Elsevier

Biotechnology Advances

Volume 56, May–June 2022, 107915
Biotechnology Advances

Research review paper
Biofilm control by interfering with c-di-GMP metabolism and signaling

https://doi.org/10.1016/j.biotechadv.2022.107915Get rights and content

Abstract

Biofilm formation and biofilm-induced biodeterioration of surfaces have deeply affected the life of our community. Cyclic dimeric guanosine monophosphate (c-di-GMP) is a small nucleotide-based signaling molecule in bacteria, which functions as a second messenger mediating a wide range of bacterial processes, such as cell motility, biofilm formation, virulence expression, and cell cycle progression. C-di-GMP regulated phenotypes are triggered by a variety of determinants, such as metabolic cues and stress factors that affect c-di-GMP synthesis, the transduction and conducting of signals by specific effectors, and their actions on terminal targets. Therefore, understanding of the regulatory mechanisms of c-di-GMP would greatly benefit the control of the relevant bacterial processes, particularly for the development of anti-biofilm technologies. Here, we discuss the regulatory determinants of c-di-GMP signaling, identify the corresponding chemical inhibitors as anti-biofilm agents, and shed light on further perspectives in the metabolic regulation of c-di-GMP through chemical and biological approaches. This review will advance the development of anti-biofilm policies applied in the industries of medicine, environment and engineering.

Introduction

Biofilms are increasingly recognized as the most successful lifestyle of microorganisms in the biosphere, including medical, engineered, and environmental contexts, which are notorious for their resistance to environmental stresses and host immune clearance, including antimicrobial compounds (Abedon, 2015; Brackman and Coenye, 2015; Chung and Toh, 2014). For example, antimicrobial tolerance often links biofilms with persistent and chronic infections, and provides ideal conditions for the acquisition or evolution of antimicrobial resistance (Roy et al., 2018). Biofilms in the built environment can cause severe biodeterioration of engineering materials (Gu et al., 1998; Liu et al., 2020; Liu et al., 2018; Wan and Gu, 2018), biofouling on the surface of the pipelines (Liu et al., 2014) or ships (Schultz et al., 2011), and other environmental issues (Lan et al., 2010; Mitchell and Gu, 2000). Bacteria can switch from free-living lifestyle to surface adapted, structured lifestyle known as biofilms, which is often regulated by the nucleotide second messenger, bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP), with inputs of a wide variety of environmental cues (Jenal et al., 2017). These environmental cues can be transduced and reflected by intracellular c-di-GMP levels, which further lead to various allosteric alterations of the c-di-GMP effectors that regulate a diversity of bacterial processes (Colley et al., 2016; Hengge, 2009; Hengge et al., 2015). High intracellular c-di-GMP levels can promote the synthesis of adhesins and exopolysaccharide substances that enhance biofilm formation, while low intracellular c-di-GMP levels can increase cell motility and cause biofilm dispersal. Therefore, intracellular c-di-GMP levels control the shift between biofilm and planktonic lifestyles of bacteria (Chua et al., 2015; Sheppard and Howell, 2016; Simm et al., 2004; Steiner et al., 2013). Furthermore, c-di-GMP regulates the virulence expression in pathogens (Hall and Lee, 2018), the production of antibiotics (Okegbe et al., 2017), cell cycle progression (Lori et al., 2015; Paul et al., 2004), and other cellular functions. Both the regulatory mechanisms that underlie molecular processes and the actions on the downstream targets that are influenced by c-di-GMP effectors require further exploration to understand the crucial role of c-di-GMP signaling in biofilm formation, which will greatly help to develop the anti-biofilm agents or technologies.

