A New Assay to Monitor the Ability of a Tuberculosis Enzyme to Convert a Stress Signal to Energy
Mentor 1
David Frick
Start Date
28-4-2023 12:00 AM
Description
Tuberculosis is one of the most ancient and dreaded infectious diseases. According to the CDC, 13 million Americans are infected with Mycobacterium tuberculosis (MTb), the bacterium that causes the disease. Similar to other respiratory diseases, tuberculosis mostly affects the lungs and spreads through airborne droplets. MTb infection is typically treated with the antibiotic’s isoniazid and rifampin, but MTb often evolves drug resistance. When stressed by drugs, MTb synthesizes “alarmones” such as the adenosine polyphosphates Ap4A and Ap5A. MTb also encodes enzymes that cleave Ap4A and Ap5A to yield ATP, which can fuel other reactions needed for proliferation. RV2985 is such a MTb protein. RV2984 is a member of the Nudix hydrolase family, which includes enzymes that hydrolyze nucleoside diphosphate coupled to another moiety, X. The goal of this project is to discover an inhibitor of RV2985 that could be used to study alarmone function and/or the role of RV2985 in the MTb lifecycle. Since RV2985 cleaves the Ap4A and Ap5A to produce ATP, we show here that diadenosine cleavage can be monitored by coupling to a reaction-catalyzed firefly luciferase. Firefly luciferase uses ATP to decarboxylate D-luciferin and emit light, which can be measured with a camera or a plate reader. This assay was optimized by varying pH, ionic strength, and substrate concentration, to maximize signal-noise and minimize well-to-well variation. The assay was also automated so that it could be used for high throughput screening and was used to show that RV2985 cleaves Ap5A with a lower Km and greater Vmax when compared to Ap4A.
A New Assay to Monitor the Ability of a Tuberculosis Enzyme to Convert a Stress Signal to Energy
Tuberculosis is one of the most ancient and dreaded infectious diseases. According to the CDC, 13 million Americans are infected with Mycobacterium tuberculosis (MTb), the bacterium that causes the disease. Similar to other respiratory diseases, tuberculosis mostly affects the lungs and spreads through airborne droplets. MTb infection is typically treated with the antibiotic’s isoniazid and rifampin, but MTb often evolves drug resistance. When stressed by drugs, MTb synthesizes “alarmones” such as the adenosine polyphosphates Ap4A and Ap5A. MTb also encodes enzymes that cleave Ap4A and Ap5A to yield ATP, which can fuel other reactions needed for proliferation. RV2985 is such a MTb protein. RV2984 is a member of the Nudix hydrolase family, which includes enzymes that hydrolyze nucleoside diphosphate coupled to another moiety, X. The goal of this project is to discover an inhibitor of RV2985 that could be used to study alarmone function and/or the role of RV2985 in the MTb lifecycle. Since RV2985 cleaves the Ap4A and Ap5A to produce ATP, we show here that diadenosine cleavage can be monitored by coupling to a reaction-catalyzed firefly luciferase. Firefly luciferase uses ATP to decarboxylate D-luciferin and emit light, which can be measured with a camera or a plate reader. This assay was optimized by varying pH, ionic strength, and substrate concentration, to maximize signal-noise and minimize well-to-well variation. The assay was also automated so that it could be used for high throughput screening and was used to show that RV2985 cleaves Ap5A with a lower Km and greater Vmax when compared to Ap4A.
