Calcium Regulation of Bacterial Virulence

Abstract

Calcium (Ca²⁺) is a universal signaling ion, whose major informational role shaped the evolution of signaling pathways, enabling cellular communications and responsiveness to both the intracellular and extracellular environments. Elaborate Ca²⁺ regulatory networks have been well characterized in eukaryotic cells, where Ca²⁺ regulates a number of essential cellular processes, ranging from cell division, transport and motility, to apoptosis and pathogenesis. However, in bacteria, the knowledge on Ca²⁺ signaling is still fragmentary. This is complicated by the large variability of environments that bacteria inhabit with diverse levels of Ca²⁺. Yet another complication arises when bacterial pathogens invade a host and become exposed to different levels of Ca²⁺ that (1) are tightly regulated by the host, (2) control host defenses including immune responses to bacterial infections, and (3) become impaired during diseases. The invading pathogens evolved to recognize and respond to the host Ca²⁺, triggering the molecular mechanisms of adhesion, biofilm formation, host cellular damage, and host-defense resistance, processes enabling the development of persistent infections. In this review, we discuss: (1) Ca²⁺ as a determinant of a host environment for invading bacterial pathogens, (2) the role of Ca²⁺ in regulating main events of host colonization and bacterial virulence, and (3) the molecular mechanisms of Ca²⁺ signaling in bacterial pathogens.

33.1. Elevated External Calcium (Ca²⁺) Regulates Adaptation of Bacterial Pathogens to Their Host Environment

33.1.1. Host-Associated Ca²⁺

In order to survive, bacteria must sense the chemical landscape of their environment and respond to it by adjusting their biological activities. Bacterial pathogens have an additional challenge of recognizing the transition between outside and inside the host and efficiently rearranging their gene expression to enable survival in the hostile host. The environment inside the host has a drastically different chemistry regulated by complex signaling systems, including one of the most versatile intracellular messengers, calcium (Ca²⁺).

Ca²⁺ signaling has been widely studied in eukaryotes [1, 2]. Ca²⁺ signaling is based on tightly regulated fluctuations in the levels of the ion in different cellular compartments, that trigger multiple molecular pathways. Whereas the cytoplasmic concentration of free Ca²⁺ is maintained at high nanomolar level, the extracellular concentration of the ion reaches millimolar levels [3–5] differing between different body fluids, tissues, and organs. Several examples are summarized in Table 33.1.

Table 33.1.

Examples of free Ca²⁺ levels in human body fluids

Body fluid	[Ca2+]	References
Joint fluids	4 mM	[26]
Plasma	1.3–1.5 mM	[23, 27–29]
Serum	0.7 to 1.4 mM.	[23, 30–34]
Saliva (in CF patients)	0.3 mM (4.8 ± 0.7 mM)	[35–1]
Nasal secretions (in CF patients)	3.1 ± 1.6 mM (4.7 ± 2.2 mM)	[42–4]
Sputum (in CF patients)	l.l mM (2.5mM)	[11]
Urine	1.6–5 mM	[45]

Since Ca²⁺ signaling regulates most essential cellular processes, slight abnormalities in Ca²⁺ homeostasis cause diseases or are a result of certain pathologies. For example, in cystic fibrosis (CF) [6, 7], different types of cells, including skin fibroblasts and bronchial epithelium cells, show elevated intracellular Ca²⁺ pools [8, 9]. In addition, abnormally elevated levels of Ca²⁺ were registered in multiple body fluids of CF patients (Table 33.1). Further, the elevation of cytosolic Ca²⁺ concentration was shown to trigger host immune responses against invading pathogens. For example, intestinal epithelial cells infected with Salmonella serotype Typhimurium require an increased cytosolic Ca²⁺ to express pro-inflammatory chemokine IL-8 [10]. Elevated Ca²⁺ in CF sputum positively correlates with the release of IL-8 in the necrotic immune cells [11]. As a part of the innate immunity defense, production of antimicrobial peptides (AMPs) by epidermal keratinocytes in response to infection by Pseudomonas aeruginosa, Staphylococcus aureus and other pathogens is induced by elevated levels of Ca²⁺ [12]. Some of the AMPs, including a family of Ca²⁺ binding EF-hand S100 family, require Ca²⁺ for their interactions with targets [13].

Some bacterial pathogens are able to alter the hosts [Ca²⁺]_in levels through activating Ca²⁺ flux across the plasma membrane and, releasing Ca²⁺ from the intracellular stores into the cytosol [10, 14–17]. These interactions can be mediated by bacterial surface associated proteins such as PilC of Neisseria meningitidis [17], FliC of P. aeruginosa and Salmonella [18], and FimH of Escherichia coli [19] or by secreted effectors, such as hemolysin A from S. aureus [20], pyocyanin and homoserine lactones from P. aeruginosa and Serratia liquefaciens [21–25]. Such alterations in the host Ca²⁺ have been shown to facilitate bacterial adherence and subsequent internalization into the host cells.

In plants, Ca²⁺ is one of the earliest signaling elements that coordinate adaptive immune responses to invading pathogenic bacteria. Cytoplasmic Ca²⁺ ([Ca²⁺]_cyt) increases in response to infecting pathogens, such as P. syringae [46]. A sustained elevation of [Ca²⁺]_cyt serves as an important early signal, which links the recognition of infection to downstream defenses including generation of reactive oxygen species (ROS) and oxidative burst [47, 48]. The ROS burst may lead to cell death preventing the pathogen establishment inside the plant [49].

Overall, Ca²⁺ is an essential component of the host environment that both responds to the presence of bacterial pathogens, and regulates specific defense mechanisms. Ca²⁺ levels in a host may signal to the invading pathogens that they are entering a host and also indicate the status of immune protection in the host. Therefore, recognizing the host Ca²⁺ level can be beneficial to the invaders and trigger their adaptation to the host environment, and lead to their increased virulence and survival of the pathogen.

