Author ORCID Identifier

https://orcid.org/0000-0003-1057-4078

Year of Publication

2022

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Medicine

Department/School/Program

Molecular and Cellular Biochemistry

First Advisor

Dr. Matthew S. Gentry

Second Advisor

Dr. Anthony P. Sinai

Abstract

Toxoplasma gondii is an opportunistic, protozoan parasite of all warm-blooded animals, infecting roughly one-third of humans worldwide. Humans acquire infections by consuming T. gondii tissue cysts in undercooked meat or from oocysts shed in cat feces. Encysted parasites convert into rapidly growing tachyzoites that disseminate throughout the body, defining the acute phase of infection. Under host immune pressure, tachyzoites convert into bradyzoites that populate tissue cysts found in CNS or muscle tissue and persist for the lifetime of the host, defining the chronic phase of infection. Tissue cysts are responsible for transmission via carnivory, but also possess the ability to reactivate into tachyzoites within their current host. In the context of immunosuppression, reactivation manifests primarily as toxoplasmic encephalitis. Current therapeutic options are poorly tolerated by many and effective only against tachyzoites. Thus, there is a need for a better understanding of the chronic infection to develop new treatments that eliminate tissue cysts or prevent reactivation.

The present work therefore examines the parasite’s utilization of amylopectin granules (AGs), a morphological feature found in bradyzoites that distinguishes them from tachyzoites. AGs, much like plant starch, are insoluble storage molecules composed of branched chains of glucose and believed to fuel bradyzoite replication, transmission, and reactivation. In plants, insoluble starch is made enzymatically accessible by a cycle of direct, reversible glucan phosphorylation. The addition of phosphate by a glucan kinase solubilizes the starch surface to facilitate enzymatic glucose release, and the subsequent removal of phosphate by a glucan phosphatase prevents hyperphosphorylation that would obstruct enzyme access. The T. gondii genome encodes these opposing enzymatic activities: the glucan, water dikinase, “TgGWD,” and the glucan phosphatase, “TgLaforin.”

The work herein, along with recent studies, strongly suggests that the historical understanding of AGs is lacking. There is mounting evidence that tachyzoites contain small and rapidly metabolized glucans that are key to T. gondii biology. Thus, an understanding of how T. gondii accesses its glucose stores throughout the asexual cycle is needed. Perturbations of glucan phosphatase activity in plants and animals result in significant defects in metabolism and glucan morphology, suggesting the need to investigate reversible phosphorylation of T. gondii AGs. To date, little has been done to characterize the enzymology or relevance of reversible glucan phosphorylation in T. gondii.

Herein, the functions and unique structures of TgLaforin and TgGWD were characterized using a wide range of biochemical and biophysical techniques. Additionally, a role for TgLaforin in T. gondii was elucidated using protein localization and knockout approaches. With these tools, it was demonstrated that TgLaforin plays a vital role in both tachyzoites and bradyzoites, linking TgLaforin to parasite metabolism, virulence, and development across the asexual cycle. The data here suggests that the tachyzoite glucan may play a previously unappreciated role in T. gondii. Thus, this work provides a unique window into the role of AGs through the first multi-disciplinary characterization of reversible glucan phosphorylation in T. gondii and establishes a framework for the development of future therapeutic approaches.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2022.56

Funding Information

This work was supported in part by National Science Foundation grants: GRFP no. 1247392 in 2017, DBI 2018007 in 2020, and MCB 1817414 in 2018.

Further support was provided by the National Institutes of Health grants R35 NS116824 in 2020, and R21 AI150631 in 2020.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Available for download on Friday, October 28, 2022

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