ABSTRACT
Enterococci are the 3rd most common cause of hospital-acquired infections and a major public health threat
due to the continuous rise of multidrug-resistant (MDR) isolates. Because the pathogenic potential of
enterococci is closely linked to their ubiquitous stress resilience, work in our lab aims to identify and dissect the
mechanisms that allow Enterococcus faecalis, the most prevalent enterococcal species of human infections, to
survive in the hostile host environment. Second messenger nucleotides such as (p)ppGpp (the effector
molecule of the stringent response) and c-di-AMP are produced by bacteria in response to internal or external
stimuli, playing major roles in the regulation of processes associated with cell homeostasis and virulence. In
addition, cyclic nucleotides such as c-di-AMP act as agonists of the innate immune response of mammalians by
stimulating a potent STING-dependent type I interferon response. Of interest, previous investigations have
shown that the c-di-AMP and (p)ppGpp signaling networks are interconnected in other bacteria, but a clear
understanding of the mechanics and physiological significance of this interaction are still lacking. In previous
studies, we discovered that E. faecalis depends on small amounts of (p)ppGpp to maintain a balanced
metabolism and to respond to external cues in an accurate and timely manner. We also found that lack of
(p)ppGpp greatly increases antibiotic sensitivity and attenuates the virulence of E. faecalis. More recently, we
showed that c-di-AMP is also critical to E. faecalis pathophysiology and that either accumulation or lack of c-
di-AMP can drastically impair the virulence potential of E. faecalis. The goals of this application are: (i) to
probe the multifaceted roles played by c-di-AMP during infection using a catheter-associated urinary tract
infection (CAUTI) mouse model that recapitulates many of the clinical characteristics of the disease in humans,
(ii) to identify and characterize the c-di-AMP binding/effector proteins in E. faecalis, and (iii) to investigate
how integration of the c-di-AMP and (p)ppGpp regulatory networks controls specific processes that promote
bacterial fitness and then determine how this association contributes to E. faecalis pathophysiology. This
conceptually innovative application builds on our extensive preliminary data and the complementary
expertise and solid track record of our research team in each specific area of this application. Significance of
the proposed studies lies in unravelling the multifaceted role of c-di-AMP in host-pathogen interactions,
uncovering the scope of c-di-AMP regulation in a major MDR pathogen, and shedding new light onto the
intricate relationship between c-di-AMP and (p)ppGpp. Given the central role of regulatory nucleotides in
bacterial pathogenesis, a better understanding of how they modulate cell physiology based on identification
and characterization of their mechanisms of action and effectors can facilitate the rational design of new
antimicrobial therapies.