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Washington University School of Medicine
Digital Commons@Becker

Open Access Publications

2017

c-di-AMP: An essential molecule in the signaling
pathways that regulate the viability and virulence of
gram-positive bacteria
Tazin Fahmi
Indiana State University

Gary C. Port
Washington University School of Medicine in St. Louis

Kyu Hong Cho
Indiana State University

Follow this and additional works at: https://digitalcommons.wustl.edu/open_access_pubs

Recommended Citation
Fahmi, Tazin; Port, Gary C.; and Cho, Kyu Hong, ,”c-di-AMP: An essential molecule in the signaling pathways that regulate the
viability and virulence of gram-positive bacteria.” Genes.8,8. 197. (2017).
https://digitalcommons.wustl.edu/open_access_pubs/6076

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Review
c-di-AMP: An Essential Molecule in the Signaling
Pathways that Regulate the Viability and Virulence of
Gram-Positive Bacteria

Tazin Fahmi 1, Gary C. Port 2,3 and Kyu Hong Cho 1,* ID

1 Department of Biology, Indiana State University, Terre Haute, IN 47809, USA;

tfahmi@sycamores.indstate.edu

2 Department of Molecular Microbiology, Washington University School of Medicine, Saint Louis, MO 63110,

USA; garyport@gmail.com
Elanco Animal Health, Natural Products Fermentation, Eli Lilly and Company, Indianapolis, IN 46285, USA

3

* Correspondence: kyuhong.cho@indstate.edu; Tel.: +1-812-237-2412

Academic Editor: Helen J. Wing
Received: 26 June 2017; Accepted: 31 July 2017; Published: 7 August 2017

Abstract: Signal transduction pathways enable organisms to monitor their external environment
and adjust gene regulation to appropriately modify their cellular processes. Second messenger
nucleotides including cyclic adenosine monophosphate (c-AMP), cyclic guanosine monophosphate
(c-GMP), cyclic di-guanosine monophosphate (c-di-GMP), and cyclic di-adenosine monophosphate
(c-di-AMP) play key roles in many signal transduction pathways used by prokaryotes and/or
eukaryotes. Among the various second messenger nucleotides molecules, c-di-AMP was discovered
recently and has since been shown to be involved in cell growth, survival, and regulation of
virulence, primarily within Gram-positive bacteria. The cellular level of c-di-AMP is maintained
by a family of c-di-AMP synthesizing enzymes, diadenylate cyclases (DACs), and degradation
enzymes, phosphodiesterases (PDEs). Genetic manipulation of DACs and PDEs have demonstrated
that alteration of c-di-AMP levels impacts both growth and virulence of microorganisms. Unlike
other second messenger molecules, c-di-AMP is essential for growth in several bacterial species as
many basic cellular functions are regulated by c-di-AMP including cell wall maintenance, potassium
ion homeostasis, DNA damage repair, etc. c-di-AMP follows a typical second messenger signaling
pathway, beginning with binding to receptor molecules to subsequent regulation of downstream
cellular processes. While c-di-AMP binds to specific proteins that regulate pathways in bacterial cells,
c-di-AMP also binds to regulatory RNA molecules that control potassium ion channel expression
in Bacillus subtilis. c-di-AMP signaling also occurs in eukaryotes, as bacterially produced c-di-AMP
stimulates host immune responses during infection through binding of innate immune surveillance
proteins. Due to its existence in diverse microorganisms, its involvement in crucial cellular activities,
and its stimulating activity in host immune responses, c-di-AMP signaling pathway has become an
attractive antimicrobial drug target and therefore has been the focus of intensive study in several
important pathogens.

Keywords:
c-di-AMP-binding proteins

c-di-AMP; Gram-positive bacteria;

c-di-AMP synthesis and degradation;

1. Introduction

Living organisms receive and process environmental stimuli through signal transduction
pathways and respond through differential regulation of various cellular processes. Cyclic nucleotides
that act as second messenger molecules play key roles in signaling pathways that sense environmental

Genes 2017, 8, 197; doi:10.3390/genes8080197

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changes such as stress, temperature, nutrition, and pH in both prokaryotes and eukaryotes [1–3].
As second messengers, these cyclic nucleotides are involved in the transmission of the signals to
effector molecules [1,4].