At the metabolic level, c-di-GMP is synthesized from two GTP molecules by diguanylate cyclases (DGCs) and is degraded by specific phosphodiesterases (EAL domain-containing PDEs) into the linear 5′-phosphoguanylyl-(3′-5′)-guanosine (pGpG) dinucleotide that is further broken down into two molecules of GMP by PDE-Bs, or is directly split into GMPs by HD-GYP domain-containing PDEs (Fig. 1). The activity of DGC is relevant to the GGDEF (Gly-Gly-Asp-Glu-Phe) domain that is named with a conserved sequence of the five essential amino acids of the active site of the enzyme (Paul et al., 2004). The EAL or HD-GYP domains are essential for the enzymatic activities of c-di-GMP-specific PDEs (Christen et al., 2005). However, proteins containing these domains show different mechanisms in diverse species. For example, certain proteins with GGDEF, EAL or HD-GYP domains are linked to other signal-sensory domains, such as Per-Arnt-Sim (PAS) domains (Galperin, 2006). Some DGCs can be allosterically regulated by c-di-GMP (as discussed below).

Inspired by whole genome sequencing and bioinformatic analysis, interest in such c-di-GMP regulatory systems has dramatically increased, when the above-mentioned domain-containing proteins were found not only ubiquitous in bacteria, but also potential in many other prokaryotic species (Galperin, 2010). Genetic and phenotypic studies showed that artificial overexpression of the GGDEF domain-containing proteins could largely enhance the synthesis of adhesins and exopolysaccharide substances and strongly inhibit the cell motility and acute virulence, whereas the overexpression of EAL domain-containing proteins resulted in the opposite phenotypes (Karatan and Watnick, 2009; Tischler and Camilli, 2004). Most importantly, current studies are now exploring the molecular functions of these specific proteins to uncover their roles in the regulatory network of c-di-GMP signaling. For example, certain degenerate GGDEF and EAL proteins have been reported to participate in the transcriptional regulation as the repressors of c-di-GMP signaling (as discussed below) (Cursino et al., 2015; Yang et al., 2014). Understanding the molecular functions of these specific proteins will not only help us elucidate the regulatory modules of c-di-GMP signaling but promote the development of corresponding chemical inhibitors to control the c-di-GMP signaling, and thereby regulate the relevant biological processes at will.

This review aims to shed light on the anti-biofilm technologies by targeting the determinants of the c-di-GMP signaling network from signal input to target output. Specifically, the metabolism of c-di-GMP with signal inputs, the transduction and conduction of signals by effectors, and the action on terminal targets will be discussed by highlighting the relevant components that take part in the regulation of the c-di-GMP signaling network, such as DGCs, PDEs and a diversity of effectors. Current chemical inhibitors will be summarized and discussed for the regulation of the c-di-GMP signaling network to advance the exploration of anti-biofilm agents and anti-biofouling technologies. These two strategies do not pose a strong selective pressure to raise antibacterial resistance of mutants since they are not based on direct microbial cell killing.

Section snippets

Regulatory determinants of c-di-GMP signaling

The control module of c-di-GMP signaling typically comprises of four components: two enzymes that catalyze the synthesis and degradation of c-di-GMP with the input of certain signals, an effector with subsequent allosteric regulation after binding with c-di-GMP, and a target that responds to the effector with the output of certain molecular substances (Fig. 2) (Hengge, 2009). Signaling among such components has genetically been defined in certain common phenotypes in model species, such as

Inhibitors of c-di-GMP signaling

Given the close relationship between the c-di-GMP signaling network and the regulation of bacterial processes, inhibition of the c-di-GMP signaling would have great significance in the development of novel antibacterial agents for medical, agricultural and food industries (Liu et al., 2014). On the basis of the regulatory determinants of c-di-GMP signaling discussed above, the novel antibacterial chemicals should mainly target the DGCs, PDEs and effectors.

Conclusions and perspectives

To summarize, c-di-GMP as a second messenger in bacteria plays a crucial role in mediating the behaviors of bacterial community, microbial biofilm formation in particular. Although the enzymes that rule the metabolism of c-di-GMP have been well identified, more studies on molecular and chemical biology should be carried out to develop an easily-controlled regulatory module of c-di-GMP for further applications in the treatment of environmental pollutants (Wu et al., 2016; Wu et al., 2015), the

Ethics approval and consent to participate

Not Applicable.