33.1.2. Ca²⁺ Triggers Life Style Switches in Bacterial Pathogens

Bacteria possess efficient regulatory systems that enable their adaptation to continuously changing environments. Regulation of gene expression is key for bacterial survival in a variety of environments. One particularly efficient and complex mechanism of surviving hostile environments is a switch between free-swimming or planktonic lifestyle to sessile life as surface-associated community, called biofilm. This transition is enabled by major molecular rearrangements ultimately enabling increased resistance, cell-cell communication and efficient metabolism [50, 51]. This mechanism is of particularly high importance to extracellular pathogenic bacteria colonizing host surfaces and surviving both host defenses and antimicrobial treatments.

There is a growing body of evidence that Ca²⁺ plays both a structural and a regulatory role in the transition to surface-associated biofilm lifestyle. Bacterial adhesion is the first step in biofilm formation, and itself is a survival mechanism, as nutrients, for example, tend to accumulate at surfaces [52]. The effect of Ca²⁺ on adhesion is partially due to electrostatic interactions, but also due to strong interactions of the surfaces with the cell structures, such as pili and fimbriae [53–55], and other macromolecules including teichoic acids, adhesins, lipopolysaccharide (LPS), and extracellular polysaccharides (EPS). It was shown that cell surface properties and their electrostatic interactions with the substratum contribute to Ca²⁺-enhanced adhesion of non-motile and motile P. aeruginosa [56]. Ca²⁺- enhanced cell adhesion to diverse host molecules and in vitro substrates, as well as cell-cell aggregation, relies on the presence of type I and type IV pili in a number of pathogens, including Xylella fastidiosa, [53], P. aeruginosa [57], Vibrio vulnificus [58], and N. gonorrhoeae [59]. The Ca²⁺ regulation of the type IV pilus is determined by its binding to pilus-biogenesis factor, PilY1, enabling pilus extension and retraction [60]. This interaction is also required for the bacterium twitching motility. By interacting with type I pili and fimbriae, Ca²⁺ modulates invasion of bacterial pathogens, such as E. coli, into host cells [19, 61].

Ca²⁺ also enhances bacterial adhesion via large cell surface Ca²⁺-binding adhesins, such as SdrC and SdrD in S. aureus [62, 63] and BapA in Paracoccus denitrificans [64]. The former contain EF hand-like motifs that bind Ca²⁺ required for protein folding. The latter belongs to repeats-in-toxin (RTX) family, containing multiple nonapeptide Ca²⁺-binding domains, secreted via Type I Secretion System (TISS), and serving a variety of functions, including cell-cell or cell-surface interactions or contributing to protection against hostile environments by forming bacterial surface (S)-layers (reviewed in [65]). In Listeria monocytogenes, elevated Ca²⁺ has been reported to stabilize the complex between the adhesin InlA (Internalin A) and the human extracellular E-cadherin domain 1 (hEC1). Once inside the host cell, where Ca²⁺ concentrations are lower, the InlA-hEC1 complex dissociates, which facilitates the liberation of the bacteria from the host cell membrane into the cytosol [66]. Ca²⁺ is required for multimerization of large adhesin LapF involved in colonization, microcolony formation, and biofilm maturation of P. putida [67–69].

Due to its interactions with surface proteins and by forming ionic bridges between negatively charged macromolecules, Ca²⁺ enhances cell aggregation and strengthens biofilm matrixes, including cell aggregation in oral Streptococci [70] and alginate cross-linking of P. aeruginosa biofilm matrix [71]. Ca²⁺ was also shown to bind extracellular DNA (eDNA), another important component of biofilm matrix, and this thermodynamically favorable binding increases bacterial aggregation in several Gram-positive and Gram-negative species, including S. aureus and P. aeruginosa. The authors concluded that Ca²⁺ did not affect DNA release [72]. However, this observation is likely species- and strain-specific [73], as Ca²⁺ was shown to induce production of P. aeruginosa pyocyanin, which promotes DNA release [74]. Furthermore, the presence of Ca²⁺ increased eDNA release, contributing to biofilm formation in Streptococcus mutans [75]. Ca²⁺ was also shown to increase the adhesive nature of P. fluorescence biofilm, but reduced its elastic properties [76].

In different bacterial species, elevated Ca²⁺ either stimulates or reduces biofilm formation. Positive regulation was observed in response to 1–10 mM Ca²⁺ in Pectobacterium carotovorum [77], Rhizobium leguminosarum [78], Pseudoalteromonas sp. [79], Shewanella oneidensis [80], P. aeruginosa [73], X. fastidiosa [81], V. cholerae [82], and V. fischeri [83]. This regulation was shown to be mediated by diverse mechanisms. For example, elevated Ca²⁺ activates the transcription of genes responsible for production of surface adhesins and EPS: alginate in P. aeruginosa [73] and P. syringae [84]; symbiosis polysaccharide (syp) or cellulose in V. fisheri. Ca²⁺-dependent hemophilic interactions of surface-associated adhesion SdrC promotes biofilm formation in S. aureus [62].

Negative regulation of biofilm by elevated Ca²⁺ was reported in S. aureus [85] and V. cholerae [86]. In V. cholerae, this regulation is mediated by negatively regulated two-component system CarSR and vps (Vibrio polysaccharide) genes. However, V. cholerae also produces Vps-independent biofilm, which is preferred under high Ca²⁺ sea water conditions, where Ca²⁺ interacts directly with the O-antigen polysaccharide [87]. S. aureus produces several surface adhesins, such as clumping factors A and B (ClfA and ClfB) [88, 89] and biofilm-associated protein (Bap) [85]. These proteins contain Ca²⁺-binding EF-hand-like motifs, and binding the ion inhibits their role in cell adhesion and biofilm formation. A point mutation in protease aureolysin (aur) gene in one of S. aureus strains led to increased activity of ClfB, required for biofilm growth under Ca²⁺-depleted conditions [90].

Some factors contributing to biofilm formation are known to be regulated by cyclic-di-GMP (c-di-GMP) (reviewed in [91]) and quorum sensing (QS) (reviewed in [92]). This raises the possibility of interconnections between c-di-GMP, QS and Ca²⁺ regulatory networks that warrant further studies.

33.1.3. Virulence Factors Regulated by Ca²⁺

Factors that enable pathogenic bacteria to cause diseases can be broadly grouped into several categories, such as penetration, colonization, damage of host cells, evasion of host defenses, and proliferation, all ultimately contributing to the developing infections. Colonization requires pathogens to establish interactions with host tissues by producing extracellular or cell-associated molecules. It may also involve communication between invaders themselves or those with commensals. The relationship between some of these factors and Ca²⁺ is discussed above. Here we outline virulence factors attributed to more invasive host-pathogen interactions that are directly or indirectly regulated by Ca²⁺.

