Understanding Primate Immunity to African Trypanosomes Through the Characterization of Trypanosome Lytic Factor Formation and Function

Author

Sara Fresard, The Graduate Center, City University of New YorkFollow

Date of Degree

9-2024

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Jayne Raper

Committee Members

Diana Bratu

Mitchell Goldfarb

Mark Field

Mark Carrington

Subject Categories

Immunology of Infectious Disease | Parasitology

Keywords

Trypanosome, APOL1, TLF, Trypanosome Lytic Factor, HDL

Abstract

African trypanosomes cause an annual loss of over 1 billion USD due to their economic and agricultural burden in Sub-Saharan Africa. These parasites are unicellular, extracellular parasites, that infect humans, cattle, and other livestock. Humans and some non-human primates are protected against most species of trypanosomes due to an innate immunity factor, Trypanosome Lytic Factor (TLF). This modified High Density Lipoprotein (HDL) contains two unique proteins, Haptoglobin Related Protein (HPR), a ligand to a trypanosome surface receptor, and Apolipoprotein-L1 (APOL1), the lytic component. Our overall goal is to use our understanding of primate immunity to create genetically modified cattle that would also be protected against trypanosomes, reducing the burden of this disease on farmers in Africa.

Two aims were addressed in this thesis to address two gaps in our knowledge of this complex: 1) characterizing how and where the proteins of TLF assemble onto the complex, and 2) elucidating the temporal mechanism of TLF-mediated lysis. Purification and characterization of TLF from human serum, murine models, and hepatocytes in culture were used to determine that TLF is generated in the liver, where APOL1, and under some conditions HPR, are assembled in/on the hepatocyte with sufficient lipid to generate a nascent, or pre-beta HDL (TLF3). This TLF3 enters circulation and accumulates lipid, cholesterol and phospholipids, from peripheral tissue to mature into TLF1. TLF1 can covert to TLF2 during an infection when the levels of IgM are higher, allowing IgM to associate with TLF1, forming TLF2. All three complexes, as well as recombinant APOL1, lyse trypanosomes.

The mechanism of APOL1-mediated lysis of trypanosomes is controversial. Flow cytometry and genetically modified trypanosome cell lines were used to elucidate the temporal mechanism of TLF-mediated lysis by the different TLF complexes, to create a consensus amongst the multiple models of lysis that have been proposed. Upon endocytosis of TLF by the trypanosome, APOL1 inserts into the endosomal membrane due to the acidic pH. Some APOL1 is trafficked to the lysosome, and some to the plasma membrane via the recycling endosome. APOL1 on the surface opens a non-selective cation channel due to the neutral pH. This channel allows sodium and calcium to influx down their concentration gradients. This influx of positive ions causes plasma membrane depolarization, triggering potassium ions to efflux and chloride ions to influx, down their concentration gradients. This ion flux results in an osmotic imbalance, which causes water to influx. This results in cell swelling, and lysis of the parasite. The influx of ions and/or plasma membrane depolarization also results in mitochondrial membrane depolarization, and potentially lysosomal swelling, though we suggest the osmotic imbalance is the overall driver of cell death. Altogether, the research presented here informs our understanding of primate TLF formation and function, and what steps should be taken in generating transgenic mammalian models expressing functional TLF that can provide robust resistance to all trypanosome species.

Recommended Citation

Fresard, Sara, "Understanding Primate Immunity to African Trypanosomes Through the Characterization of Trypanosome Lytic Factor Formation and Function" (2024). CUNY Academic Works.
https://academicworks.cuny.edu/gc_etds/5991