The original second messenger molecule discovered was cyclic adenosine monophosphate
(cAMP) through its role in metabolic pathways of eukaryotes, specifically, mammalian hormonal
regulation [5,6]. Since its initial discovery, cAMP has been found to act as a second messenger in a
variety of organisms including many bacterial species and has diverse activity including roles in biofilm
formation, expression of virulence factors, motility, and carbon metabolism [7]. Subsequently, several
additional cyclic nucleotide second messengers have been discovered including cyclic guanosine
monophosphate (cGMP), cyclic di-guanosine monophosphate (c-di-GMP), cyclic di-adenosine
monophosphate (c-di-AMP), and cyclic guanosine monophosphate-adenosine monophosphate
(cGAMP) in a wide variety of organisms [2,8,9]. cGMP and c-di-GMP have been well characterized,
particularly in Gram-negative bacteria. cGMP is involved in chemotaxis and UV stress-response, while
c-di-GMP facilitates the transition from motile phase to adhesive phase and the expression of fimbriae
in bacteria [3].

c-di-AMP is a new addition to the growing list of second messenger nucleotides and has
since been identified in Gram-positive bacteria including Listeria monocytogenes, Bacillus subtilis,
Staphylococcus aureus, and Streptococcus spp., and in a few Gram-negative bacteria including
Chlamydia trachomatis and Borrelia burgdorferi [1,10–16]. c-di-AMP has been implicated in diverse
essential cellular processes including cell wall and membrane homeostasis, regulation of potassium ion
channels, DNA damage repair, and sporulation (Table 1). Though c-di-AMP has been shown to play a
critical role in many human pathogenic bacteria, neither its environmental stimuli nor the mechanisms
controlling the regulation of cellular physiology and virulence are well understood [15,17].

Table 1. The function of cyclic di-adenosine monophosphate (c-di-AMP) and its synthesis and
degradation enzymes in bacteria.

Bacterium

Function of c-di-AMP

c-di-AMP
Synthesis
Enzyme

c-di-AMP
Degrading
Enzyme

Phenotype Involved in
an Altered Level of
c-di-AMP

Ref.

Bacillus subtilis

DisA, CdaA,
and CdaS

GdpP and
PgpH

DisA binds to DNA and
maintains DNA integrity.
CdaS regulates
sporulation. CdaA
regulates cell wall
synthesis and ion
channel homeostasis.

Listeria
monocytogenes

Regulates cell wall
homeostasis, resistance
to acid, and carbon
metabolism.

CdaA (DacA)

PdeA (GdpP
homolog) and
PgpH

DisA mutation: decreased
DNA integrity CdaA
mutation: impaired
potassium ion channel
system, weakened cell
wall, increased resistance
to antibiotics CdaS
mutation: delayed
sporulation.

Phosphodiesterase (PDE
mutation: cell wall defects,
increased resistance to
antibiotics, low survival
rate, sensitivity towards
acid stress, altered
interferon-ß stimulation in
host cells.

[17–20]

[21]

Mycobacterium
tuberculosis

Functions are not fully
understood yet, but
DisA is predicted to be
involved in DNA repair.

MtbDisA (DisA
ortholog)

MtbPDE (Pde2
ortholog)

PDE mutation: reduced
virulence.

[22]

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Table 1. Cont.

Bacterium

Function of c-di-AMP

c-di-AMP
Synthesis
Enzyme

c-di-AMP
Degrading
Enzyme

Phenotype Involved in
an Altered Level of
c-di-AMP

Staphylococcus
aureus

Regulates cell wall
synthesis, cell size, and
potassium ion channel
homeostasis.

CdaA

GdpP and Pde2
ortholog

Streptococcus
mutans

Regulates biofilm
formation by binding to
receptor proteins.

CdaA

PdeA (GdpP
ortholog) and
Pde2

Streptococcus
pneumoniae

Maintains potassium ion
channel homeostasis.