Bacterial invasion is commonly facilitated by the production and secretion of molecules that cause enzymatic or non-enzymatic damage to the host cells [93, 94]. A number of secreted enzymes are known to be regulated by Ca²⁺ in bacterial pathogens. For example, in P. aeruginosa, Ca²⁺ promotes the production of extracellular proteases LasB, LasA, PrpL (protease IV), and AprA [73, 95–97]. In the case of elastase LasB, Ca²⁺ not only increases the production of the protein, but modulates its processing, export, stability and enzymatic activity [95, 98, 99]. The enzymatic activity covers a wide repertoire of substrates, including elastin, collagen, and human immunoglobulins, underlining the significance of the protein and its Ca²⁺ regulation in P. aeruginosa pathogenicity. The alkaline protease A (AprA) binds Ca²⁺ through its C-terminal RTX domain, enabling folding of both C- and N-terminal proteolytic domains, which is required for stable conformation and enzymatic activity of the protease [73, 80, 96]. Both AprA and LasB are capable of degrading exogenous flagellin monomers under Ca²⁺-replete condition, which prevents flagellin-mediated immune recognition and killing of P. aeruginosa via complement-mediated phagocytosis [99, 100]. The Ca²⁺-enhanced production of the two proteases with anti-flagellin activity provides a robust strategy for P. aeruginosa to escape the detection by the complement system.

Our earlier studies showed that production of pyocyanin, the extracellular redox cycling compound and a virulence factor of P. aeruginosa [101, 102] is up-regulated in response to elevated Ca²⁺ [73]. Pyocyanin is found in pulmonary fluids of CF patients and shown to disrupt Ca²⁺ homeostasis of the host epithelial cells [21, 103], potentially contributing to a further increase of extracellular host Ca²⁺ and therefore induction of Ca²⁺-regulated virulence.

Toxins represent one of the most powerful strategies of bacterial pathogens to conquer a host. Ca²⁺ modulates the production, secretion, and function of several toxins in a number of pathogens. In E. coli, Ca²⁺ is required for Hemolysin A (HlyA) binding to erythrocytes [104]. Binding Ca²⁺ causes conformational change in the toxin increasing its surface hydrophobicity and promoting the irreversible binding to the lipid bilayer of erythrocytes. This interaction preludes and ensures the lytic effect [105]. In V. cholerae, Ca²⁺ enhances bile salt-dependent activation of virulence. The mechanism relies on Ca²⁺ promoting the bile salt-induced activation of transmembrane virulence regulator TcpP, which then induces the production of major virulence factors, including toxin-coregulated pilus (TCP) [106]. The presence of Ca²⁺ has been reported to be essential for the toxicity of anthrax-edema toxin (composed of protective antigen and edema factor) produced by Bacillus anthracis [107]. The edema factor has adenylate cyclase activity synthesizing cAMP. Once in the host cytosol, the edema factor produces cAMP, which causes a rapid influx of Ca²⁺. The accumulation of cAMP in the cytosol requires the presence of extracellular Ca²⁺. As a potent inhibitor of immune response, accumulated cAMP leads to suppression of proinflammatory cytokines, phagocytosis and bactericidal activity of leukocytes thereby facilitating the survival of bacteria in the host [107, 108].

On the other hand, elevated host Ca²⁺ may have a negative regulatory effect on virulence and thus contribute to host defenses. One example is a cell wall degrading enzyme endopolygalacturonase (PehA) that is down-regulated by high (10–30 mM) levels of Ca²⁺ in a plant pathogen Pectobacterium carotovorum. This prevents the pathogen from infecting the plant [109].

Overall, Ca²⁺ regulates many virulence factors of invading bacterial pathogens, which stresses the importance of a detailed understanding of Ca²⁺ regulatory pathways in these pathogens.

33.1.4. Ca²⁺-Regulated Secretion Systems

Most bacteria can respond to and manipulate their environment through the secretion of extracellular proteins. Bacterial secreted proteins are often involved in breakdown of macromolecules, such as polysaccharides or polypeptides to simple sugars or amino acids that the bacteria can take up and utilize as carbon, nitrogen, and energy sources. Secreted proteins may also act as virulence factors, as in the case for the proteases described above, LasA, LasB, PrpL, and AprA, which modulate immune effectors and degrade elastic tissues [73, 95, 96, 99, 100]. Pathogenic bacteria also use protein secretion to kill other cells, including eukaryotic cells [110] and, in some cases, competing bacteria within biofilm communities [111]. Extracellular Ca²⁺ concentration plays a direct or indirect signaling role in many of the bacterial protein secretion systems.

Bacteria use at least six different strategies to secrete proteins (termed: T1SS to T6SS) reviewed in [112]. The T1SS transports specific proteins directly from the cytoplasm to the extracellular medium, with no apparent periplasmic intermediate. The Ca²⁺ requiring protease, AprA [113], is secreted by the T1SS, composed of three components, AprDEF, which include cytoplasmic ATPase, an inner membrane protein component, and an outer membrane protein [114]. These proteins form a molecular complex, dedicated to the export of AprA [113]. AprA accumulates in the biofilms of P. aeruginosa under Ca²⁺-replete conditions, but not under Ca²⁺ − limiting conditions [73]. The other protease virulence factors described above that require Ca²⁺ for activity or structural integrity, LasA, LasB, and the PrpL [98], are secreted by the T2SS [115–117]. The T2SS is a general pathway for secretion of a variety of extracellular proteins. In the T2SS, proteins with N-terminal export signal peptides, are first exported to the periplasm by either the Sec export machinery or the twin-arginine translocation (TAT) system [118, 119]. Sec, exports proteins in an unfolded state, then folds the proteins into their three dimensional confirmation in the periplasm, with the help of proteins such as disulfide bond isomerase, DsbA. The TAT system exports folded proteins into the periplasm. Once in the periplasm, proteins are secreted across the outer member via the secretion apparatus. For example, in P. aeruginosa the secretion apparatus is composed of the Xcp proteins (XcpA and XcpP-Z) or the homologous system, Hxc (composed of HxcP-Z) [112]. In addition to secretion of enzymes into the extracellular medium, the T2SS also plays a role in generation of certain types of pili, the Type IV pilus, which plays role in bacterial attachment and movement along surface. In twitching motility, bacteria move along surfaces by extension and retraction of the pili, through polymerization and depolymerization of the pilin subunits. Some bacteria, including E. coli and several Vibrio spps, requires Ca²⁺ for structural integrity of the major pseudopilin subunit, GspG [120].

