Fall Research Expo 2021

Antimalarial Drug Research

 

In this project, I worked to induce a mutation in the DXR enzyme, another vital enzyme in the MEP pathway. Mutations in the gene encoding DXR had previously been found in the lab in parasites resistant to fosmidomycin (FSM), a DXR inhibitor. We aimed to determine the impact of this mutant DXR on enzyme function and sensitivity to fosmidomycin. To do this, we induced the same mutation in Escherichia coli DXR, which has close homology to the P. falciparum DXR, and transfected wild-type E. coli with a plasmid containing the mutated protein. The next steps would be to grow the mutated protein using the transfected E. coli. Next, we would harvest cells, purify the protein, then evaluate its enzymatic activity and quantify its resistance to FSM.

PRESENTED BY
PURM - Penn Undergraduate Research Mentoring Program
College of Arts & Sciences 2023
Advised By
Audrey John
Chief of the Division of Pediatric Infectious Diseases at Children's Hospital of Philadelphia.
Join Andrew for a virtual discussion
PRESENTED BY
PURM - Penn Undergraduate Research Mentoring Program
College of Arts & Sciences 2023
Advised By
Audrey John
Chief of the Division of Pediatric Infectious Diseases at Children's Hospital of Philadelphia.

Comments

I'm interested in why you chose to introduce the plasmid to E. coli DXR mutants over P. falciparum directly. What's the benefit of using E. coli specifically? Also, major props to you for the PyMOL models of EcDXR and PfDXR! 

You mentioned growing parasites under the presence of the drug, but how exactly do you do that? Is it similar to growing bacteria or animal cells, or is it entirely different? Great job! 

It is very similar to other types of cell cultures. These parasites feed on nutrients they get from red blood cells. So to grow the parasites, we seed infected red blood cells in a culture with fresh blood where the parasites can grow, multiply, and infect other blood cells. To grow these parasites under the presence of the drug, we pipet a small concentration of the drug into the culture so as not to kill all the parasites in that culture. This allows the already resistant parasites in the culture to be selected out. Once these parasites have multiplied , we can harvest them and sequence their DNA to try and see what mutations led to the resistance. This helps us learn more about the way the drug and its target enzyme function.