Consent for publication

Not Applicable.

Availability of data and material

Not Applicable.

Funding

This work was supported by the National Natural Science Foundation of China (Grant No. 32100101 and 92051103).

Authors' contributions

X.L. and J.-D.G wrote, reviewed, and approved the manuscript with inputs from all co-authors.

Declaration of Competing Interest

There are no conflicts of interest to declare.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant No. 32100101 and 92051103). We appreciate the international reviewers for their constructive suggestions to the preprint of this paper.

References (108)

  • D.A. Ryjenkov et al.

    The PilZ domain is a receptor for the second messenger c-di-GMP

    J. Biol. Chem.

    (2006)
  • D.C. Sheppard et al.

    Biofilm exopolysaccharides of pathogenic fungi: lessons from bacteria

    J. Biol. Chem.

    (2016)
  • S. Srey et al.

    Biofilm formation in food industries: a food safety concern

    Food Control

    (2013)
  • A. Sundriyal et al.

    Inherent regulation of EAL domain-catalyzed hydrolysis of second messenger c-di-GMP

    J. Biol. Chem.

    (2014)
  • N. Tschowri et al.

    Tetrameric c-di-GMP mediates effective transcription factor dimerization to control Streptomyces development

    Cell

    (2014)
  • A. Winkler et al.

    Characterization of elements involved in allosteric light regulation of phosphodiesterase activity by comparison of different functional BlrP1 states

    J. Mol. Biol.

    (2014)
  • S.T. Abedon

    Ecology of anti-biofilm agents II: bacteriophage exploitation and biocontrol of biofilm bacteria

    Pharmaceuticals

    (2015)
  • P. Aldridge et al.

    Role of the GGDEF regulator PleD in polar development of Caulobacter crescentus

    Mol. Microbiol.

    (2003)
  • J.B. Andersen et al.

    Identification of small molecules that interfere with c-di-GMP signaling and induce dispersal of Pseudomonas aeruginosa biofilms

    npj Biofilms Microb.

    (2021)
  • A.E. Baker et al.

    PilZ domain protein FlgZ mediates c-di-GMP-dependent swarming motility control in Pseudomonas aeruginosa

    J. Bacteriol.

    (2016)
  • A.S. Ball et al.

    Quorum sensing gene regulation by LuxR/HapR master regulators in vibrios

    J. Bacteriol.

    (2017)
  • C. Baraquet et al.

    Cyclic diguanosine monophosphate represses bacterial flagella synthesis by interacting with the Walker a motif of the enhancer-binding protein FleQ

    Proc. Natl. Acad. Sci.

    (2013)
  • T.R. Barends et al.

    Structure and mechanism of a bacterial light-regulated cyclic nucleotide phosphodiesterase

    Nature

    (2009)
  • N. Barraud et al.

    Nitric oxide signaling in Pseudomonas aeruginosa biofilms mediates phosphodiesterase activity, decreased cyclic di-GMP levels, and enhanced dispersal

    J. Bacteriol.

    (2009)
  • A. Basu Roy et al.

    Diguanylate cyclase NicD-based signalling mechanism of nutrient-induced dispersion by Pseudomonas aeruginosa

    Mol. Microbiol.

    (2014)
  • D. Bellini et al.

    Crystal structure of an HD-GYP domain cyclic-di-GMP phosphodiesterase reveals an enzyme with a novel trinuclear catalytic iron centre

    Mol. Microbiol.

    (2014)
  • S. Beyhan et al.

    Identification and characterization of cyclic diguanylate signaling systems controlling rugosity in Vibrio cholerae

    J. Bacteriol.

    (2008)
  • E. Bordeleau et al.

    C-di-GMP riboswitch-regulated type IV pili contribute to aggregation of Clostridium difficile

    J. Bacteriol.

    (2014)
  • G. Brackman et al.

