Exploring the Links Between Nucleotide Signaling and Quorum Sensing Pathways in Regulating Bacterial Virulence

Abstract

The survival of all organisms depends on implementation of appropriate phenotypic responses upon perception of relevant environmental stimuli. Sensory inputs are propagated via interconnected biochemical and/or electrical cascades mediated by diverse signaling molecules, including gases, metal cations, lipids, peptides, and nucleotides. These networks often comprise second messenger signaling systems in which a ligand (the primary messenger) binds an extracellular receptor, thereby altering the intracellular concentration of a second messenger molecule which ultimately modulates gene expression through interaction with various effectors. The identification of intersections of these signaling pathways, such as nucleotide second messengers and quorum sensing, provides new insights into the mechanisms by which bacteria use multiple inputs to regulate cellular metabolism and phenotypes. Further investigations of the overlap between bacterial signaling pathways may yield new targets and methods to control bacterial behavior, such as biofilm formation and virulence.

Graphical Abstract

While bacteria play essential roles in many aspects of human health, as evidenced by the growing body of work on the human microbiome,^1–2 bacterial infections also can wreak havoc, particularly if the infectious microbes are antibiotic resistant. Currently within the United States, nearly 2 million people develop hospital-acquired infections each year, the majority of which are antibiotic resistant and result in nearly 100,000 deaths. In addition, antibiotic resistant infections cost the United States between 21–34 million dollars each year, resulting in a financial strain on the health care system.^3–5 Development of new and effective antibiotic treatments requires a detailed understanding of the signaling mechanisms that bacteria use to respond to stress, which can lead to a variety of phenotypic changes that result in infections. In addition, gaining insight into the differences in sensing and signaling pathways between infectious and symbiotic bacteria may identify novel pathways that regulate cell decision-making, allowing us to potentially develop new therapeutics to target bacterial infections without harming the commensal populations.

A major challenge in our current understanding of bacterial signaling pathways is the interconnection of numerous signaling molecules. While historically there was a focus on bacterial two component signaling (sensor histidine kinase phosphorylates a response regulator that changes transcription of genes),⁶ it is now apparent that this simple depiction of linear signaling pathways is incomplete. Instead, bacteria have evolved to sense environmental inputs, often using seemingly redundant sensing proteins, and translate these inputs into the production of their own signaling molecules, which include nucleotide-based second messengers and quorum sensing autoinducers.^7–8 Elucidating the mechanisms by which these bacterial signaling molecules control phenotypic effects and the points within downstream pathways at which the signaling molecules intersect presents a major challenge for the field. However, the results of this work have broad implications for both our fundamental understanding of bacterial physiology and for the rational identification of targets for new compounds that modulate bacterial behavior.

While the links between nucleotide second messengers and quorum sensing have been reviewed previously by the Waters and Sintim groups,^9–10 in this Perspective, we highlight recent advances in our understanding of the interconnection of signaling pathways within Gram-negative bacteria, focusing particularly on nucleotide-based signaling molecules and quorum sensing autoinducers, and the effects on biofilm formation and other virulence-related bacterial phenotypes.

