Abstract

Staphylococcus aureus is the leading cause of skin and soft tissue infections in the United States and leads to diseases such as bacteremia, infective endocarditis, and pneumonia. Beta-lactam antibiotics have been the primary treatment since their first use in the 1940s; however, antibiotic overuse has led to the development of resistant strains such as methicillin-resistant S. aureus (MRSA). In addition to antibiotic resistance, MRSA has unique defenses against the host immune system; one example is its resistance to nitric oxide (NO·) produced by host phagocytes. The S. aureus response to NO· is complex and involves many regulatory pathways. The second messenger cyclic-di-AMP plays a major role in the NO· stress response, but the specific details remain unclear. C-di-AMP is produced through the condensation of two molecules of ATP by the diadenylate cyclase enzyme DacA. Our lab previously found that elevated levels of c-di-AMP, due to overexpression of dacA, cause a small growth defect during aerobic growth, but this defect worsens during NO· stress. Thus, having too much c-di-AMP is especially toxic during NO· stress. In this project, we aimed to investigate the consequences of depleting c-di-AMP levels during NO· stress. To reduce expression of the dacA gene, we created knockdown strains using CRISPR interference. We verified that dacA expression was reduced more than 10-fold in all knockdown strains via quantitative real-time PCR. When dacA knockdown strains were grown aerobically, they exhibited a major growth defect relative to wild-type. However, when exposed to NO· stress, this defect decreased relative to wild-type, suggesting that reduced c-di-AMP levels have a lesser impact during NO· stress. Future work will include RNA sequencing to provide insight into changes in gene expression associated with the mutation.

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