The type III secretion system (T3SS), encoded on pathogenicity islands of many pathogenic bacteria, delivers effector protein toxins directly into the cytosol of eukaryotic cells during infection. The toxins, including enzymes such as ADP-ribosyltransferases, phospholipases, or adenylate cyclases, disrupt such host cell activities as actin remodeling, and gene regulation [112]. Perhaps the most interesting role of Ca²⁺ in secretion of bacterial virulence factors is its direct role in expression regulation (activation or repression) of the genes encoding the secretion apparatuses. It has been known for many years that expression of the T3SS genes is induced by either host-cell contact or by chelation of Ca²⁺ from the medium (low [Ca²⁺]) [121, 122]. The T3SS forms a complex needle-like structure that is related to the bacterial flagella basal body [123]. The T3SS includes inner and outer membrane ring structures, and cytoplasmic protein components that dock to the inner membrane ring. The needle-like structure protrudes from the basal body, punctures the host cell membrane, and secretes toxins directly into the host cells. For this reason, the T3SS has also been termed the injectosome.

Regulation of expression of the T3SS gene clusters by host cell contact has been well characterized in P. aeruginosa [124, 125]. Transcription of the T3SS in P. aeruginosa is controlled by the transcriptional activator, ExsA, an AraC/XylS-type regulator. ExsA is inhibited by a cascade of protein-protein interactions that prevent ExsA binding to the DNA. The cascade involves interactions of ExsA, ExsD, ExsC, and ExsE. Expression of the T3SS genes is induced when ExsE is translocated from the cell through the T3SS, ultimately titrating the anti-activator, ExsD away from the ExsA and allowing transcription. If translocation of ExsE through the T3SS is functional (e.g. during host cell contact or at low Ca²⁺), transcription of the TS33 genes is activated. If ExsE builds up in the cell due to lack of host cell contact, then further transcription of the TS33 genes is inhibited.

Another role of Ca²⁺ in regulation of T3SS was recently shown to involve the Ca²⁺-sensor protein, LadS [126]. LadS is a hybrid membrane-bound sensor, containing both a histidine kinase domain and a periplasmic Ca²⁺-binding DISMED2 domain. Broder et al. [126] mutated potential Ca²⁺-binding residues in P. aeruginosa and found that the resulted T3SS gene expression became insensitive to Ca²⁺ conditions. Ca²⁺ binding to the LadS DISMED2 domain is the first step in a regulatory cascade that responds to external Ca²⁺. The cascade involves two component system GacCS, two small regulatory RNAs, RsmY and RsmZ, and the RNA binding protein, RsmA. Ultimately, binding of RsmA to specific mRNA sequences results in gene regulation at the translational level [126].

Assembly of the T3SS is a dynamic process that responds to external Ca²⁺. Using Yersinia entercolitica, Diepold et al. [127] tagged components of the T3SS with fluorescent reporter proteins, so that they could image the membrane and cytosolic components. Using Fluorescence correlation spectroscopy, they calculated the diffusion rates of the T3SS cytoplasmic components under inducing (low Ca²⁺) and non-inducing (high Ca²⁺) conditions, as they assembled. They found that the rate of diffusion of the cytoplasmic components changed with the external Ca²⁺ concentration and proposed this as a novel mechanism for the role of Ca²⁺ in regulation of T3SS assembly.

The switch between expression of T3SS (low Ca²⁺) to more recently discovered type T6SS [128] may correlate with the switch from acute to chronic infections of P. aeruginosa [126]. The T6SS also uses direct injection of effector proteins into other cells, which may be host cells, or other competing bacteria [111]. However, rather than being evolutionarily related to the flagella basal body, the T6SS appears related to bacteriophage tail-associated proteins [129]. The tail-spike is used to puncture the membranes of cells, and the effector molecules at the tip of the spike are injected directly into the cytosol. Regulation of expression of T6SS is not well characterized and the role of Ca²⁺ in regulation of T6SS is not well known. However, Allsopp et al. [130] using a transposon mutagenesis screening approach identified the RNA binding protein, RsmA as a primary component involved in the regulation of all the three T6SS gene operons of P. aeruginosa. Therefore, a common link in the inverse regulation of T3SS and T6SS involves the RNA binding protein RsmA.

33.2. Molecular Mechanisms of Ca²⁺ Responses in Pathogens

33.2.1. Two Component Systems

As discussed above, Ca²⁺ levels differ within a host, fluctuate during disease progression, and thus form a complex signaling landscape for invading pathogens. Therefore, sensing host Ca²⁺ is an important task enabling pathogens to efficiently adjust to the host environment by triggering the expression of genes responsible for virulence and resistance. Bacteria accomplish this in part by employing two-component regulatory systems (TCS). A traditional TCS consists of a sensor kinase and a response regulator. The sensor kinase is usually embedded into the inner membrane with the sensor region often facing the periplasm. Upon signal binding, the sensor kinase auto-phosphorylates followed by phosphorylating the response regulator, typically regulating transcription of a set of target genes [131–133].

Several Ca²⁺ sensing TCSs have been identified. Some of them are positively and some are negatively regulated by elevated Ca²⁺ (Table 33.2). To test whether the relationship with the ion is defined by the recognition sequence within the TCS sensor, we aligned the sensor sequences of the TCS experimentally shown to be regulated by Ca²⁺. Based on the Clustal Omega alignment, a maximum likelihood tree was constructed using MEGA4 algorithm [134, 135] (Fig.33.1). The distinct grouping of positively and negatively regulated sensors supports the idea of sequence-dependent relationship with Ca²⁺.

Table 33.2.

