Cyclic di-AMP signalling affects cell division and penicillin susceptibility in Streptococcus pneumoniae.

Resistance to {beta}-lactam antibiotics in the human pathogen Streptococcus pneumoniae is mainly attributed to the acquisition of mutated versions of pbp2x, pbp2b and pbp1a, encoding functional penicillin binding proteins with reduced affinity to {beta}-lactams. This enables the bacterium to synthesise peptidoglycan required for cell division in the presence of higher {beta}-lactam concentrations. In addition, it has recently been shown that increased levels of the second messenger molecule cyclic di-AMP, resulting from mutations that compromise the cyclic di-AMP degrading phosphodiesterase Pde1, contribute to decreased {beta}-lactam susceptibility in this species. A link between cyclic di-AMP levels and beta-lactam resistance has also been reported in several other Gram-positive bacteria. Here, we further investigated this link in S. pneumoniae and present evidence supporting that cyclic di-AMP could be involved in regulation of cell division. We confirm that elevated cyclic di-AMP levels decreased penicillin susceptibility and cell size in pde1 mutants. Next, we show that low cyclic di-AMP levels had the opposite effects in which cells became more susceptible to penicillin and displayed an elongated morphology with multiple incomplete septa. Using fluorescence microscopy and bacterial two-hybrid assays, we found that proteins involved in cyclic di-AMP synthesis (CdaA, CdaR) and degradation (Pde1) were enriched around the division site and that they most probably interact with each other. Finally, we identified changes to the cell wall stem peptide composition of {Delta}pde1 cells. Together our findings indicate that cyclic di-AMP could function as a regulator of the division machinery.