c-di-GMP and QS

Bacterial adaptation and virulence are regulated by interconnected nucleotide signaling and quorum sensing circuits. Among the most essential signaling molecules in bacteria is the second messenger bis-(3’-5’)-cyclic dimeric guanosine monophosphate (c-di-GMP).¹¹ Initially linked to the allosteric control of cellulose biogenesis in Gluconacetobacter xylinum over 30 years ago,¹² c-di-GMP governs an intricate regulatory network orchestrating the transition from the motile planktonic state to sessile biofilm communities.¹³ Cyclic-di-GMP is biosynthesized from two GTP molecules by diguanylate cyclases (DGCs), which contain a conserved GGDEF amino acid motif in the active site. The activity of some DGCs also is subject to allosteric inhibition via c-di-GMP binding to a remote site on the enzyme.¹⁴ Hydrolysis of c-di-GMP to the dinucleotide 5’-phosphoguanylyl-(3’-5’)-guanosine (pGpG) is catalyzed by specific phosphodiesterases (PDEs), which are characterized by conserved EAL or HD-GYP motifs in the active site.¹³ The aforementioned active site residues are essential for catalytic activity, and genes encoding GGDEF-, EAL-, and HD-GYP-containing enzymes are conserved across bacteria, with many species expressing multiple functional diguanylate cyclases and c-di-GMP-specific phosphodiesterases.¹¹ For example, the E. coli K-12 genome encodes 12 DGCs and 13 PDEs. Among these 25 proteins, six contain domains homologous to both the GGDEF- and EAL-containing families, but lack one of the two associated enzymatic activities, while one such dual-domain-containing protein exhibits both DGC and PDE activity. In addition, four proteins in E. coli K-12 are homologous to either the GGDEF- and/or EAL-containing families, but are devoid of both DGC and/or PDE activity.¹⁵ These enzymatically inactive proteins nonetheless mediate biofilm production and motility in E. coli through interactions with other proteins and small regulatory RNAs (sRNAs). In certain cases, these macromolecular interactions are controlled allosterically by c-di-GMP or GTP binding to the degenerate GGDEF or EAL site.¹¹ Moreover, catalytically competent GGDEF- and EAL-containing enzymes also engage in this complex interaction network to generate spatially-resolved pools of c-di-GMP which vary over the growth curve, facilitating activation of specific c-di-GMP-binding effectors with exquisite spatial and temporal control to mediate distinct aspects of motility and biofilm formation.^{11, 16} These effectors include both proteins structured mRNA elements known as riboswitches which undergo c-di-GMP-specific conformational changes to regulate translation of certain transcripts.¹¹ Families of cyclic-di-GMP-responsive proteins include PilZ, PelD, and FleQ effectors, along with certain enzymatically inactive GGDEF- and EAL-family proteins, as previously discussed herein.¹¹ Signaling cascades mediated by c-di-GMP are responsive to environmental factors such as oxygen availability,¹⁷ light,¹⁸ aminoglycosides,¹⁹ and nutrient deprivation.²⁰ Perception of these diverse inputs relies on various GGDEF- and EAL-family proteins, as many contain a sensory domain which enables integration of the signal via modulation of the associated DGC or PDE activity.¹¹

These diverse nucleotide-driven prokaryotic signaling cascades also intersect with bacterial quorum sensing (QS) networks, which involve inter- and intraspecies gene regulation via secreted autoinducer molecules in a cell density-dependent fashion.²¹ QS in Gram-negative bacteria relies primarily on diffusible acyl-homoserine lactones (AHLs), while Gram-positive taxa utilize cyclic peptide quormones. In addition, 4,5-dihydroxy-2,3-pentanedione (autoinducer-2 [AI-2]) regulates quorum interactions both within and across bacterial species.²² The paradigmatic Gram-negative AHL quorum sensing system initially was discovered in Vibrio fischerii nearly 40 years ago,²³ and later emerged as a key facet of symbiosis between the bacteria and the Euprymna scolopes squid symbiont.²⁴ A single bacterial species may produce multiple AHL molecules with different acyl moieties which typically are sensed in a species-specific fashion.^25–26 AHLs are biosynthesized by the LuxI-type enzymes, which catalyze lactonization of S-adenosylmethionine (SAM) followed by N-acylation with various fatty acids.^27–28 The resulting AHL signal modulates QS gene expression via interaction with the LuxR-type transcription factors.²⁹

In contrast to the highly specific nature of AHL-mediated QS, AI-2 regulates bacterial physiology across diverse species, with over 500 bacterial taxa encoding homologs of the LuxS AI-2 synthase.³⁰ Additionally, the periplasmic LuxP- and LsrB-family AI-2 receptors are widely conserved across bacteria,³⁰ further demonstrating the significance of AI-2 as an interspecies quormone. AI-2 biosynthesis involves initial cleavage of SAM by LuxS to produce 4,5-dihydroxy-2,3-pentanedione,³¹ which can undergo cyclization and hydration to yield different diastereomeric tetrahydrofurans.³² These distinct forms of AI-2 bind either the LuxP- or LsrB-family receptor to regulate gene expression through a phosphorylation cascade.³⁰

