Research review paperBiofilm control by interfering with c-di-GMP metabolism and signaling
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
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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.
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