Publication in: Spring 2023 Issue

Two-Pronged Approach to Investigating Regulation of the NO· Stress Response in S. aureus
Noela Moraga
Faculty Mentor(s):
Melinda Grosser
Abstract / Summary:
Methicillin-resistant Staphylococcus aureus is a Gram-positive bacterium and an opportunistic pathogen. It causes infections such as skin abscesses, sepsis, urinary tract infections, and meningitis. Due to antibiotic resistance, S. aureus is a major public health concern. One of the contributing factors to its survival in a host is cyclic di-adenosine monophosphate (c-di-AMP),. This second messenger regulates many aspects of cellular function in Gram-positive bacteria, including maintaining osmotic pressure and cell wall homeostasis. In S. aureus, an enzyme called DacA generates c-di-AMP from two ATP molecules. C-di-AMP is degraded into 5’-pApA by the phosphodiesterase enzyme GdpP. The deletion of DacA causes growth arrest and sensitization to antibiotics, which indicates the importance of c-di-AMP for bacterial survival. Most c-di-AMP quantification methods require purification of cell lysates; however, to understand how dynamic changes in c-di-AMP are related to antimicrobial resistance and stress responses, it will be useful to have a fluorescence-based biosensor that can be monitored in real-time in live cells. In this project, we are adapting and testing a fluorescence resonance energy transfer (FRET),-based c-di-AMP biosensor created for Listeria monocytogenes for use in S. aureus. The biosensor includes cyan fluorescent protein (CFP), and yellow fluorescent protein (YFP), linked by a truncated c-di-AMP binding protein (lmo0553), from L. monocytogenes. When c-di-AMP is present at high levels, lmo0553 changes conformation to bring CFP and YFP into close proximity, allowing FRET and a change in emission spectra to occur. We are testing the expression of the biosensor from the S. aureus plasmid pCN52, with expression driven by either the constitutive rpsJ or rpoD promoters. The resulting vector will be tested in S. aureus strains overexpressing DacA or GdpP and in conjunction with an ELISA to calibrate a standard curve relating FRET results to c-di-AMP concentration.
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