Studies conducted within the past decade have begun to elucidate the interplay between c-di-GMP signaling and QS (Fig. 1). In the pandemic Vibrio cholerae biotype E1, distinct QS-mediated pathways regulated by the hydroxtridecanone cholerae autoinducer-1 (CAI-1) and AI-2 converge to modulate biofilm formation in a c-di-GMP-dependent fashion. A random genetic reporter screen in V. cholerae E1 identified four putative HD-GYP c-di-GMP-specific PDEs that are induced at high cell density by the LuxR-family regulator HapR in response to a phosphorylation cascade initiated by CAI-1 and AI-2 binding to cognate sensor kinases CqsS and LuxP/Q, respectively.³³ Upon induction of PDE expression by HapR, the intracellular c-di-GMP level decreases, which attenuates transcription of the biofilm-associated vps gene cluster.^33–35 In contrast, the pathogenic V. cholerae C1 biotype lacks the HapR transcriptional regulator, but similar antagonistic links exist between CAI-1/AI-2 QS and c-di-GMP-induced biofilm formation. In the C1 biotype, transcription of GGDEF protein Vca0930 is positively regulated by the quorum regulatory sRNAs (Qrr), which repress translation of the HapR regulator in HapR⁺ V. cholerae strains, further demonstrating the inverse relationship between c-di-GMP and QS.³⁶ In addition, the VieSAB QS circuit also regulates biofilm production in the hapR-deficient C1 strain in response to an unknown QS signal. The VieSAB system induces transcription of EAL PDE VieA, resulting in c-di-GMP hydrolysis and concomitant inhibition of biofilm production at high cell density when the unknown autoinducer is abundant.³⁷ In the related Vibrio parahaemolyticus, which can cause gastrointestinal illness, the scrABC operon modulates biofilm formation and motility by linking QS to the c-di-GMP pool. ScrA, a predicted class V pyridoxal phosphate-dependent aminotransferase, produces an unknown diffusible signal that binds the periplasmic receptor ScrB. The ScrB receptor forms a complex with bifunctional GGDEF/EAL protein ScrC, which functions as a c-di-GMP PDE in the presence of ScrAB to up-regulate swarming and inhibit production of extracellular matrix polymers.³⁸

Figure 1.

Overview of intersection of c-di-GMP and quorum sensing pathways in P. aeruginosa (purple), V. cholerae (orange), and B. cenocepacia (green). Arrows represent effects or signaling molecule/transcript levels that are promoted by the input, while flat ended lines represent effects/concentrations that are inhibited by the input.

In the opportunistic pathogen Pseudomonas aeruginosa, AHL-mediated QS similarly decreases the c-di-GMP concentration via induction of Tyr phosphatase TbpA by AHL-bound LasR. This inhibition of c-di-GMP synthesis likely occurs due to TbpA-catalyzed dephosphorylation and concomitant inactivation of GGDEF-containing DGC TbpB. In accord with these data, depletion of TbpA in P. aeruginosa increases the c-di-GMP level, stimulates biofilm formation, and reduces motility.³⁹ Furthermore, reducing the c-di-GMP concentration through overexpression of PDE YhjH induces transcription of both the rhl and pqs QS regulons in P. aeruginosa PAO1, as compared to transcript levels in PAO1 constitutively expressing the DGC WspR to elevate intracellular c-di-GMP.⁴⁰ Importantly, the rhl and pqs systems are mediated by AHL and quinolone autoinducers, respectively,^41–43 and up-regulate transcription of several virulence factors, including rhamnolipids, PQS, and pyocyanin.^44–45 In agreement with the gene expression analyses, production of these rhl- and pqs-controlled virulence factors increased upon depletion of c-di-GMP, resulting in elevated pathogenesis against murine macrogphages, relative to P. aeruginosa with increased c-di-GMP (Fig. 1).⁴⁰ Despite the antagonistic relationship between QS and c-di-GMP signaling in P. aeruginosa, a high-throughput screen of microbial extracts identified the cyclopentenone terrein (produced by the fungus Apsergillus terreus) as an inhibitor of both c-di-GMP synthesis and QS synthesis/signaling.⁴⁶ Additional experiments using QS reporter strains and LC-MS/MS quantitation revealed that terrein antagonizes the LasR and RhlR receptors in P. aeruginosa and inhibits production of OdDHL, BHL, and PQS²⁸ – the three major pseudomonad QS autoinducers.^{43, 47–50}

