Cyclic di-AMP signaling in Listeria monocytogenes
- Author(s): Whiteley, Aaron Thomas
- Advisor(s): Portnoy, Daniel A
- et al.
The Gram positive facultative intracellular pathogen Listeria monocytogenes is both ubiquitous in the environment and is a facultative intracellular pathogen. A high degree of adaptability to different growth niches is one reason for the success of this organism. In this dissertation, two facets of L. monocytogenes, growth and gene expression have been investigated. The first portion examines the function and necessity of the nucleotide second messenger c-di-AMP, and the second portion of this dissertation examines the signal transduction network required for virulence gene regulation.
Through previous genetic screens and biochemical analysis it was found that the nucleotide second messenger cyclic di-adenosine monophosphate (c-di-AMP) is secreted by the bacterium during intracellular and extracellular growth. Depletion of c-di-AMP levels in L. monocytogenes and related bacteria results in sensitivity to cell wall acting antibiotics such as cefuroxime, decreased growth rate, and decreased virulence. We devised a variety of bacterial genetic screens to identify the function of this molecule in bacterial physiology. The sole di-adenylate cyclase (encoded by dacA) responsible for catalyzing synthesis of c-di-AMP in L. monocytogenes could not be deleted by conventional methods. However, ∆dacA mutants could be obtained by flanking dacA with loxP sites and expressing the site-specific recombinase Cre. All of the ∆dacA mutants generated by this novel method on conventional medium harbored suppressor mutations that bypassed the essential functions of c-di-AMP in multiple ways. Characterization of ∆dacA suppressor mutations revealed cross-talk between c-di-AMP and (p)ppGpp, another nucleotide second messenger that slows growth in response to amino acid starvation as part of the stringent response. Depletion of c-di-AMP in rich media resulted in an increase in (p)ppGpp, which was toxic to the bacterium. Whereas (p)ppGpp is essential for growth in nutrient poor synthetic medium, c-di-AMP was found to be essential only in rich medium and genome sequencing of ∆dacA mutants constructed in synthetic medium revealed no suppressor mutations.
Synthetic medium thus provided a tool for generating ∆dacA mutants in combination with targeted mutations identified from our suppressor analysis. These mutants were further analyzed for growth in rich medium or resistance to cefuroxime. Suppressor mutations in the oligopeptide permease and glycine betaine osmolyte importer showed that peptides from rich medium were toxic to ∆dacA mutants due to a dysregulation of osmotic pressure. These defects in osmotic pressure could be overcome by addition of salt to the medium, which allowed for recovery of ∆dacA on rich medium and ameliorated sensitivity of ∆dacA to cefuroxime. To identify how c-di-AMP regulated intracellular osmotic pressure, we screened for suppressor mutations that overcome growth on rich media and cefuroxime resistance. Suppressor mutations from this screen indicated that c-di-AMP inhibits pyruvate carboxylase in order to limit carbon flux into the TCA cycle when acetyl-CoA levels are high. Increased flux through the TCA cycle was only toxic when citrate synthase was present, implying that accumulation of citrate is toxic to ∆dacA mutants. These findings demonstrate a role of c-di-AMP in balancing central metabolism and TCA cycle intermediates for optimal growth on rich media, resistance to cefuroxime, and virulence.