Sensors from two-component regulatory systems (TCS) regulated by Ca²⁺

Name	GenBank ID	Stimuli	Ca2+-dependent regulatory targets	Regulated Phenotype	Organism	References
Positively Regulated by Elevated Ca2+
CarS	AAG06044.1	Ca2+	carP, carO	Virulence	P. aeruginosa	[153]
CvsS	AAO56858.1	Ca2+	hspR, algU	Biofilm formation, cellulose production, virulence, T3SS	P. syringae	[84]
AtoS	AEH68827.1	Ca2+ SpermidineHis-tamineAcetoacetate	Ato operon	Synthesis of PHB-PP, motility	E. coli	[154–157]
VicK	PNM00564.1	Ca2+, Mn2+, sucrose	atlA operon	eDNA release, response to host immunity, attachment, and biofilm formation	S. mutans	[91, 158, 159]
Negatively Regulated by Elevated Ca2+
PhoQ	AIB53821.1	Mg2+,Ca2+, acetate		LPS modification	E. coli	[137, 147]
PhoQ	P0DM80.1	Mg2+,Ca2+		LPS modification, SOS response	S. enterica	[136, 145, 160]
PhoQ	AEU17992.1	Mg2+,Ca2+		LPS modification	P. aeruginosa	[137, 141, 143]
PehS	BAJ11971.1	Ca2+	pehA	Virulence	P. carotovorum	[148]
CiaH	P0A4I6.1	Ca2+	ciaX	Ca2+ mediated cell functions and biofilm production	S. pneumoniae	[161]
CarS	2,614,773	Ca2+	vps	Vps-dependent biofilms, antibiotic resistance	V. cholerae	[86, 162]

Fig. 33.1. Molecular Phylogenetic analysis of Ca²⁺regulated TCS.

To analyze the sequences of Ca²⁺ regulated TCS sensors, we first determined the sensor regions by selecting the periplasmic loop of the proteins based on TMHMM analyses. After the 11 sequences were aligned, all positions containing gaps and missing data were eliminated. The phylogenetic relationship of the sensor regions was inferred by using the Maximum Likelihood algorithm based on the JTT matrix-based model [1] in MEGA7 [2]. The tree with the highest log likelihood (−2667.30) is shown. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site

The TCSs that are negatively regulated by Ca²⁺ have been studied in more details. PhoPQ is a well-characterized TCS, found in a variety of Gram-negative pathogens, such as P. aeruginosa, E. coli, Yersinia pestis, Shigella flexneri and S. enterica [82, 131, 136–138]. PhoPQ systems regulate multiple virulence factors, resistance and motility. The PhoPQ regulon includes the SOS response of S. enterica [139], and the arn operon of P. aeruginosa, which is responsible for lipid A modifications and increased resistance to antimicrobial peptides [138, 140–144]. Interestingly, PhoPQ systems are also repressed by elevated Mg²⁺, and two-distinct Mg²⁺ and Ca²⁺ binding regions were identified in PhoQ sensor of S. enterica [136]. These regions are conserved in other PhoQ homologs [145, 146]. While the PhoPQ systems share overall sequence similarly, they differ in their responses. Thus, mutating Ca²⁺-binding residues in PhoQ have less of an impact on transcriptional regulation in E. coli than in S. enterica [146]. PhoQ interactions with ligands also differ in different species: Mg²⁺ binds to PhoQ dimer causing destabilization and preventing signal transduction in P. aeruginosa PhoQ (PaPhoQ), but not in E. coli PhoQ (EcPhoQ) [137]. Finally, PhoQ may respond to additional ligands such as acetate in the E. coli PhoQ [147]. The TCS most closely grouped with PhoPQ is PehSR from a plant pathogen Pectobacterium carotovorum (Fig.33.1). This system regulates the production of a secreted endopolygalacturonase, PehA which is required for initial invasion of the pathogen into a host [109, 148–150]. Homologs of PehA, although not yet characterized, have been identified in another plant pathogen Erwinia chrysanthemi [151, 152].

Another TCS negatively regulated by elevated Ca²⁺ is CarSR from V. cholera [86]. CarSR regulates Vibrio polysaccharide (vps), the main matrix component of vps-dependent biofilms. Although elevated Ca²⁺ decreases the formation of vps- dependent biofilms, it increases the formation of vps-independent biofilm in V. cholerae, as well as production of bile-salt-dependent virulence factors required for colonization of the gut [82, 163, 164]. The mechanisms for this positive Ca²⁺ regulation of V. cholerae virulence are not known and potentially involve an alternative TCS that is positively regulated by Ca²⁺. To predict such a TCS, we performed BLASTP alignment of the sensor region from the positively regulated by Ca²⁺ P. aeruginosa CarS against the V. cholerae genome and identified a putative Ca²⁺ responsive sensor protein CSC11701.1. The close grouping of its sensor sequence with P. aeruginosa CarS and P. syringae CvsS supports its potentially positive regulation by Ca²⁺ (Fig.33.1).

An atypical TCS, CiaHR, was identified in Streptococcus mutans. CiaHR con- tains a third component, CiaX, a small protein, which upon binding Ca²⁺, interacts with the sensor portion of CiaH and prevents the phosphor-relay. The system was shown to regulate antibiotic resistance, biofilm formation, eDNA uptake, as well as stress response [161, 165–167]. Interestingly, CiaH grouped closely with the positively regulated sensors, indicating a possibility that CiaH itself may be regulated by Ca²⁺. In addition, S. mutans biofilm formation and eDNA release can be positively regulated in response to Ca²⁺ via the TCS VicKR [91, 159, 168, 169]. However, VicK is more distantly related to the positively regulated Ca²⁺ sensors, which could be attributed to its potential to respond to other stimuli (Fig.33.1).