CdaA

GdpP and Pde2

Streptococcus
pyogenes

Streptococcus
suis (SS2)

Mycoplasma
pneumoniae

Regulates cell wall
homeostasis and
virulence gene
expression.

Promotes biofilm
formation and increases
virulence.

Predicted to regulate
potassium import
through binding of KtrC.

CdaA
(SpyDacA)

GdpP and Pde2
ortholog

CdaA

GdpP and Pde2
ortholog

CdaM

PdeM

gdpP deletion: smaller cell
size, increased
peptidoglycan
cross-linking, increased
resistance against cell wall
and membrane targeting
antibiotics, impaired
potassium ion channel
system.

cda deletion: Increased
sensitivity to hydrogen
peroxide and enhanced
polysaccharide synthesis.
pdeA deletion: Increased
biofilm formation.

PDE mutation: Impaired
ability of long chain
formation, decreased
growth, and imbalance in
the potassium ion channel.

gdpP deletion: Impaired
biogenesis of SpeB,
decreased virulence and
increased antibiotic
resistance.

gdpP deletion: Reduced
growth and reduced
biofilm formation.

cdaM and pdeM: essential
for growth.

[23,24]

Ref.

[17]

[25]

[26]

[27]

[28]

c-di-AMP was first identified in a study of the DNA repair mechanism in Thermotoga maritima [29],
and later, the same role of c-di-AMP in DNA repair was identified in B. subtilis [18,29]. In a separate line
of research, c-di-AMP was isolated from the cytosol of L. monocytogenes-infected host cells that were
producing elevated levels of interferon β [16]. Interferon β is secreted by immune cells in response to
infection by microorganisms, and it induces the innate immune response in the host. The correlation
between the presence of c-di-AMP and the higher level of interferon β expression indicated that
c-di-AMP plays a role in eliciting an immune response in the infected host. Because many human
pathogenic Gram-positive bacteria such as Mycobacterium tuberculosis, Streptococcus pyogenes, S. aureus,
and L. monocytogenes produce c-di-AMP, the signaling pathway has recently become an attractive drug
target [30]. c-di-AMP is essential for the growth of many Gram-positive bacteria such as S. aureus,
L. monocytogenes and B. subtilis, so complete depletion of c-di-AMP by deleting the diadenylate cyclase
genes in those bacteria leads to the lethal phenotype under standard lab growth conditions unless a
special medium is provided, or a suppressor mutation develops [31,32]. On the contrary, the deletion
of the diadenylate cyclase gene in Streptococcus mutans, cdaA was not lethal, suggesting that some
bacteria can survive in the absence of c-di-AMP [24].

Interestingly, although some bacteria such as B. subtilis and L. monocytogenes produce both
c-di-AMP and c-di-GMP, S. aureus and S. pyogenes are unable to synthesize c-di-GMP since they
lack c-di-GMP synthesizing enzymes [26]. Although most second messenger molecules utilize similar
mechanisms in their signaling pathways, their contributions to cell physiology and function differ
greatly. Each of the second messenger nucleotides bind to different sets of proteins or RNA molecules,
which thereby regulate distinct cellular processes [17]. c-di-AMP works in the signaling pathway in

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a manner similar to other second messenger molecules such as c-di-GMP, cAMP, and cGMP, but the
environmental stimuli and detailed mechanisms are not yet known.

2. Synthesis of c-di-AMP

In contrast to the variety of GGDEF domain-containing proteins that synthesize c-di-GMP [33],
only a few c-di-AMP synthesizing enzymes have thus far been discovered in bacteria and
archaea. These c-di-AMP synthesizing enzymes are found primarily in Gram-positive Firmicutes
and Actinobacteria but are also present in some Gram-negative bacteria including Bacteroidetes,
Deltaproteobacteria, and Cyanobacteria [3]. Moreover, the signaling pathways for c-di-GMP and
c-di-AMP rarely co-exist in the same organisms. For example, Staphylococcus, Streptococcus, and
Corynebacterium species do not contain functional c-di-GMP synthesizing enzymes [17,34,35]. Notable
exceptions to this rule include Bacillus, Clostridium, Listeria, Mycobacterium, and Streptomyces, which
produce both signaling molecules [3,32].

