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.

Grant Number: 5F31AI181098-02
NIH Institute/Center: NIH
Principal Investigator: Anna Brogan