The TCS that are positively regulated by elevated Ca²⁺ were shown to be involved in regulating virulence and resistance factors in response to Ca²⁺. Earlier, our group identified a TCS that is positively regulated by Ca²⁺ in P. aeruginosa. It was named it Calcium Regulated Senor/ Regulator, CarSR [153]. This system regulates at least two identified targets, CarO and CarP, involved in Ca²⁺–induced production of virulence factors pyocyanin and pyoverdine and contributes to the regulation of the intracellular Ca²⁺ homeostasis and tolerance to elevated Ca²⁺. Recently, another Ca²⁺-induced TCS was found in plant pathogen P. syringae, named Calcium, Virulence, and Swarming Sensor and Regulator, CvsSR. CvsSR is required for P. syringae pathogenicity in plants by enhancing transcription of genes for T3SS and small RNAs while repressing alginate and flagella biosynthesis [84]. In E. coli, synthesis of the polyhydoxybutyrate polyphosphate (PHB-PP) Ca²⁺ channels is positively regulated by the AtoSC TCS. These non-proteinaceous Ca²⁺ channels play a role in eDNA uptake and folding of the outer membrane protein OmpA [154, 170, 171]. In addition, AtoSC regulates other processes, such as motility in response to acetoacetate, histamine, and spermidine [155–157]. This may be reflected in its distant grouping from CarSR and CvsSR (Fig.33.1).

In summary, bacterial pathogens utilize multiple TCS to recognize changes in the environmental Ca²⁺ and adjust their transcriptional activity. These systems are versatile as they evolved to enable bacterial adaptations to multi-variant environments. Understanding the regulation by TCS is challenged by several factors: the presence of multiple systems in one organism, sensors responding to different stimuli, and additional components involved in signal recognition [172–175]. Different TCS may also cross-talk enabling multiple signals to control similar responses, as in the case of SypFG, proposed to mediate Ca²⁺ induction of biofilm formation in V. fischeri [83]. The sensing portion of SypF, however, was not required for Ca²⁺ induction, and involved an alternative sensor kinase RscS phosphorylating SypF in response to Ca²⁺ [83]. A much better understanding of the TCS signaling networks triggered by Ca²⁺ is needed to fully appreciate their role in Ca²⁺-mediated communication between invading pathogens and their hosts.

33.2.2. Ca²⁺ Sensors

In eukaryotes, members of the calmodulin superfamily are the best studied Ca²⁺ sensors [176, 177]. Calmodulin (CaM) contains two canonical EF-hand motifs coordinating Ca²⁺ binding [178, 179]. Upon binding Ca²⁺, CaM undergoes conformational changes enabling binding and activating diverse target proteins [180]. Therefore, searches for components of Ca²⁺ signaling networks in bacteria have focused on proteins with EF hands [181]. In addition, proteins that play roles in translocating or buffering Ca²⁺ have been studied [182–188]. A number of calmodulin-like proteins have been reported based on their sequence and structure similarity to CaM and binding to anti-calmodulin antibodies. Several of these Ca²⁺ sensors are summarized in Table 33.3. The first proposed bacterial Ca²⁺ sensor was CasA, from Rhizobium etli. CasA has three pairs of EF hand domains, similarly to eukaryotic calbindin and calretinin [189]. CasA mediates Ca²⁺-dependent symbiotic relationship between R. etli and its plant host. Our group identified a homolog of CasA in P. aeruginosa and named it EfhP (EF-hand protein) [190]. EfhP is required for maintaining Ca²⁺_in homeostasis and involved in Ca²⁺ regulation of P. aeruginosa virulence. Our current studies verified the ability of this protein to selectively bind Ca²⁺ and undergo Ca²⁺–dependent conformational changes supporting its role as a Ca²⁺ sensor (Kayastha et al. in preparation). Another EF hand protein, proposed to function as a Ca²⁺-sensor, is CabD from Streptomyces coelicolor. CabD contributes to Ca²⁺ regulation of aerial mycelium formation. CabD has high- and low-affinity Ca²⁺-binding sites, suggesting their distinct roles in mediating Ca²⁺-regulatory and buffering roles, respectively [177].

Table 33.3.

Ca²⁺ Sensors

Protein name/GenBank ID	Organism	Function/Properties	Domain	References
EfhP	P. aeruginosa	Regulates Ca2+ induced virulence and intracellular Ca2+ homeostasis	One pair of EF hands	Kayastha et al. (in preparation)
AAG07494.1
CasA	R. etli	Mediates Ca2+ dependent symbiosis with leguminous host	Three pairs of EF hands	[189]
AF288533
CabD	S. coelicolor	Affects formation of aerial mycelium	Two pairs of EF hands	[177]
3AKA_A
Protein S	M. xanthus	Required for assembly of spore coat	Beta-gamma crystalline fold	[191]
WP_020477824
CAMLP	M. tuberculosis	Activates NAD kinase and phosphodiesterase upon Ca2+ binding	Single EF hand motif	[192, 193]
NP_215727
CAMLP	M. smegmatis	Activates phosphodiesterase	Single EF hand motif	[194]
AY319523.1
CALP	B. subtilis	Activates phosphodiesterase in Ca2+ dependent manner		[195]
YP_004243569
CLP	B. pertussis	Activates adenylate cyclase in Ca2+ dependent manner		[196]
LadS	P. aeruginosa	Ca2+ dependent phosphor-relay to GacSA	Histidine kinase, 7 transmembrane, DISMED2	[197, 198]
AAG07361

CaM-like proteins have been reported in Mycobacteria and are suggested to play a role in sensing Ca²⁺ [199]. The CaM-like protein, Rv1211 of M. tuberculosis binds Ca²⁺ through its single EF hand domain and stimulates the activities of NAD kinase and phosphodiesterase (PDE), targets that are similar to those of eukaryotic CaM. Reduced expression of this protein has been shown to impair M. tuberculosis growth and survival in macrophages, suggesting its importance during infection [192, 193]. A homologous CaM-like protein from M. smegmatis has been shown to stimulate phosphodiesterase activity and regulate the metabolism of phospholipids [200] supporting the role of this protein as a Ca²⁺ sensor.

Protein S from Myxococcus xanthus is a member of ßγ-crystalline family. This protein shares structural similarity to CaM and binds Ca²⁺, which is required for the protein assembly on the surface of the spores [201]. A more recent report showed that the protein’s Ca²⁺-binding site forms a high charge density pocket similar to that in calsequestrin and human Hsp70. However it is still not clear whether the Ca²⁺-induced conformational changes in protein S play roles in signaling events [191].

