Molecular basis of cyclic-di-AMP regulation in Gram-positive bacteria

ABSTRACT Bacteria are surrounded by a cell envelope that is essential for growth, integrity, and pathogenesis. The envelope and the biogenesis pathways that build it are also the target of many of our most effective antibiotic and vaccine therapies. Although envelope assembly pathways have been studied for decades and most of the factors

involved have been identified, a gap in our knowledge is how bacteria monitor the envelope for defects and coordinate their repair. This proposal focuses on the nucleotide second messenger cyclic-di-AMP (c-di-AMP) and how bacteria modulate its levels in response to envelope defects to facilitate repair. c-di-AMP principally

controls K+ and osmolyte transporters. It is hypothesized that when c-di-AMP levels are reduced turgor pressure increases; conversely when c-di-AMP levels are increased turgor decreases. Importantly, mutations that alter the levels of this second messenger are associated with antibiotic resistance. Although much is known about the

intracellular targets of c-di-AMP, the signals that modulate changes in the cellular pool of the cyclic dinucleotide are largely unknown. The major and most highly conserved c-di-AMP synthase, CdaA, forms a membrane complex with its hypothesized regulator CdaR. The central hypothesis of this proposal is that Gram-positive

bacteria use CdaR to sense changes in the cell envelope and homeostatically adjust c-di-AMP synthesis. In preliminary studies, I found that levels of c-di-AMP increase in response to cell wall defects. Separately, I discovered that CdaR's extracytoplasmic domain is required for cell envelope integrity in the presence of cell

wall defects. In Aim 1, I will investigate the hypothesis that CdaR monitors the cell envelope for defects and adjusts c-di-AMP levels, and thereby cytoplasmic turgor pressure, in response. The objective of Aim 2 is to identify specific stimuli sensed by a second more broadly conserved extracytoplasmic domain on CdaR and then

elucidate the molecular mechanism of signal sensation and transduction. This proposal will address important and outstanding questions related to what stimuli modulate the intracellular cyclic nucleotide pools as well as the molecular mechanisms by which Gram-positive bacteria monitor and respond to changes in their cell envelope.

My findings will enable the development of drugs which could alter c-di-AMP levels to inhibit growth or re- sensitize drug-resistant bacteria to frontline antibiotics. These findings are of particular relevance for the treatment of methicillin resistant S. aureus (MRSA) whose high level of methicillin tolerance has been tied to

high levels of c-di-AMP. In addition, the proposed research and training plan will provide me the skills, knowledge, and experience to become a successful independent investigator.