Abstract

Staphylococcus aureus is a ubiquitous Gram-positive human pathogen with antibiotic resistant characteristics. It is the culprit for a majority of skin and soft tissue infections (SSTIs), which can lead to more serious infections such as endocarditis. The success of S. aureus can be traced to several key virulence traits, including resistance to nitric oxide (NO·), a crucial component of the human innate immune response. S. aureus promotes a metabolic state in the presence of NO· to continue replication and detoxify NO·. Furthermore, S. aureus utilizes a second messenger signaling system that relies on the small molecule cyclic di-adenosine monophosphate (c-di-AMP), which regulates cell wall size and other basic cellular processes such as respiration, there is evidence that it may also play a role in NO· resistance. Developing methods to detect concentrations of c-di-AMP during cell stress will lead to further understanding of the role this second messenger plays during the pathogenesis of S. aureus. On a cellular level, c-di- AMP binds to protein and RNA effectors that alter transcription, translation, or enzyme function. Linking c-di-AMP- responsive protein or RNA effectors to conditionally-fluorescent molecules has been demonstrated as a viable strategy for c-di-AMP detection in another pathogen, Listeria monocytogenes. We constructed and tested several genetically encoded biosensor plasmid constructs in S. aureus using a microplate reader. Our data evaluate the efficacy of each biosensor for detecting changes in c-di-AMP levels that are induced by genetic manipulation. We show that differences can be detected, but further optimization is needed to improve sensitivity. Understanding the conditions under which c-di-AMP levels fluctuate in S. aureus ultimately creates a stronger understanding of its function in bacteria and role in NO· resistance in Staphylococcus aureus. Additionally, the successful creation of c-di-AMP biosensors in S. aureus will allow future screens of chemical compounds that alter c-di-AMP levels in bacteria as potential new drugs.

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