Recently, a hybrid histidine kinase LadS was shown to trigger a Ca²⁺-induced switching between acute and chronic type of virulence in P. aeruginosa [198]. As discussed above, LadS belongs to a unique class of bacterial sensors that possess histidine kinase, seven transmembrane, and the periplasmic DISMED2 domain. The latter was shown to bind Ca²⁺ via Asp80 and Asp90 residues and activate the kinase activity leading to phosphorelay cascade [197, 198]. In contrast to typical sensors of TCS that phosphorylate partnered response regulators, LadS phosphorylates the TCS GacSA and thus activates GacS/Rsm pathway responsible for the global regulation of chronic infection-type virulence and tolerance [88]. In silico analysis of the sequence conservation of LadS showed that this protein is unique to Pseudomonas. Interestingly, during searches for homologous DISMED2 domains among selected bacterial pathogens, we identified several proteins including putative alkaline phosphatase synthesis sensors CJK91172.1 and CJK40304.1, and a membrane protein with GGDEF domain, CJK88170.1, in S. pneumoniae. The GGDEF domain is known to act as a diguanylate cyclase responsible for synthesis of cyclic-di-GMP, a second messenger mainly regulating biofilm formation [202]. In addition to DISMED2 domain, these proteins contain the two residues (Asp80 and Asp90) required for Ca²⁺ recognition, suggesting they may sense and respond to Ca²⁺.

Pathogenic bacteria possess Ca²⁺-sensing proteins that play an important role in modulating their pathogenicity. However, despite evidence of the significance of Ca²⁺ regulation in bacterial physiology and virulence, the knowledge on Ca²⁺ sensors and regulators in bacteria is still limited. Further studies are needed to determine whether these proteins sense the changes in the intracellular Ca²⁺ and thus enable Ca²⁺ to serve as second messenger.

33.3. Components of Intracellular Ca²⁺ Signaling in Pathogenic Bacteria Mediating Ca²⁺ Regulation of Virulence

A number of studies have shown that bacterial metabolic processes respond to elevated Ca²⁺ (reviewed in [203–205]). Some of these processes are regulated by extracellular Ca²⁺ levels and are mediated by TCS. However, other processes may respond to the transient changes in the intracellular Ca²⁺ (Ca²⁺_in), thus implicating Ca²⁺ as a second messenger. Although the latter still requires experimental proof, here we discuss the components of Ca²⁺_in signaling network that have been shown to play a role in Ca²⁺ regulation, supporting the idea of a regulatory role of Ca²⁺_in in bacteria.

Similarly to eukaryotic cells, bacteria maintain their basal [Ca²⁺]_in at high nanomolar level and generate transient changes in [Ca²⁺]_in in response to diverse stimuli [206–210]. These stimuli include a variety of extracellular factors, such as Ca²⁺_ex, pH, mechanical stimulation, intermediates of carbohydrate metabolism, and oxidative stress; all factors potentially to be encountered upon entering a host [206, 208, 210–213]. A number of proteins have been shown to contribute to the maintenance of Ca²⁺_in homeostasis and to the generation of Ca²⁺_in fluctuations [203, 206]. However, it is still not clear whether bacteria have an intracellular source of Ca²⁺ for these fluctuations (e.g. a compartment for storing and releasing Ca²⁺ into the cytoplasm) or if they rely on influx of extracellular Ca²⁺. E. coli accumulates Ca²⁺ in the periplasm to millimolar levels when grown in the presence of millimolar extracellular Ca²⁺ [207]. It is possible that the periplasmic Ca²⁺ may be released into the cytoplasm and used for generating intracellular Ca²⁺ transients. However, further mechanistic studies are imperative.

33.3.1. Ca²⁺ Channels

Several types of Ca²⁺ transporters have been identified in bacteria. The transporters contribute to the regulation of virulence and host-pathogen interactions. First, the poly-β-hydroxybutyrate polyphosphate (PHB-PP) in E. coli forms non-proteinaceous Ca²⁺ channels and translocates Ca²⁺ into the cytoplasm [214]. In addition, PHB-PP channels are required for Ca²⁺-dependent genetic competence, which plays a key role in uptake of foreign DNA, eventually enhancing bacterial adaptation to the host environment and resistance [215]. The production of the corresponding PHB-PP synthases was shown to be induced by elevated extracellular Ca²⁺ and several other stimuli via Ca²⁺-dependent TCS AtoSC [154].

Another type of Ca²⁺ channels, a pH-dependent Ca²⁺ leak channel, YetJ was identified in B. subtilis [216]. This protein contains a BAX-1 inhibitor domain homologous to the one in a Ca²⁺ leak channel found in the endoplasmic reticulum (ER) membrane. Eukaryotic channels containing transmembrane BAX inhibitor-1 motif (TMBIM) mediate Ca²⁺_in homeostasis and apoptosis [217]. Interestingly, this highly conserved domain has been identified in a number of bacterial proteins. Initially, this domain was identified in E. coli protein YccA, and was shown to play a role in biofilm maturation [99]. While more research is needed to determine the roles of YetJ and YccA in B. subtilis and E. coli physiology, our group recently identified another homolog of the channel in P. aeruginosa [Guragain et al. in preparation]. We named it CalC for Ca²⁺ Leak Channel and determined that the protein is responsible for generating transient changes in [Ca²⁺] in the P. aeruginosa cytoplasm in response to extracellular Ca²⁺. Transcriptional profiling of the mutant strain with disrupted calC revealed that the responses to elevated Ca²⁺ were impaired, particularly for genes encoding virulence factors and biofilm determinants [Guragain et al. in preparation]. This work provides experimental proof of the regulatory role of Ca²⁺_in transients in bacterial responses to Ca²⁺. Furthermore, homology searches identified a number of putative BAX-1 Ca²⁺ leak channels in bacterial pathogens including S. pneumoniae, P. carotovorum, Coexiella burnetti, S. enterica, and H. pylori, indicating the conserved nature of the Ca²⁺ leak channel in bacterial pathogens, and possibly suggesting a role in the pathogenic life style.

