c-di-AMP Is Essential for the Virulence of Enterococcus faecalis

Second messenger nucleotides are produced by bacteria in response to environmental stimuli and play a major role in the regulation of processes associated with bacterial fitness, including but not limited to osmoregulation, envelope homeostasis, central metabolism, and biofilm formation. In this study, we uncovered the biological significance of c-di-AMP in the opportunistic pathogen Enterococcus faecalis by isolating and characterizing strains lacking genes responsible for c-di-AMP synthesis (cdaA) and degradation (dhhP and gdpP). Using complementary approaches, we demonstrated that either complete loss of c-di-AMP (Delta cdaA strain) or c-di-AMP accumulation (Delta dhhP, Delta gdpP, and Delta dhhP Delta gdpP strains) drastically impaired general cell fitness and virulence of E. Wallis. In particular, the Delta cdaA strain was highly sensitive to envelope-targeting antibiotics, was unable to multiply and quickly lost viability in human serum or urine ex vivo, and was virtually avirulent in an invertebrate (Galleria mellonella) and in two catheter-associated mouse infection models that recapitulate key aspects of enterococcal infections in humans. In addition to evidence linking these phenotypes to altered activity of metabolite and peptide transporters and inability to maintain osmobalance, we found that the attenuated virulence of the AcciaA strain also could be attributed to a defect in Delta cdaA pilus production and activity that severely impaired biofilm formation under both in vitro and in vivo conditions. Collectively, these results demonstrate that c-di-AMP signaling is essential for E. faecalis pathogenesis and a desirable target for drug development.

Automated summary

The study reveals that c-di-AMP signaling is crucial for the pathogenesis of Enterococcus faecalis, and loss or accumulation of c-di-AMP significantly impairs cell fitness and virulence. Additionally, the attenuated virulence of the Delta cdaA strain is attributed to defective pilus production and impaired biofilm formation.