Binding of Cyclic Di-AMP to the Staphylococcus aureus Sensor Kinase KdpD Occurs via the Universal Stress Protein Domain and Downregulates the Expression of the Kdp Potassium Transporter

Abstract

Nucleotide signaling molecules are important intracellular messengers that regulate a wide range of biological functions. The human pathogen Staphylococcus aureus produces the signaling nucleotide cyclic di-AMP (c-di-AMP). This molecule is common among Gram-positive bacteria and in many organisms is essential for survival under standard laboratory growth conditions. In this study, we investigated the interaction of c-di-AMP with the S. aureus KdpD protein. The sensor kinase KdpD forms a two-component signaling system with the response regulator KdpE and regulates the expression of the kdpDE genes and the kdpFABC operon coding for the Kdp potassium transporter components. Here we show that the S. aureus KdpD protein binds c-di-AMP specifically and with an affinity in the micromolar range through its universal stress protein (USP) domain. This domain is located within the N-terminal cytoplasmic region of KdpD, and amino acids of a conserved SXS-X20-FTAXY motif are important for this binding. We further show that KdpD2, a second KdpD protein found in some S. aureus strains, also binds c-di-AMP, and our bioinformatics analysis indicates that a subclass of KdpD proteins in c-di-AMP-producing bacteria has evolved to bind this signaling nucleotide. Finally, we show that c-di-AMP binding to KdpD inhibits the upregulation of the kdpFABC operon under salt stress, thus indicating that c-di-AMP is a negative regulator of potassium uptake in S. aureus.IMPORTANCE Staphylococcus aureus is an important human pathogen and a major cause of food poisoning in Western countries. A common method for food preservation is the use of salt to drive dehydration. This study sheds light on the regulation of potassium uptake in Staphylococcus aureus, an important aspect of this bacterium's ability to tolerate high levels of salt. We show that the signaling nucleotide c-di-AMP binds to a regulatory component of the Kdp potassium uptake system and that this binding has an inhibitory effect on the expression of the kdp genes encoding a potassium transporter. c-di-AMP binds to the USP domain of KdpD, thus providing for the first time evidence for the ability of such a domain to bind a cyclic dinucleotide.

New Insights into the Cyclic Di-adenosine Monophosphate (c-di-AMP) Degradation Pathway and the Requirement of the Cyclic Dinucleotide for Acid Stress Resistance in Staphylococcus aureus

“…These include expelling cytoplasmic protons through the F 1 F 0 -ATPase, producing alkaline compounds to neutralize the cytoplasmic pH, and importing osmolytes such as choline or proline (46, 47). Microarray data from S. aureus cultured in medium acidified to pH 4.5 revealed that besides genes encoding urease and proton efflux pumps, oxidative stress response genes and the kdp and opuC operons, encoding the c-di-AMP receptor proteins KdpD and OpuCA, are up-regulated (45, 48–51). Although an intriguing correlation, understanding the mechanism through which elevated c-di-AMP levels contributes to the acid tolerance in S. aureus or other microorganisms awaits future studies.…”

Cyclic di-AMP Released from Staphylococcus aureus Biofilm Induces a Macrophage Type I Interferon Response

“…However, the role of c-di-AMP in the major human pathogen S. aureus has been limited to mediating ion homeostasis by regulating channel activity (11,36,37). In this study, we investigated an extracellular role for c-di-AMP during S. aureus biofilm growth and regulation of macrophage activation and functional activity.…”

“…(29,30). Cytosolic c-di-AMP is sensed by both the endoplasmic reticulum resident protein stimulator of interferon genes (STING) (31-33) and the helicase DDX41 (34); however, the individual contributions of these proteins in sensing c-di-AMP remain unclear.While several intracellular proteins function as c-di-AMP receptors in S. aureus (11,(35)(36)(37)(38), the spatial and temporal production as well as any extracellular role for c-di-AMP during biofilm development has yet to be determined. Furthermore, the ability of extracellular c-di-AMP to trigger type I IFN production in macrophages has not yet been demonstrated and may serve as a potential mechanism to polarize macrophages to an anti-inflammatory state characteristic of biofilm infections (3,(5)(6)(7)(8).…”

Versatile modes of cellular regulation via cyclic dinucleotides

“…C-di-AMP has been shown to bind to the RCK_C domain of another S. aureus transporter, the cation-proton antiporter CpaA, and to the KdpD sensor histidine kinase, which typically controls the expression of another potassium uptake system, as well as several virulence factors in the pathogen 18,19 .…”

“…Interestingly, c-di-AMP can be both essential, which makes it unique among second messengers, and toxic, when overproduced 12–14 . Similarly to c-di-GMP, c-di-AMP controls a spectrum of cellular processes, including gene expression 15 , DNA repair 11 , cell wall synthesis 16 , metabolism 17 and potassium homeostasis (Figure 2) 12,18,19 .…”