Mechanosensitive channels (MSC) are large, non-selective channels that usually allow the passage of ions in response to mechanical or osmotic stress (reviewed in [205, 218, 219]). MSC are found in a variety of different bacteria including human and plant pathogens [220–222]. In B. subtilis, MSC SpoVAC releases Ca-dipicolinic acid complex, which is required for spore formation [223]. Mechanical stress in E. coli was shown to cause an increase in [Ca²⁺]_in leading to altered gene expression [212]. However, the MSC, MscL in this organism did not impact Ca²⁺ in homeostasis [224], raising a possibility of an alternative Ca²⁺ channel responding to mechanical stress. Our studies with P. aeruginosa identified several Ca²⁺ transporters including a putative MSC encoded by PA4614, which contributed to the restoration of the [Ca²⁺]_in basal level and the regulation of Ca²⁺-induced swarming motility [206].

33.3.2. Ca²⁺ Pumps

Elevated levels of free Ca²⁺_in can be toxic to bacterial cells, and the recovery to the basal [Ca²⁺]_in is critical for re-sensitizing cells for the next wave of [Ca²⁺]_in signaling. Therefore, the mechanisms of Ca²⁺ efflux are of high importance for cellular survival and for Ca²⁺_in regulation. Underlining their physiological significance, multiple families of efflux transporters have evolved and been shown to play a role in bacterial physiology and virulence (reviewed in [203]). The first group includes two types (P and F) of ATPases that couple Ca²⁺ export to ATP hydrolysis [60, 225, 226]. These proteins are highly conserved and were identified in diverse bacterial pathogens. In addition to translocating Ca²⁺, or likely because of it, some of these proteins are important in diverse bacterial processes related to survival in a host. For example, CaxP plays a role in host colonization by S. pneumoniae [227], CtpE of M. smegmatis contributes to cell surface integrity [228], and PA3920 and PA2435 of P. aeruginosa mediate Ca²⁺ regulation of swarming motility [206]. The second group includes ion exchange transporters coupling Ca²⁺ export to co-transport of other ions. Although many of these transporters have been identified in bacterial pathogens (reviewed in [203]), there is little evidence yet about their role in virulence.

33.3.3. Predicting Novel Components of Ca²⁺ Signaling Network

To expand our knowledge on the components of Ca²⁺_in signaling network in pathogenic bacteria, we aimed to predict novel Ca²⁺-recognizing or translocating proteins based on their homology to well-characterized components of eukaryotic Ca²⁺ network. For this, we selected well-characterized eukaryotic Ca²⁺-binding proteins, whose homologs in bacteria have not been reported. All sequence alignments were carried out using NCBI BLASTP [76, 229]. CarR, a Ca²⁺ sensor that is a G protein-coupled receptor, plays an essential role in fluctuating intracellular Ca²⁺ homeostasis in response to minute changes in [Ca²⁺]_ex and other stimuli [230–234]. The protein contains three cooperative Ca²⁺ binding sites. To our surprise, four (underlined) out of five (in bold) residues required for Ca²⁺ binding (RXXEXXEEAEERD) were found in a large number of bacterial proteins involved in a variety of life-sustaining functions, including recruitment of replisome in S. aureus [235], cell division protein FtsA in E. persicina [236], putative Rhs toxin and DNA recombination regulation system in E. coli [237–239], and putative pili assembly gene in Enterobacter cloacae. Another eukaryotic Ca²⁺ signaling protein is RyR1, which is a Ca²⁺ channel known to release Ca²⁺ stored in the sarcoplasmic reticulum into the cytoplasm [240]. Sequence homology searches against bacterial genomes identified only a small fragment of the protein as aligning with bacterial proteins. This region happened to be located within the lining of the pore required for Ca²⁺ sensitivity [240–242]. Four (underlined) out of five residues E3893, H3895, E3967, Q3970, T5001 required for Ca²⁺ binding were found with similar spacing in several putative ABC transporters of pathogenic bacteria, including S. aureus and S. pneumoniae. We also detected these residues in putative bestrophin transporters of many bacterial species including P. aeruginosa and E. coli, in an orphan transcriptional regulator unique to a P. aeruginosa clinical isolate, and in several enzymes including a putative purine phosphatase of P. aeruginosa. The discovery of a putative Ca²⁺ binding site in bacterial bestrophin channels is particularly interesting, since human bestrophin is a Ca²⁺-gated potassium channel [243]. However, the only characterized bacterial bestrophin channel (in K. pneumoniae) was shown to not require Ca²⁺ for its function [244] nor did it contain the Ca²⁺-binding site found in the human bestrophin. This raises a possibility of two types of bestrophin channels in bacteria, Ca²⁺-dependent and Ca²⁺-independent. Interestingly, the eukaryotic bestrophin has been demonstrated to possess multiple splicing variants: with and without Ca²⁺ binding region [245]. Overall, these findings suggest that a significantly greater number of bacterial processes are likely regulated by Ca²⁺ than already known. The fact that most of these predicted Ca²⁺- binding proteins were detected in bacterial pathogens suggests the importance of Ca²⁺ regulation in their physiology and, possibly, interactions with hosts, whose vital processes are regulated by Ca²⁺.

33.4. Concluding Remarks

Bacteria have very dynamic and complex responses to Ca²⁺. Over the past 10 years, the evidence that bacteria utilize Ca²⁺ for signaling has grown, yet important pieces are still missing. An area that needs study is on intracellular Ca²⁺ signaling. While a number of Ca²⁺ sensors and Ca²⁺-dependent regulatory systems have been shown to regulate essential functions, most of the findings are of correlative nature with no direct experimental evidence linking the changes in the intracellular Ca²⁺ to the regulation of transcription or translation. Even less is known about how the amplitude and the frequency of intracellular Ca²⁺ signals modulate the response. An interesting aspect is the conservation of many Ca²⁺-binding domains in eukaryotes and bacteria indicating an evolutionary lineage between Ca²⁺ signaling networks in these domains of life. One technical problem, that is worth mentioning, is the disregard for the presence of Ca²⁺ in commonly used rich bacteriological growth media, such as LB or BHI. Consequently, Ca²⁺ regulation of the resultant bacterial phenotypes may be underestimated. Overall, Ca²⁺ signaling in bacteria is an exciting and quickly developing field, which is providing not only the fundamental understanding of bacterial life and evolution but also generating insights into the regulation of bacterial pathogenicity.