c-di-AMP Is a New Second Messenger in Staphylococcus aureus with a Role in Controlling Cell Size and Envelope Stress Rebecca M. Corrigan 1 , James C. Abbott 2 , Heike Burhenne 3 , Volkhard Kaever 3 , Angelika Gru ¨ ndling 1 * 1 Section of Microbiology, Imperial College London, London, United Kingdom, 2 Department of Life Sciences, Imperial College London, London, United Kingdom, 3 Institute of Pharmacology, Hannover Medical School, Hannover, Germany 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 di adenylate 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. Citation: Corrigan RM, Abbott JC, Burhenne H, Kaever V, Gru ¨ ndling A (2011) c-di-AMP Is a New Second Messenger in Staphylococcus aureus with a Role in Controlling Cell Size and Envelope Stress. PLoS Pathog 7(9): e1002217. doi:10.1371/journal.ppat.1002217 Editor: Ambrose Cheung, Dartmouth Medical School, United States of America Received March 11, 2011; Accepted July 8, 2011; Published September 1, 2011 Copyright: ß 2011 Corrigan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Wellcome Trust grant WT084483, the Medical Research Council grant G071212 and the ‘‘LiSta-LTA’’ European Research Council grant 260371 to A.G. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: a.grundling@imperial.ac.uk Introduction Staphylococcus aureus is a very prevalent human pathogen that permanently colonizes the nares and skin of approximately 20% of the population, while another 60% are colonized transiently [1]. Infections caused by this pathogen are becoming increasingly more difficult to treat due to its resistance to antibiotic therapy. Where once methicillin was the antibiotic of choice, now only around 60% of S. aureus isolates remain sensitive to this drug. There has also been a rise in the number of community acquired methicillin resistant S. aureus (CA-MRSA) cases in recent years often resulting in severe skin and soft tissue infections as well as invasive diseases such as sepsis, necrotizing pneumonia or osteomyelitis [2,3]. The ability of S. aureus to cause such a wide range of diseases depends on many factors and is, in part, due to the diverse functions that are linked to its cell envelope. A myriad of proteins are embedded in this structure that allow bacteria to take up nutrients and adhere to diverse surfaces or niches within the human host. It also protects bacteria from environmental insults and at the same time allows the cells to sense and respond to changes in their surroundings, a function crucial for the survival of this pathogen in the host. In addition, the cell wall helps bacteria to maintain their shape and functions to counteract the high internal turgor pressure. Because the cell envelope has such essential functions, it also forms a weak point of the cell, as the inhibition of enzymes required for its synthesis is often lethal or leads to virulence defects. Therefore, this structure has been, and remains, an attractive target for therapeutic interventions. A typical cell wall of Gram-positive bacteria consists of proteins, peptidoglycan (PG) and the cell wall polymers wall teichoic acid (WTA), which is covalently linked to PG, and lipoteichoic acid (LTA), a polymer anchored to the outside of the bacterial membrane via a lipid moiety [4,5,6]. Synthesis of these cell wall components is highly coordinated and any mistakes can lead to cell lysis and death. From studies on the Gram-positive model organism Bacillus subtilis, it has emerged that PG and WTA synthesis enzymes form multi-protein complexes, which are further linked in this organism with cytoplasmic cell shape determining proteins, thereby coordinating and physically linking extracellular and intracellular synthesis processes [7,8,9,10]. PLoS Pathogens | www.plospathogens.org 1 September 2011 | Volume 7 | Issue 9 | e1002217