Characterization of a novel cell wall-associated nucleotidase of Enterococcus faecalis that degrades extracellular c-di-AMP

Enterococcus faecalis is a prolific opportunistic pathogen responsible for a range of life-threatening infections, notorious for its ability to withstand environmental stressors. Second messengers are small molecules that relay signals in response to stimuli and are thought to be crucial for bacteria like E. faecalis to modulate their adaptation to stress. The second messenger nucleotide c-di-AMP has emerged as an essential bacterial signaling molecule due to its impact on physiological processes, including adaptation to osmotic stress, cell wall homeostasis, antibiotic tolerance, and virulence. In addition, c-di-AMP is a pathogen-associated molecular pattern (PAMP) molecule that can trigger a potent stimulation of the host immune system. In previous work, we identified and characterized the enzymes responsible for the synthesis and degradation of intracellular c-di-AMP in E. faecalis, demonstrating that maintenance of c-di-AMP homeostasis is vital for its fitness and virulence. In addition to the intracellular enzymes that regulate c-di-AMP levels, a small number of bacteria encode surface-associated nucleotidases that cleave extracellular c-di-AMP and are potentially associated with immune evasion. Here, we characterize a novel and unique cell wall-anchored nucleotidase, termed EecP (E. faecalis extracellular c-di-AMP phosphodiesterase), which features duplicated catalytic domains and degrades extracellular c-di-AMP. Through competition experiments, we show that EecP likely uses c-di-GMP, and to a lesser extent AMP, as additional substrates. While a ΔeecP strain failed to display relevant phenotypes under most in vitro conditions, it exhibited increased susceptibility to killing by phagocytic cells, a phenotype at least partly associated with cGAS-STING immune signaling. NanoString analysis revealed distinct innate immune signatures in murine macrophages infected with the parent strain OG1RF or ΔeecP, uncovering differential expression of host targets known to be impacted by c-di-AMP, as well as novel targets. Using two murine infection models, we show that the impact of eecP deletion and the consequent buildup of extracellular c-di-AMP on E. faecalis pathogenesis might depend on the site of infection. Notably, disseminated infection was more severe in mice infected with ΔeecP, suggesting that extracellular c-di-AMP influences infection outcomes, likely through modulation of host immune responses.