell traversal. We determined the structure of PvCelTOS and observed similarity to proteins that bind and disrupt cell membranes, including HIV-1 gp41 and the Mycobacterium toxin ESAT-6. The structural similarity of CelTOS to these proteins informed the hypothesis that CelTOS binds and disrupts host and vector cell plasma membranes to enable the exit of parasites out of invaded cells during traversal. Subsequent investigations revealed that CelTOS specifically binds phosphatidic acid, a lipid predominantly present within the inner leaflet of cell plasma membranes, and disrupts liposomes composed of this lipid by pore-formation. Furthermore, microinjection of CelTOS into Xenopus oocytes damaged the plasma membrane and resulted in leakage of the cytosol. Taken together, we have identified CelTOS as the first Plasmodium protein that enables the exit of malaria parasites from host and vector cells.

Given that CelTOS enables the progression of parasites through stages in the mosquito vector and human host, it is a unique malaria vaccine candidate that may block both malaria transmission and infection. Immunization with CelTOS elicits antibodies that have been shown to inhibit malaria infection in mice. CelTOS antibodies also block ookinete development into oocysts, an important step in sporozoite development within mosquitoes. We investigated the mechanism of antibody-mediated inhibition of CelTOS by identifying the epitopes in CelTOS targeted by neutralizing and non-neutralizing antibodies. The crystal structures of CelTOS in complex with neutralizing antibodies delineate residues that are targeted by antibodies that block CelTOS-mediated membrane disruption. Identification of neutralizing epitopes in CelTOS will inform the design of immunogens that elicit potent neutralizing antibodies.

In summary, our findings reveal CelTOS targets the inner leaflet of host and vector cell plasma membranes by pore formation which may enable the exit of parasites during cell traversal. Additionally, we have identified epitopes in Plasmodium CelTOS that are targeted by antibodies, which begins to elucidate the mechanism of antibody-mediated inhibition of CelTOS. These findings provide a foundation for exploring the biophysical mechanism of CelTOS including the mechanisms of lipid binding, membrane insertion, and oligomerization within membranes to form pores. Also, the approaches used in this study may be applied to elucidate the role of putative pore-forming proteins, such as SPECT2 and MAOP, which mediate other steps in cell traversal. Finally, by determining the structure of CelTOS and developing an in vitro method for testing CelTOS activity, this study will inform the design and screening of therapeutics and vaccines which target CelTOS to protect against malaria.

Language

English (en)

Chair and Committee

Niraj H. Tolia

Committee Members

Thomas J. Brett, Daved H. Fremont, Daniel E. Goldberg, Audrey R. Odom John,

Comments

Permanent URL: https://doi.org/10.7936/K7ZP45K1

Available for download on Thursday, August 17, 2119

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