Coordinated regulation of osmotic imbalance by c-di-AMP shapes ß-lactam tolerance in Group B Streptococcus
Streptococcus agalactiae is among the few pathogens that have not developed resistance to ß-lactam antibiotics despite decades of clinical use. The molecular basis of this long-lasting susceptibility has not been investigated, and it is uncertain whether specific mechanisms constraint the emergence of resistance. In this study, we first report a conserved role of the signaling nucleotide cyclic-di-AMP in the sensitivity of S. agalactiae to ß-lactam. Specifically, we demonstrate that inactivation of the phosphodiesterase GdpP confers ß-lactam tolerance. Characterizing the signaling pathway revealed an antagonistic regulation by the transcriptional factor BusR, which is activated by c-di-AMP and negatively regulates ß-lactam susceptibility. Furthermore, we show that simultaneous inhibition of osmolyte transporters activity and transcription by c-di-AMP has an additive effect, sustaining ß-lactam tolerance. Finally, transposon mutagenesis for ß-lactam reduced susceptibility reveals a convergent pattern of mutations, including in the KhpAB small RNA chaperone and the protein S immunomodulator. Overall, our findings suggest mechanisms that may foster antibiotic resistance in S. agalactiae and demonstrate that c-di-AMP acts as a turgor pressure rheostat, coordinating an integrated response to cell wall weakening due to ß-lactam activity. Overall design: To investigate the c-di-AMP signaling pathway in Group B Streptococcus, we generated deletion mutants for the c-di-AMP phosphodiesterase GdpP (?gdpP) and for the c-di-AMP regulated transcriptional repressor BusR (?busR) For comparative transcritomic analysis, ?gdpP and ?busR mutants were generated in two wild-type strains: NEM316 (capsular serotype III, CC-23) and BM110 (capsular serotype III, CC-17) RNA are purified from exponentialy growing cultures (OD600 = 0.5) in rich media (THY) incubated at 37°C in static condition. Biological triplicate (Replicate 1, 2, and 3) are done on different days.