Abstract

Infectious diseases have emerged as the second leading cause of human death worldwide. Staphylococcus aureus, a Gram-positive bacterium, causes a variety of life threatening infectious diseases such as skin and soft tissue infections (SSTIs), osteomyelitis, pneumonia, and endocarditis. Moreover, S. aureus is unique compared to other bacterial species in that it is able to resist a variety of antibiotics and essential components of the host innate immune system, such as nitric oxide (NO·). In bacteria, NO· inhibits respiration and many metabolic pathways and can cause significant cellular damage, but S. aureus successfully adapts, detoxifies NO·, and alters its metabolism to allow for continued growth, unlike other bacterial species. S. aureus uses a second messenger called cyclic di-adenosine monophosphate (c-di-AMP) to regulate cell processes like stress response, cell wall size, and respiration. The small molecule c-di-AMP can function as an allosteric regulator of specific target proteins within the cell and is synthesized by the protein DacA and degraded by the phosphodiesterase enzyme GdpP. A previous transposon screen study in vivo on a community-acquired methicillin resistance Staphylococcus aureus strain (CA-MRSA) indicated that several genes associated with c-di-AMP signaling exhibited decreased fitness during NO· stress, including the gene SAUSA300_0730, encoding a hypothetical protein with a known second-messenger binding domain. However, it still is not well understood how c-di-AMP signaling relates to NO· stress. Here, we manipulated c-di-AMP levels by over- expressing the enzymes dacA and gdpP under an inducible promoter and found that high c-di-AMP levels are toxic during NO· stress. Furthermore, to examine the function of SAUSA300_0730 during NO· stress, site-directed mutagenesis was performed to create a deletion mutant, which exhibited slightly decreased fitness relative to wild- type. Altogether, our results suggest that proper regulation of c-di-AMP signaling is critical for the overall success of S. aureus under nitrosative stress. These findings may lead to identifying potential antibiotic targets to alter this signaling pathway.

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