c-di-AMP is synthesized from ATP or ADP by cyclase domain-containing proteins known as
diadenylate cyclases (DACs) (Figure 1). DAC enzymes catalyze the synthesis of a single molecule
of c-di-AMP from two molecules of ATP or ADP through a condensation reaction [3,14,32,34,35].
Structural and functional analysis of individual DAC domains and operons have enhanced the current
understanding of the functions of the various classes of these proteins [32,35]. The DAC domain
was first identified by X-ray crystallography in DisA (DNA integrity scanning protein A), which
functions as a DNA check point protein scanning the chromosome for lesions [18,29]. Later, DAC
domain-containing proteins were found in many bacterial and archaeal species [34]. Four classes
of DACs have been identified: DisA, CdaA, CdaS, and CdaM. While most organisms contain only
one type of DAC, some contain multiple enzymes including Clostridium spp., which contain two
types of DACs (CdaA and DisA) and B.subtilis, which has DisA, CdaA, and CdaS [36]. Several
Gram-positive human pathogenic bacteria including S. pyogenes, S. pneumoniae, and L. monocytogenes
encode CdaA [14,16,25] while M. tuberculosis expresses MtDisA, a DisA homolog [11,37]. Previous
studies have demonstrated that DAC mutant strains display altered physiologies such as loss of
resistance to heat, salt, and DNA-damaging molecules due to the synthesis of a weak cell wall, making
the bacteria vulnerable to its environment [15,32]. The impact of DAC on numerous aspects of cell
physiology highlights the essential nature of DACs in bacteria [15,19,29,32].

All DAC domain proteins possess conserved motifs, most commonly DGA and RHR motifs, which
perform the cyclase activity in the c-di-AMP synthesis reactions [38,39]. However, these domains do not
share any structural or amino acid similarities with the GGDEF domain found in c-di-GMP-catalyzing
enzymes suggesting these signaling pathways evolved independently [40]. Structural analysis of the
three types of DACs have demonstrated that the DAC domains in CdaA and CdaS share 40% amino
acid identity, while the DAC domain in the DisA protein is more distantly related, sharing only 19%
identity with CdaA and CdaS [32,34]. The association of neighboring genes with each class of DAC
points to the physiological role of the c-di-AMP. For example, the association of cdaA with a gene
that encodes an enzyme synthesizing a cell wall building block (glmM) suggests a role for CdaA and
c-di-AMP in the maintenance of cell wall homeostasis [32,34].

2.1. DisA

DisA, crystalized from Thermotoga maritima, was the first protein with a DAC domain to be
characterized [29]. DisA is found in both Gram-negative and Gram-positive bacteria but is particularly
prevalent in Gram-positive spore-forming bacteria such as Bacillus and Clostridium species. X-ray
crystallography revealed that the DAC domains of two tetrameric DisA molecules interact to
form a stable octameric structure. The direct contact of DAC domains is necessary for c-di-AMP
production [29]. The carboxyl end of DisA enzyme contains a DNA binding domain, and the amino
terminal end possesses a globular domain with catalytic activity. These two domains are connected by
a helical domain [18]. RadA, a DNA repair protein in B. subtilis, is encoded together with DisA in a

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conserved operon, and their close genetic proximity suggests the involvement of DisA in maintaining
DNA integrity [18,32]. Indeed, DisA mutant strains are less able to repair damaged DNA than the
wild type [32,34]. It turned out that DisA is a checkpoint protein that directly regulates DNA repair
mechanisms by responding to DNA damage [18]. During sporulation in B. subtilis, DisA scans DNA
and detects damage of chromosomal breaks via its DNA-binding domain, thereby signaling a stop
in the sporulation process [17,18]. After repairing the DNA damage, c-di-AMP signals to restart the
sporulation pathway [18]. Both high and low c-di-AMP levels impair the DNA binding activity of
DisA [18].