Furthermore, virulence factor production in P. aeruginosa is tuned by the intersecting Las, Rhl, and Pqs QS circuits in response to different starvation conditions.⁵¹ Chemical genetics studies employing QS inhibitors specific for each QS receptor have elucidated crucial functions for the Rhl and Pqs systems in activating pyocyanin, rhamnolipid, and elastase B production under phosphate and iron limitation. Conversely, LasR is dispensable for synthesis of these virulence factors upon nutrient depletion.⁵² While this study did not investigate links between P. aeruginosa QS and c-di-GMP, prior work in Pseudomonas fluorescens revealed that phosphate depletion induces expression of the c-di-GMP-specific PDE RapA via the PhoBR two-component phosphate-sensing system, resulting in attenuated biofilm formation.⁵³ Intriguingly, P. aeruginosa PhoB activates the Rhl and Pqs systems in response to phosphate limitation,^54–55 suggesting that such conditions impact both QS and c-di-GMP signaling in pseudomonads. In addition, the PhoB transcription factor modulates the synthesis of c-di-GMP and AI-2 in E. coli in response to polyphosphate abundance.⁵⁶ These findings suggest interplay between QS and c-di-GMP in mediating the response to phosphate depletion in P. aeruginosa and in other opportunistic pathogens, alluding to the potential of disrupting these transduction pathways to attenuate biofilm formation and virulence factor production.

Additional studies have identified antagonistic relationships between c-di-GMP dependent regulation and different QS systems in the opportunistic pathogen Burkholderia cenocepacia. In this organism, virulence gene expression is regulated by AHL, as well as cis-2-dodecanoic acid (Burkholderia diffusible signal factor; BDSF).^57–58 BDSF is synthesized by RpfF (which bears sequence similarity to enoyl-CoA hydratase) and activates virulence gene expression through activation of receptor RpfR, a multi-domain PAS-GGDEF-EAL protein.⁵⁹ Binding of the BDSF signal to the PAS domain allosterically stimulates activity of the EAL domain to modulate swarming, biofilm formation, and virulence via c-di-GMP degradation (Fig. 1).⁶⁰ BDSF-bound RpfR also induces transcription of cepI,³⁷ encoding N-octanoyl homoserine lactone (OHL) synthase,⁵⁷ demonstrating that BDSF regulates concentrations of both OHL and c-di-GMP to coordinate gene expression in B. cenocepacia. Electrophoretic mobility shift experiments and two-hybrid assays elucidated that RpfR activates cepI expression through association with the Fis-family transcriptional regulator GtrR, thereby increasing the affinity of GtrR for cepI promoter DNA.⁶¹ Interestingly, the intracellular concentration of c-di-GMP further regulates cepI transcription, as c-di-GMP binds to the RpfR EAL domain and reduces the affinity of the GtrR-RpfR complex for target promoter DNA in vitro, constituting a complex feedback mechanism through which BDSF down-regulates the c-di-GMP level and stimulates OHL synthesis, while c-di-GMP attenuates OHL production.⁶¹ Subsequent transcriptional profiling experiments in B. cenocepacia expressing RpfR with a mutated EAL domain revealed over 100 differentially expressed transcripts relative to the WT control, demonstrating the global transcriptional impact of converging QS and c-di-GMP signals which act through receptor RpfR.⁶² Expression of QS regulons and other virulence-associated transcripts generally was down-regulated in the EAL mutant and pathogenicity was attenuated in a nematode model, indicating that accumulation of c-di-GMP represses virulence factor production in B. cenocepacia.⁶² While the clinical efficacy of disrupting QS and c-di-GMP signaling remains largely untested, the aforementioned studies depict the importance of understanding the antagonistic cross-talk between these fundamental signaling mechanisms to optimize anti-virulence and anti-biofilm therapies.

3’,5’-cAMP and QS

QS-mediated regulation also intersects with adenosine 3’,5’-cyclic monophosphate (3’,5’-cAMP)-dependent signaling (Fig. 2), another important nucleotide second messenger system in numerous bacterial species. In Escherichia coli, 3’,5’-cAMP primarily regulates carbon catabolism via the 3’,5’-cAMP receptor protein (CRP), a transcription factor that interacts with RNA polymerase to induce expression of over 100 genes.^63–64 The activity of adenylate cyclase (CyaA) in E. coli is modulated by carbon-source availability; an abundance of glucose indirectly inhibits CyaA-catalyzed 3’,5’-cAMP synthesis, whereas glucose limitation activates cyclase activity and increases intracellular 3’,5’-cAMP.⁶³ Stimulation of CyaA is mediated by phosphorylated glucose transport protein IIA (IIA^glc) which accumulates during glucose starvation, as phosphoryl-IIA^glc participates in phosphate transfer to glucose during uptake.⁶³ This cyclic nucleotide also mediates expression of virulence- and biofilm-associated genes in other bacterial taxa,⁶⁵ and regulates cell division in the archaeon Halobacterium salinarum.⁶⁶

Figure 2.

