Exploring the relationship between the two-component system CroRS and the second messenger c-di-AMP in regulating β-lactam resistance in Enterococcus faecalis

The opportunistic pathogen Enterococcus faecalis, which is responsible for serious hospital-acquired infections, has a high level of intrinsic antibiotic resistance, especially to β-lactam antibiotics. E. faecalis uses the CroRS two-component system to control antibiotic resistance against cell-wall targeting antibiotics, such as the β-lactam ampicillin. A distinct phenotype was observed upon deletion of croRS, including a high susceptibility towards β-lactams, low overall growth rate, lager cell size and a thinned cell wall. However, previous genome profiling studies exploring the CroRS regulon did not show the exact genes responsible for the CroRS dependent β-lactam resistance. To gain further insights into the mechanistic link between CroRS regulation and β-lactam resistance, we here used experimental evolution to restore ampicillin resistance in a croRS deletion strain. The four resulting strains exhibited a 10-fold increase in ampicillin resistance, which is equivalent to that of the wild type. Genome sequencing showed that the only mutations common to all evolved lines were in genes encoding enzymes responsible for the synthesis and degradation of the second messenger c-di-AMP. Consistent with this, deletion of croRS led to a marked increase in c-di-AMP levels, while the evolved strains exhibited a decrease in c-di-AMP levels similar to the wild type. This observation may begin to explain the sensitivity of the croRS-deleted strain, because in other Gram-positive bacteria excessive c-di-AMP production has been shown to coincide with β-lactam sensitivity. Curiously, in the ΔcroRS background single gene deletion of tow of the phosphodiesterases fully restores β-lactam resistance. These deletions also improved the overall growth rate of the croRS-deleted strain. While we do not yet know the mechanistic basis for this effect, our findings clearly show that a tight correlation between c-di-AMP concentration and β-lactam resistance exists in E. faecalis. These findings will help to improve our understanding of how CroRS regulation controls β-lactam tolerance in E. faecalis.