2.2. CdaA

Among the three DACs, CdaA, sometimes referred to as DacA, is the most common as it is
found in a wide variety of bacteria including notable human pathogens S. aureus, S. pneumoniae,
S. pyogenes, and L. monocytogenes [17,39]. c-di-AMP produced by CdaA has been shown to be involved
in maintaining cell wall homeostasis as well as controlling potassium ion channel activity [32]. The cdaA
gene is often located immediately upstream of the gene encoding GlmM, a critical peptidoglycan
biosynthetic enzyme [32,41]. Bacteria that rely solely upon CdaA for c-di-AMP production develop
weakened cell walls, which manifests as a gain in antibiotic resistance when c-di-AMP levels are
altered. Furthermore, alterations in c-di-AMP production results in reduced potassium ion channel
activity (see KtrA below) [41,42]. In B. subtilis, CdaA directly regulates cell wall homeostasis through
interaction with GlmM [38]. The CdaA encoding operon also encodes a regulatory protein CdaR,
and the interaction of CdaA and CdaR regulates potassium ion channel activity [19,38]. Due to its
crucial role in multiple physiological processes, the deletion of the cdaA gene is often unsuccessful or
only possible under certain circumstances (acquisition of secondary suppressor mutations or specific
growth conditions), thus making CdaA an attractive novel target for antibacterial drugs.

The third type of DAC, CdaS, is found only in the spore-forming Bacillus species and one
Clostridium species [32] and is expressed exclusively during spore germination [19]. CdaS-mutant
strains have a two-fold decreased germination rate compared with wild-type strains, but the
mechanism of CdaS-dependent regulation of sporulation has yet to be revealed. CdaS contains two
N-terminal α-helices that are linked to the C-terminal DAC domain [19]. CdaS forms a hexamer that
displays relatively low catalytic activity, but a truncated CdaS engineered to lack one or both helices
results in a monomer that is hyper-active in c-di-AMP production, indicating that the N-terminal
helices are involved in hexamer formation as well as regulation of enzymatic activity.

2.3. CdaS

2.4. CdaM

The fourth class of c-di-AMP synthesizing enzyme, CdaM, has been recently identified in
Mycoplasma pneumoniae through pull-down assay [28]. CdaM only exists in M. pneumoniae, and
it is closely related to the DAC domain of CdaS present in B. subtilis [28]. CdaM mutant strains were
unable to grow, indicating that c-di-AMP is essential for the survival of M. pneumoniae, [28]. In this
bacterium, c-di-AMP also regulates potassium ion transportation. c-di-AMP binds to KtrC, and this
interaction interrupts the activity of the low-affinity potassium ion transporter, KtrCD [28].

Genes 2017, 8, 197

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Figure 1. Synthesis and degradation of cyclic di-adenosine monophosphate (c-di-AMP). Diadenylate
cyclase (DAC) enzymes synthesize c-di-AMP through a condensation reaction of two ATP or two
ADP molecules. c-di-AMP binds to specific target proteins, thereby regulating the functions of
downstream proteins within a variety of cellular pathways. To maintain appropriate levels of c-di-AMP,
phosphodiesterases (PDEs) degrade c-di-AMP into pApA, which further degrades into AMP [22,43].

3. c-di-AMP Degradation

Bacteria utilize both synthesis and degradation enzymes to regulate the cellular level of second
messenger molecules. The c-di-AMP hydrolyzing enzyme phosphodiesterase (PDE) was first identified
in B. subtilis and was subsequently found in S. aureus, L. monocytogenes, and Streptococcus species [44,45].
PDE enzymes degrade c-di-AMP, converting it into the linear form of phosphoadenyl adenosine
(pApA), which can then be further degraded into two molecules of AMP [22,43]. Three classes of PDE
are involved in c-di-AMP degradation; GdpP, Pde2 and PgpH [44,45]. The presence of each class of
PDE varies by bacterial species. Some bacteria, such as L. monocytogenes, encode GdpP and PgpH
whereas others, such as Streptococcus and Staphylococcus species, possess GdpP and Pde2 [44]. The PDE
enzymes appear to be stimulated by internal and external stimuli, but the specific stimuli and detailed
mechanisms are not yet known.