Cross-talk between cAMP and quorum sensing pathways in E. coli (dark green), P. aeruginosa (purple), and Vibrio species (orange). Arrows represent effects or signaling molecule/transcript levels that are promoted by the input, while flat ended lines represent effects/concentrations that are inhibited by the input.

In contrast to the generally antagonistic regulatory relationship between QS- and c-di-GMP-dependent cascades, 3’,5’-cAMP positively regulates AI-2 signaling in both vibrios and pseudomonads. In the halophilic and sepsis-inducing Vibrio vulnificus, transcription of the AI-2-binding global transcriptional regulator SmcR (homologous to Vibrio fiscerhi LuxR)⁶⁷ is induced by the 3’,5’-cAMP-CRP complex.⁶⁸ Additionally, the related pathogen Vibrio cholerae up-regulates production of the CAI-1 quormone in a CRP-dependent fashion. The resulting increase in CAI-1 synthesis induces expression of hapR, encoding a transcriptional activator of haemagglutinin (HA)/protease. Accordingly, deletion of either crp or cqsA (encoding CAI-1 synthase) decreases production of HA/protease.⁶⁹ In addition, the reduced HapR level in the crp and cqsA mutants elevates biofilm formation and cholera toxin (CT) production,⁶⁹ in agreement with the repressive role of HapR on genes encoding exopolysaccharides and CT (Fig. 2).^70–71 Collectively, these findings demonstrate that 3’,5’-cAMP-CRP elevates CAI-1 synthesis, resulting in up-regulated HA/protease production and attenuated synthesis of CT and biofilm.⁶⁹ Thus, while inhibition of 3’,5’-cAMP and/or CAI-1 signaling is an attractive method to repress HA/protease expression, such a strategy likely would stimulate production of CT and biofilm, highlighting important considerations in developing clinically effective inhibitors of virulence and biofilm formation. These studies reveal that catabolite repression modulates pathogenicity in vibrios via regulation of CAI-1 and AI-2 in V. cholerae and V. vulnificus, respectively. Similarly, the lasR promoter in Pseudomonas aeruginosa contains the prototypical CRP-binding consensus sequence, and subsequent β-galactosidase reporter experiments revealed that P. aeruginosa Vfr (homologous to E. coli CRP) induces lasR expression, linking 3’,5’-cAMP-dependent regulation to AHL signaling (Fig. 2).⁷² Interestingly, separate research identified an inverse correlation between 3’,5’-cAMP and c-di-GMP levels in P. aeruginosa,⁷³ further demonstrating the overlapping regulatory features which tune the balance between virulence gene expression and biofilm production.

E. coli CRP also regulates QS via modulation of AI-2 signaling. However, 3’,5’-cAMP-CRP inhibits AI-2 production by indirectly repressing luxS expression, indicating that antagonistic links exist between 3’,5’-cAMP and autoinducer production in certain taxa.⁷⁴ In addition, 3’,5’-cAMP-CRP directly activates transcription of the lsr operon, thereby increasing AI-2 uptake (Fig. 2);⁷⁴ thus 3’,5’-cAMP links the intracellular AI-2 level to carbon catabolism. Intriguingly, enterohemorrhagic E. coli O157:H7 deficient for the luxS AI-2 synthase exhibit decreased swimming motility and reduced production of the translation inhibitor Shiga toxin 2 (Stx2), demonstrating important functions for AI-2 signaling and/or SAM metabolism (from which AI-2 is derived) in host colonization and virulence factor expression.⁷⁵ Additional research in E. coli has identified complex interactions between 3’,5’-cAMP, c-di-GMP, AHL-type QS, and AI-2-mediated QS. Although E. coli does not produce AHL autoinducers due to the lack of a LuxI homologue, it does express a LuxR-type AHL receptor (SdiA) which can intercept AHLs produced by other taxa.⁷⁶ The SdiA regulon includes the EAL-encoding gene ydiV, which is induced through direct binding of SdiA to the promoter, indicating that E. coli modulates c-di-GMP signaling in response to extraneous AHLs in multi-species bacterial communities. Furthermore, SdiA and YdiV converge to up-regulate the 3’,5’-cAMP concentration in E. coli through unknown mechanisms, presumably resulting in global transcriptional changes mediated by 3’,5’-cAMP-bound CRP (Fig. 2). The intracellular AI-2 level also is regulated cooperatively by SdiA and YdiV, as the ∆sdiA-ydiV double mutant exhibits decreased expression of the AI-2 receptor lsrR and the lsrACDBFG operon involved in AI-2 uptake.⁷⁷ These discoveries reveal the convergence of multiple nucleotide and QS signaling systems in the integration of environmental and ecological factors to tune global gene expression in E. coli. Thus, bacterial behavior is coordinated not only by interplay between individual nucleotide and QS systems, but also by regulatory hierarchies linking different nucleotide-driven pathways and distinct QS circuits.

