Publication in: Spring 2023 Issue

Quantifying cyclic di-AMP in Staphylococcus aureus during stress using a competitive ELISA
Carissa Her
Faculty Mentor(s):
Melinda Grosser
Abstract / Summary:
Staphylococcus aureus, a Gram-positive bacterium, is one of the leading causes of life-threatening bacterial infection in the US, partly due to its antimicrobial resistance. Diseases like pneumonia, skin and soft tissue infections, sepsis, and endocarditis may arise from a serious staph infection. S. aureus uses a second messenger called cyclic di-adenosine monophosphate (c-di-AMP), to regulate many cellular functions such as peptidoglycan synthesis, stress response, and more. Our data show that high c-di-AMP levels can be toxic during nitric oxide (NO·), stress. Others have shown that low c-di-AMP levels result in reactive oxygen species (ROS), toxicity in S. aureus. Therefore, maintaining normal c-di-AMP is essential for proper cell functions, especially during stress. In this research, we aim to optimize a competitive enzyme-linked immunosorbent assay (ELISA), to quantify specific concentrations of c-di-AMP in S. aureus, with the ultimate goal of understanding how c-di-AMP levels change during stress and what levels are optimal. For assay development, we used three strains: S. aureus containing a vector control of an anhydrotetracycline (aTc),-inducible plasmid (pRMC2),, and S. aureus overexpressing the genes encoding either DacA (produces c-di-AMP), or GdpP (hydrolyzes c-di-AMP), from pRMC2. Growth conditions and lysate purification methods were tested with these strains in conjunction with a commercial c-di-AMP ELISA kit, and a protocol was developed to successfully detect different concentrations of c-di-AMP in S. aureus. Currently, we are using this new assay combined with stepwise induction of DacA or GdpP via aTc (in the above strains), to determine the precise concentration range of c-di-AMP that can be tolerated in the presence of stressors. We are also using ELISA to calibrate a fluorescence resonance energy transfer (FRET),-based biosensor for c-di-AMP, another means of measuring c-di-AMP in S. aureus in real-time. Long-term, these findings may contribute to the development of drugs that alter signaling pathways of antibiotic-resistant bacteria.
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