3.1. GdpP

The best-characterized PDE, GdpP, contains the catalytic DHH/DHHA1 domain (DHH stands
for Asp-His-His) [44,45]. This domain is primarily found in phosphatases and phosphodiesterases that
regulate the phosphorylation of proteins and breakdown of phosphodiester bonds, respectively [35].
The DHH/DHHA1 domain in GdpP cleaves c-di-AMP into pApA [45]. GdpP is highly specific for
c-di-AMP and has been observed to have only very weak enzymatic activity toward c-di-GMP [16,34,45].
GdpP and GdpP homologs have been identified in most microorganisms that produce c-di-AMP thus
far [45]. The operon containing the gdpP gene appears to be evolutionarily conserved amongst all
GdpP producing bacteria as gdpP is co-transcribed with the genes of ribosomal protein L9 (RpL9)
and a DNA replication protein, DnaC [36,46]. Thus, the co-expression of GdpP, RpL9, and DnaC
likely leads to a decrease in the c-di-AMP level during cell growth and replication [36,46]. GdpP also
possesses a PAS (Per-Arnt-Sim) domain that is involved in phosphodiesterase inhibiting activity. PAS
domains are present in many signaling protein molecules and play critical roles as sensory domains in
signal transduction pathways [45]. The PAS domain in GdpP can bind to heme, which inhibits the

Genes 2017, 8, 197 5 of 16 c-di-AMP levels are altered. Furthermore, alterations in c-di-AMP production results in reduced potassium ion channel activity (see KtrA below) [41,42]. In B. subtilis, CdaA directly regulates cell wall homeostasis through interaction with GlmM [38]. The CdaA encoding operon also encodes a regulatory protein CdaR, and the interaction of CdaA and CdaR regulates potassium ion channel activity [19,38]. Due to its crucial role in multiple physiological processes, the deletion of the cdaA gene is often unsuccessful or only possible under certain circumstances (acquisition of secondary suppressor mutations or specific growth conditions), thus making CdaA an attractive novel target for antibacterial drugs. 2.3. CdaS The third type of DAC, CdaS, is found only in the spore-forming Bacillus species and one Clostridium species [32] and is expressed exclusively during spore germination [19]. CdaS-mutant strains have a two-fold decreased germination rate compared with wild-type strains, but the mechanism of CdaS-dependent regulation of sporulation has yet to be revealed. CdaS contains two N-terminal α-helices that are linked to the C-terminal DAC domain [19]. CdaS forms a hexamer that displays relatively low catalytic activity, but a truncated CdaS engineered to lack one or both helices results in a monomer that is hyper-active in c-di-AMP production, indicating that the N-terminal helices are involved in hexamer formation as well as regulation of enzymatic activity. 2.4. CdaM The fourth class of c-di-AMP synthesizing enzyme, CdaM, has been recently identified in Mycoplasma pneumoniae through pull-down assay [28]. CdaM only exists in M. pneumoniae, and it is closely related to the DAC domain of CdaS present in B. subtilis [28]. CdaM mutant strains were unable to grow, indicating that c-di-AMP is essential for the survival of M. pneumoniae, [28]. In this bacterium, c-di-AMP also regulates potassium ion transportation. c-di-AMP binds to KtrC, and this interaction interrupts the activity of the low-affinity potassium ion transporter, KtrCD [28]. Figure 1. Synthesis and degradation of cyclic di-adenosine monophosphate (c-di-AMP). Diadenylate cyclase (DAC) enzymes synthesize c-di-AMP through a condensation reaction of two ATP or two ADP molecules. c-di-AMP binds to specific target proteins, thereby regulating the functions of downstream proteins within a variety of cellular pathways. To maintain appropriate levels of c-di-AMP, phosphodiesterases (PDEs) degrade c-di-AMP into pApA, which further degrades into AMP [22,43]. Genes 2017, 8, 197

7 of 17

enzymatic activity of GdpP [47]. GdpP also contains a degenerated c-di-GMP synthesizing domain,
GGDEF. However, this domain is not able to synthesize c-di-GMP, but, rather, is involved in the
degradation of ATP [45]. A mutation in the GGDEF domain causes reduced catalytic activity of GdpP,
thus demonstrating a role of the GGDEF domain in the regulation of GdpP catalytic activity [45].

