c-di-AMP Is a New Second Messenger in Staphylococcus aureus with a Role in Controlling Cell Size and Envelope Stress

Abstract

The cell wall is a vital and multi-functional part of bacterial cells. For Staphylococcus aureus, an important human bacterial pathogen, surface proteins and cell wall polymers are essential for adhesion, colonization and during the infection process. One such cell wall polymer, lipoteichoic acid (LTA), is crucial for normal bacterial growth and cell division. Upon depletion of this polymer bacteria increase in size and a misplacement of division septa and eventual cell lysis is observed. In this work, we describe the isolation and characterization of LTA-deficient S. aureus suppressor strains that regained the ability to grow almost normally in the absence of this cell wall polymer. Using a whole genome sequencing approach, compensatory mutations were identified and revealed that mutations within one gene, gdpP (GGDEF domain protein containing phosphodiesterase), allow both laboratory and clinical isolates of S. aureus to grow without LTA. It was determined that GdpP has phosphodiesterase activity in vitro and uses the cyclic dinucleotide c-di-AMP as a substrate. Furthermore, we show for the first time that c-di-AMP is produced in S. aureus presumably by the S. aureus DacA protein, which has diadenylate cyclase activity. We also demonstrate that GdpP functions in vivo as a c-di-AMP-specific phosphodiesterase, as intracellular c-di-AMP levels increase drastically in gdpP deletion strains and in an LTA-deficient suppressor strain. An increased amount of cross-linked peptidoglycan was observed in the gdpP mutant strain, a cell wall alteration that could help bacteria compensate for the lack of LTA. Lastly, microscopic analysis of wild-type and gdpP mutant strains revealed a 13–22% reduction in the cell size of bacteria with increased c-di-AMP levels. Taken together, these data suggest a function for this novel secondary messenger in controlling cell size of S. aureus and in helping bacteria to cope with extreme membrane and cell wall stress.

Systematic identification of conserved bacterial c-di-AMP receptor proteins

“…Cultures were grown to an OD 600 of 0.5-0.7, protein expression induced with 0.5 mM IPTG, and incubated overnight at 16°C. Protein purifications were performed by nickel affinity and size exclusion chromatography as previously described (11,39). Protein concentrations were determined by A 280 readings.…”

“…Many Gram-positive bacteria, including the important human pathogens Staphylococcus aureus (11), Streptococcus pyogenes (12), Listeria monocytogenes (5), and Mycobacterium tuberculosis (13), produce c-di-AMP, and it is likely that c-di-AMP is also synthesized by several Gram-negative bacteria and a subset of archaea (14). c-di-AMP is synthesized by DisA_N domain-containing diadenylate cyclases DacA, DisA, and YojJ and degraded by the phosphodiesterase enzyme GdpP (4,5,11,(15)(16)(17)(18).…”

“…c-di-AMP is synthesized by DisA_N domain-containing diadenylate cyclases DacA, DisA, and YojJ and degraded by the phosphodiesterase enzyme GdpP (4,5,11,(15)(16)(17)(18). A variety of different phenotypes have been linked to altered c-di-AMP levels; an increase in c-di-AMP levels correlates with increased acid resistance (16,19) and altered antibiotic resistance, including an increase in methicillin resistance in S. aureus (11,18,20). Most notable, however, are the findings that L. monocytogenes (5) and Bacillus subtilis (18) cannot grow in the absence of c-di-AMP, showing that in contrast to other signaling nucleotides, c-di-AMP controls essential cellular pathways.…”

Genome-wide screen for genes involved in eDNA release during biofilm formation by Staphylococcus aureus

“…Disruption of any of these three genes would lead to increased de novo nucleotide synthesis. We also identified another gene involved in purine nucleotide metabolism, gdpP, which encodes a phosphodiesterase that cleaves the second messenger cyclic-di-AMP (15). Additionally, members of the pyruvate dehydrogenase complex pdhA, pdhB, and pdhC were identified, disruption of which could lead to an increase in pyruvate metabolism by pyruvate formate lyase (Pfl), thereby increasing cellular formate levels.…”

Small molecule inhibitor of lipoteichoic acid synthesis is an antibiotic for Gram-positive bacteria

“…Characterization of LtaS in Bacillus anthracis, Bacillus subtilis, and Listeria monocytogenes confirmed that membrane proteins with pfam00884 domains are indeed responsible for the synthesis of polyglycerol-phosphate LTA (7). Where examined, ltaS mutants exhibited diminished viability, increased cell size, and altered morphology (16)(17)(18)(19). These results suggested that LtaS of Gram-positive bacteria may represent an extracellular target for the development of antibiotics against drugresistant Gram-positive bacteria.…”