Connections Between Inhibition Of Atp Production And Fatty Acid Uptake In Procyclic Trypanosoma Brucei Brucei

Olivia Walkowiak

School Name

Governor's School for Science and Math

Grade Level

12th Grade

Presentation Topic

Microbiology

Presentation Type

Mentored

Mentor

Mentor: Dr. Paul; Department of Genetics and Biochemistry, Clemson University

Oral Presentation Award

2nd Place

Abstract

Trypanosoma brucei spp. are blood-borne parasites transmitted by the bite of a tsetse fly that cause Human African Trypanosomiasis (HAT), also known as African Sleeping Sickness, in humans and a wasting disease called Nagana in cattle. HAT is a major problem in over twenty-four countries in West and Central Africa. Of all infected individuals, less than 10% received any treatment. These parasites are dependent on acquiring fatty acids from their hosts. In order to better understand fatty acid uptake in T. brucei, we examined the energy requirements for this process using the tsetse fly midgut procyclic form of T. brucei brucei, the sub-species that infects cattle. Energy for endocytosis comes from ATP production. We investigated the relationship between ATP levels and fatty acid uptake. We developed a luminescence-based assay using Cell Titer GLO (Promega) to record intracellular ATP levels, which were normalized to cell number using a Coomassie-based Protein Assay (Bio-Rad). This assay was validated using a serial dilution of cells. ATP production was limited using a mixture of the metabolic inhibitors sodium azide and 2-deoxy glucose. Fatty acid uptake was measured using a BODIPY C-12 fluorescence assay. Inhibition of ATP production caused no difference in fatty acid uptake. Fatty acid uptake in T. brucei brucei does not appear to be solely dependent on high ATP levels. These results suggest that the primary mechanism of fatty acid uptake in T. brucei brucei is diffusion. This research may help identify and evaluate novel methods to treat HAT and Nagana.

Location

Owens 110

Start Date

4-16-2016 9:30 AM

COinS
 
Apr 16th, 9:30 AM

Connections Between Inhibition Of Atp Production And Fatty Acid Uptake In Procyclic Trypanosoma Brucei Brucei

Owens 110

Trypanosoma brucei spp. are blood-borne parasites transmitted by the bite of a tsetse fly that cause Human African Trypanosomiasis (HAT), also known as African Sleeping Sickness, in humans and a wasting disease called Nagana in cattle. HAT is a major problem in over twenty-four countries in West and Central Africa. Of all infected individuals, less than 10% received any treatment. These parasites are dependent on acquiring fatty acids from their hosts. In order to better understand fatty acid uptake in T. brucei, we examined the energy requirements for this process using the tsetse fly midgut procyclic form of T. brucei brucei, the sub-species that infects cattle. Energy for endocytosis comes from ATP production. We investigated the relationship between ATP levels and fatty acid uptake. We developed a luminescence-based assay using Cell Titer GLO (Promega) to record intracellular ATP levels, which were normalized to cell number using a Coomassie-based Protein Assay (Bio-Rad). This assay was validated using a serial dilution of cells. ATP production was limited using a mixture of the metabolic inhibitors sodium azide and 2-deoxy glucose. Fatty acid uptake was measured using a BODIPY C-12 fluorescence assay. Inhibition of ATP production caused no difference in fatty acid uptake. Fatty acid uptake in T. brucei brucei does not appear to be solely dependent on high ATP levels. These results suggest that the primary mechanism of fatty acid uptake in T. brucei brucei is diffusion. This research may help identify and evaluate novel methods to treat HAT and Nagana.