3.2. PgpH

Another class of phosphodiesterase, PgpH, was first identified in L. monocytogenes, and now
appears to be widespread throughout multiple bacterial phyla [44,48]. PgpH possesses a catalytic
histidine-aspartate domain (HD) in its C-terminus that binds to c-di-AMP with high specificity and
degrades it into 5’-pApA. HD is composed of two subdomains and two active sites that require iron
for their catalytic activity [44]. In addition to an HD domain, PgpH also possesses an N-terminal
extracellular domain and seven transmembrane helices [44,48]. In L. monocytogenes, PgpH and PdeA,
a homolog of GdpP, regulate the intracellular level of c-di-AMP in a cooperative manner. However,
these enzymes are regulated by different external stimuli. PgpH expresses preferably in broth culture
while PdeA is preferentially expressed during intracellular infection of eukaryotic host cells [44].
L. monocytogenes mutants lacking either PgpH or PdeA exhibit slight growth defects compared to the
wild type. However, mutants lacking both pdeA and pgpH exhibit higher levels of c-di-AMP, which is
detrimental to bacterial growth and contributes to reduced virulence of L. monocytogenes in a mouse
model of infection, indicating the cooperative c-di-AMP degradative activity of these two enzymes [44].
The double mutant also elicits increased interferon (IFN) – β during intracellular infection of host
cells [48]. Upon infection, L. monocytogenes induces the expression of β -interferon and co-regulated
genes following stimulation of the cytosolic surveillance pathway (CSP), STING, and DDX41 pathways
in the host immune system [21]. In a screen designed to determine the Pathogen Associated Molecular
Patterns (PAMPs) recognized by the infected mammalian cells, c-di-AMP was identified [16]. Indeed,
bacterial mutants that secrete more c-di-AMP lead to an increase in β-interferon secretion [21].

3.3. Pde2

Pde2 is a recently discovered c-di-AMP degrading enzyme containing a DHH/DHHA1 domain. It
was first identified in S. pneumoniae [43,49] and its enzymatic activity and structures have been studied
in S. aureus [50], Mycobacterium spp. [51–53], and Borrelia burgdoferi [54]. M. pneumoniae, B. burgdorferi,
and M. tuberculosis contain only PDEs of the Pde2 type. Pde2 is a cytoplasmic protein that can degrade
c-di-AMP but preferentially hydrolyzes linear pApA to AMP as demonstrated in S. aureus [43,50].
Deletion of the pde2 gene leads to a rapid increase of intracellular pApA levels compared to the
wild type. pApA and c-di-AMP concentrations are interconnected as pApA inhibits the c-di-AMP
hydrolyzing activity of GdpP [50]. Thus, Pde2 plays a key role in maintaining the homeostasis of
intracellular pApA and c-di-AMP levels that are crucial for the growth and survival of bacteria.

4. c-di-AMP Binding Molecules

Since the diverse roles of c-di-AMP in cell signaling pathways depend upon its binding to target
molecules, the investigation into the identification, structure, and function of c-di-AMP binding
molecules is important to reveal the mechanisms of c-di-AMP activity in various signaling pathways.
However, unlike c-di-GMP for which hundreds of binding proteins have been identified [32,33,55],
a few c-di-AMP binding proteins have been discovered so far in bacteria (Table 2) [17]. Multiple
strategies have been successfully employed to identify c-di-AMP binding proteins including
UV-crosslinking of 32P-labelled c-di-AMP with a library of purified proteins [55]. However, the
most common strategy involves the use of c-di-AMP affinity columns to isolate binding proteins
from bacterial cytoplasmic extracts followed by gel electrophoresis and mass-spectroscopy [17,25,48].
c-di-AMP binding is typically confirmed via standard affinity assays such as surface plasmon
resonance (SPR) or pulse chase analysis [55], or more recently through an adaption of the differential
radial capillary action of ligand assay (DRaCALA) first used to study c-di-GMP binding [17,48].