c-di-GMP and 3’,5’-cAMP

The intersection between the nucleotide signals c-di-GMP and 3’,5’-cAMP (Fig. 3) is typically antagonistic and associated with changes in biofilm formation. To date, the changes in biofilm formation has been demonstrated to typically be caused by altered transcript levels that encode for proteins involved in the various nucleotide metabolic pathways, rather than through direct binding to cNMP metabolic enzymes. For instance, in Vibrio cholerae, 3’,5’-cAMP binding to CRP inhibits biofilm formation through repression of biofilm-associated gene expression.⁷⁸ The dysregulated genes include those encoding a number of diguanylate cyclases and c-di-GMP phosphodiesterases, as well as two transcriptional regulators (vpsR and vpsT) that control expression of the vps genes that encode for the biosynthetic machinery to produce Vibrio polysaccharide (VPS), a key component of biofilms (Fig. 3).^79–80 The DGC-encoding gene cdgA was identified as the key target of cAMP-CRP mediated biofilm inhibition, as cdgA up-regulates transcription of genes encoding proteins involved in VPS biosynthesis and matrix protein production.⁸¹

Figure 3.

Intercommunication between c-di-GMP and cAMP pathways in P. aeruginosa (purple), V. cholerae (orange), and K. pneumoniae (magenta). Arrows represent effects or signaling molecule/transcript levels that are promoted by the input, while flat ended lines represent effects/concentrations that are inhibited by the input.

Additionally, 3’,5’-cAMP and c-di-GMP cooperate to regulate expression of gbpA in V. cholerae, which encodes a protein that promotes attachment to aquatic surfaces and host epithelial cells in the intestine.⁸² Depleting c-di-GMP levels leads to increased gbpA transcription but requires the presence of the adenylate cyclase (encoded by cya) and the 3’,5’-cAMP receptor CRP, demonstrating that gpbA transcription is under CRP control (Fig. 3). Although reduced c-di-GMP concentration results in increased 3’,5’-cAMP synthesis and gbpA expression, levels of crp transcript and protein do not change and c-di-GMP does not interfere with binding of 3’,5’-cAMP-CRP to the gpbA promoter.⁸³ As c-di-GMP levels do not globally impact the CRP regulon, the mechanism of gbpA transcriptional regulation by c-di-GMP and 3’,5’-cAMP-CRP is unclear. However, these data suggest an unknown mediator protein governs the expression of specific CRP transcriptional targets in a c-di-GMP-dependent fashion and highlight the intersection of c-di-GMP/cAMP signaling at the level of transcriptional control.

The links in Vibrio between biofilm formation, c-di-GMP, and cAMP are mirrored in other bacterial species. Studies in Pseudomonas aeruginosa demonstrated similar indirect intersecting mechanisms of biofilm regulation by c-di-GMP and cAMP. Binding of cAMP to Vfr, the P. aeruginosa homologue of CRP, results in up-regulated levels of the biofilm-related response regulator phospho-AlgR, which induces expression of PilY1, a protein involved in pilus assemblies and required for adhesion to surfaces (Fig. 3).⁸⁴ Expression of PilY1 then activates the diguanylate cyclase SadC, increasing c-di-GMP levels and further promoting biofilm formation.^85–87

Similar linkages between c-di-GMP and cAMP at the transcriptional levels are found within Klebsiella pneumoniae, highlighting the generality of the antagonistic effects. Expression of type 3 fimbriae (encoded by mrkABCDF), which facilitate biofilm formation on surfaces,^88–89 is positively regulated by c-di-GMP through the mrkHIJ operon, which encodes a c-di-GMP-binding PilZ protein that induces fimbriae expression,^90–91 a LuxR-type regulator,⁹⁰ and a c-di-GMP phosphodiesterase,⁹² respectively. Expression of the phosphodiesterase MrkJ results in decreased c-di-GMP levels and repression of mrkABCDF transcription and type 3 fimbriae expression.⁹² In contrast, decreased cAMP levels (caused by increased glucose concentration) results in increased MrkA production, while addition of exogenous cAMP abrogates this effect.⁹³ Taken together, recent work has suggested that c-di-GMP and cAMP bind cognate effectors to elicit opposing effects on biofilm formation through differential transcriptional regulation of the type 3 fimbriae operon (Fig. 3). While the mechanisms by which these cyclic nucleotide pathways interconnect are still relatively unknown, these types of studies suggest that additional proteins that monitor 3’,5’-cAMP and c-di-GMP levels may occur in many Gram-negative bacteria.