    Quorum sensing inhibitors as anti-biofilm agents

    Curr. Pharm. Des.

    (2015)
  • P.K. Brown et al.

    MlrA, a novel regulator of curli (AgF) and extracellular matrix synthesis by Escherichia coli and Salmonella enterica serovar typhimurium

    Mol. Microbiol.

    (2001)
  • C. Chan et al.

    Structural basis of activity and allosteric control of diguanylate cyclase

    Proc. Natl. Acad. Sci.

    (2004)
  • S.-H. Chou et al.

    Diversity of c-di-GMP-binding proteins and mechanisms

    J. Bacteriol.

    (2015)
  • S.L. Chua et al.

    C-di-GMP regulates Pseudomonas aeruginosa stress response to tellurite during both planktonic and biofilm modes of growth

    Sci. Rep.

    (2015)
  • P.Y. Chung et al.

    Anti-biofilm agents: recent breakthrough against multi-drug resistant Staphylococcus aureus

    Pathogens Dis.

    (2014)
  • D. Cohen et al.

    Oligoribonuclease is a central feature of cyclic diguanylate signaling in Pseudomonas aeruginosa

    Proc. Natl. Acad. Sci.

    (2015)
  • B. Colley et al.

    SiaA/D interconnects c-di-GMP and RsmA signaling to coordinate cellular aggregation of Pseudomonas aeruginosa in response to environmental conditions

    Front. Microbiol.

    (2016)
  • L. Cursino et al.

    Characterization of the Xylella fastidiosa PD1671 gene encoding degenerate c-di-GMP GGDEF/EAL domains, and its role in the development of Pierce’s disease

    PLoS One

    (2015)
  • Y. Deng et al.

    Cis-2-dodecenoic acid receptor RpfR links quorum-sensing signal perception with regulation of virulence through cyclic dimeric guanosine monophosphate turnover

    Proc. Natl. Acad. Sci.

    (2012)
  • A. Duerig et al.

    Second messenger-mediated spatiotemporal control of protein degradation regulates bacterial cell cycle progression

    Genes Dev.

    (2009)
  • M. Fazli et al.

    The CRP/FNR family protein Bcam1349 is a c-di-GMP effector that regulates biofilm formation in the respiratory pathogen Burkholderia cenocepacia

    Mol. Microbiol.

    (2011)
  • M. Fazli et al.

    Regulation of Burkholderia cenocepacia biofilm formation by RpoN and the c-di-GMP effector BerB

    MicrobiologyOpen

    (2017)
  • S. Fernicola et al.

    In silico discovery and in vitro validation of catechol-containing sulfonohydrazide compounds as potent inhibitors of the diguanylate cyclase PleD

    J. Bacteriol., JB.

    (2015)
  • S. Fernicola et al.

    Synthesis of triazole-linked analogues of c-di-GMP and their interactions with diguanylate cyclase

    J. Med. Chem.

    (2015)
  • K. Furukawa et al.

    Identification of ligand analogues that control c-di-GMP riboswitches

    ACS Chem. Biol.

    (2012)
  • G.M. Gadd

    Geomicrobiology of the built environment

    Nat. Microbiol.

    (2017)
  • M.Y. Galperin

    Structural classification of bacterial response regulators: diversity of output domains and domain combinations

    J. Bacteriol.

    (2006)
  • C.L. Hall et al.

    Cyclic-di-GMP regulation of virulence in bacterial pathogens

    Wiley Interdiscip. Rev.: RNA

    (2018)
  • R. Hengge

    Principles of c-di-GMP signalling in bacteria

    Nat. Rev. Microbiol.

    (2009)
  • R. Hengge et al.

    Bacterial signal transduction by c-di-GMP and other nucleotide second messengers

    J. Bacteriol.

    (2015)
  • R. Hengge et al.

    Systematic nomenclature for GGDEF and EAL domain-containing cyclic di-GMP turnover proteins of Escherichia coli

    J. Bacteriol.

    (2016)
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