The second messenger c-di-AMP regulates osmotic and pH adaptation in Staphylococcus aureus under aerobic and anaerobic conditions

Gillis, Annika [UCL] Gründling, Angelika [Section of Microbiology and Medical Research Council Centre for Molecular Bacteriology and Infection, Imperial College London, London]

Cyclic nucleotides that act as second messenger molecules play key roles in signaling pathways involved in rapid responses to changing environments. Cyclic di-adenosine monophosphate (c-di-AMP) is a secondary messenger that is mainly produced by Gram-positive bacteria, including many human pathogens. Changes in the intracellular concentration of this signaling nucleotide has been implicated in diverse physiological roles including potassium ion transport, cell wall biosynthesis, biofilm formation and resistance to some antibiotics. In Staphylococcus aureus, c-di-AMP is synthesized from two molecules of ATP by a single diadenylate cyclase enzyme, namely DacA, and is degraded by two phosphodiesterase enzymes, called GdpP and Pde2, to linear 5’-phosphadenylyl-adenosine (pApA). Pde2 can further degrade pApA to two molecules of AMP. Recently, it was shown that DacA is required for the growth of S. aureus in rich media and under standard laboratory conditions, but it is dispensable for viability under anaerobiosis. Therefore, in this work we investigated the role of c-di-AMP in the aerobic and anaerobic growth of S. aureus under salt and pH stresses. The growth of the MRSA USA300 strain LAC* (WT) and several isogenic mutants for the dacA, gdpP and pde2 genes, was assessed in media containing increasing concentrations of either NaCl or KCl and pH raging from 4.5 to 10.5. We found that DacA is likely to be important for S. aureus viability under both aerobic and anaerobic conditions when growing in media with high salt concentrations (>2 M), particularly in NaCl. Unexpectedly, high levels of c-di-AMP seemed to be detrimental under anaerobic conditions in presence of elevated salt concentrations (>1.5 M), as both phosphodiesterase GdpP and Pde2 are essential for S. aureus viability under these conditions. Furthermore, GdpP and Pde2 are required for anaerobic growth when exposed to low pH. To investigate in more detail de contribution of c-di-AMP to the osmotic adaptation of S. aureus growing under aerobiosis or anaerobiosis, suppressor strains for LAC* dacA and LAC* gdpPpde2 were obtained with improved growth in 2M KCl under aerobic conditions and in 2M NaCl under anaerobiosis, respectively. Currently, a whole-genome-sequencing approach is being employed to further identify the mutations restoring the viability of the suppressor strains. Taken together, these findings indicate that changes in the concentration of the c-di-AMP, mediated by DacA, GdpP and Pde2 enzymes, are critical for osmotic and pH adaptation in S. aureus under both aerobic and anaerobic conditions.