(p)ppGpp, QS, and c-di-GMP

The stringent response in bacteria is activated in response to stress conditions, including nutrient deprivation (amino acid starvation, iron limitation, etc.) and heat shock, and is controlled through production of the nucleotide signal guanosine 5’-tri/diphosphate 3’-diphosphate ((p)ppGpp) by Rel/Spo homologues and small alarmone synthetase proteins (SAS).⁹⁴ Increased (p)ppGpp levels regulate transcription of numerous genes, typically upregulating genes related to stress responses and down-regulating genes related to growth.⁹⁵ As (p)ppGpp exhibits far-reaching regulatory roles, recent work demonstrating that the stringent response can modulate biofilm formation and quorum sensing hints at (p)ppGpp potentially having a role as a “master” second messenger (Fig. 4).⁹⁶

Figure 4.

Regulation of other nucleotide and quorum sensing pathways by (p)ppGpp in V. cholerae (orange), P. aeruginosa (purple), and P. atrosepticum (blue). Arrows represent effects or signaling molecule/transcript levels that are promoted by the input, while flat ended lines represent effects/concentrations that are inhibited by the input.

Within P. aeruginosa, triggering the stringent response through membrane perturbation leads to increased RelA expression, which regulates the stringent response through (p)ppGpp production, resulting in elevated levels of the AHL synthase genes las and rhl (Fig. 4).⁹⁷ These data demonstrate a direct link between stringent response induction and quorum sensing and suggest that (p)ppGpp levels are above quorum sensing in the signaling hierarchy.⁹⁸ Furthermore, studies in Pectobacterium atrosepticum, a Gram-negative soft rot plant pathogen, have demonstrated that inhibiting the stringent response through supplementation with nutrients or deletion of the (p)ppGpp synthase genes (ΔrelAΔspoT) decreases production of P. atrosepticum AHL (3-oxo-hexanoyl-L-homoserine lactone (OHHL)) and plant cell wall degradation enzymes, which are key virulence factors for infection of plant hosts (Fig. 4).⁹⁹ The effects of (p)ppGpp on AHL-dependent virulence factor production are mediated by increasing expression of rsmB, an antagonist of RsmA, which represses transcription of plant cell wall degrading enzymes. These studies directly link (p)ppGpp signaling and downstream pathways to modulation of quorum sensing molecules and virulence.

These results have been extended to the effects on biofilm formation in Vibrio cholerae, where experiments have demonstrated that (p)ppGpp levels (modulated through deletion of rel genes) control biofilm formation. The rel deletion strains exhibit decreased transcript levels of the vps genes involved in Vibrio polysaccharide production, a major component of Vibrio biofilms (Fig. 4).¹⁰⁰ While many further studies remain to be done to further link (p)ppGpp signaling to other cellular pathways, these early results highlight intriguing links to quorum sensing and biofilm formation and suggest that the stringent response may be a potential target for modulating multiple, key bacterial pathways involved in infection.

Links between primary nucleotide metabolism, c-di-GMP, ppGpp, and QS

Recent pharmacological and genetic studies in different bacterial taxa have elucidated associations between primary nucleotide metabolism, biofilm formation, and virulence factor production. Inhibition of the essential purine biosynthesis enzyme 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylase with the purine analog azathioprine reduces biofilm production in E. coli by depleting purine nucleotide pools and consequently decreasing the c-di-GMP concentration.¹⁰¹ Interestingly, de novo pyrimidine synthesis and salvage also influence biofilm production, as disruption of any UMP biosynthesis gene attenuates curli amyloid synthesis in E. coli.¹⁰² Furthermore, the polymeric composition of the E. coli biofilm is modulated by the availability of pyrimidine bases; an abundance of pyrimidines favors UMP synthesis through nucleotide salvage and up-regulates cellulose production via allosteric activation of DgcQ by UTP. Conversely, reliance on de novo pyrimidine synthesis decreases cellulose biogenesis due to allosteric inhibition of DgcQ by N-carbamoyl L-Asp (an intermediate in de novo pyrimidine synthesis).^102–103 These findings illustrate the therapeutic potential of modulating nucleotide metabolism to intercept c-di-GMP signaling and disrupt biofilm formation.

In addition, a genome-wide transposon screen in P. aeruginosa identified novel associations between uracil metabolism and QS-regulated processes, including biofilm formation and virulence gene expression.¹⁰⁴ Specifically, disruption of de novo UMP biosynthesis genes (but not other pyrimidine or purine biosynthesis genes) down-regulates biofilm formation, and exogenous uracil reverses the hypo-biofilm phenotype. Moreover, disruption of de novo UMP biosynthesis attenuates transcription of genes involved in all three primary pseudomonad QS systems (Rhl, Las, and Pqs), as well as virulence-associated genes linked to chemotaxis, the type II secretion system, phenazine synthesis, and protease production. Thus, disruption of pyrimidine metabolism is a promising anti-virulence strategy, and this concept is further validated by the anti-biofilm and anti-QS activity of the antimetabolite 5-fluorouracil against P. aeruginosa.¹⁰⁴

Furthermore, the second messenger guanosine 3’,5’-bis(diphosphate) (ppGpp), which induces the stringent response upon nutrient depletion,^105–106 inhibits E. coli adenylosuccinate synthetase in vitro.¹⁰⁷ This enzyme catalyzes GTP-dependent O⁶-phosphorylation of IMP and subsequent condensation with L-Asp to produce adenylosuccinate,¹⁰⁸ suggesting that ppGpp directly represses de novo purine synthesis to conserve resources during starvation. Interestingly, crystallographic studies of adenylosuccinate synthetase identified guanosine 2’,3’-cyclic monophosphate 5’-diphosphate (5’-pp 2’,3’-cGMP) as a high-affinity ligand for the GTP binding site (with affinity exceeding that of ppGpp). While the biological relevance of the 5’-pp 2’,3’-cGMP-adenylosuccinate synthetase interaction requires further investigation, studies in our group have identified 2’,3’-cyclic nucleotide monophosphates (2’,3’-cNMPs) as novel modulators of diverse bacterial processes, including biofilm formation,¹⁰⁹ potentially through regulation of primary nucleotide metabolism. Collectively, these discoveries suggest that multiple nucleotide signals converge to modulate the cellular response to nutrient deprivation.

Outlook and Conclusions

In addition to the nucleotides discussed above, recent studies have identified novel nucleotide-based regulators that likely intersect with the signaling pathways described above. Specifically, our group is focused on determining the functions of 2’,3’-cyclic nucleotide monophosphates (2’,3’-cNMPs) in bacterial signaling. Recent work has elucidated that 2’,3’-cNMPs play a role in E. coli biofilm formation,¹⁰⁹ and ongoing studies employing (bio)chemical manipulation of 2’,3’-cNMP levels have identified functions for 2’,3’-cNMPs in regulating virulence-associated processes, potentially by modulating c-di-GMP signaling and primary nucleotide metabolism. Future efforts seek to elucidate the interactions of 2’,3’-cNMPs with other signaling pathways in controlling behavior in E. coli and in other Gram-negative bacteria. Furthermore, additional nucleotide second messengers are emerging as important regulators of prokaryotic physiology, such as diadenosine tetraphosphate (Ap4a), a side product of tRNA aminoacylation, which is found throughout all kingdoms of life.¹¹⁰ Recent genetic and phenotypic studies in P. fluorescens suggest that Ap4A stimulates GTP synthesis, ultimately increasing the c-di-GMP level and promoting surface adhesion .¹¹¹ These findings warrant further investigation into potential links between Ap4A and additional facets of nucleotide signaling and quorum sensing. Discovering new nucleotide messengers and determining how signaling molecules intersect must be studied in order to combat the rising antibiotic resistance and biofilm formation of bacteria. By understanding how bacteria utilize nucleotide messengers to regulate phentoypes will allow us to find new drug targets for antibiotics and to reduce biofilm formation in clinical settings. Furthermore, to tackle the increasing number of infections and agricultural damage caused by bacteria, work must be done to understand how bacterial signaling intersects with eukaryotic cells to cause infectious diseases.