Controlling Biofilm Development Through Cyclic di-GMP Signaling

Abstract

The cyclic di-GMP (c-di-GMP) second messenger represents a signaling system that regulates many bacterial behaviors and is of key importance for driving the lifestyle switch between motile loner cells and biofilm formers. This review provides an up-to-date summary of c-di-GMP pathways connected to biofilm formation by the opportunistic pathogen P. aeruginosa. Emphasis will be on the timing of c-di-GMP production over the course of biofilm formation, to highlight non-uniform and hierarchical increases in c-di-GMP levels, as well as biofilm growth conditions that do not conform with our current model of c-di-GMP.

Keywords: Biofilm; Cyclic di-GMP; Diguanilate cyclase; Nosocomial infection; Phosphodiesterase; Second messenger.

Figure 1.. Surface behavior regulation in P. aeruginosa.

(A) The Wsp chemosensory pathway. The Wsp system are composed of a methyl-accepting protein (WspA), a CheR-like methyltransferase (WspC), a CheB-like methylesterase (WspF), two CheW homologues (WspB and WspD), a CheA homologue (WspE), and a diguanylate cyclase (WspR). The association of surfaces leads to the activation of WspA, which promotes the autophosphorylation of WspE. WspE phosphorylates its two response regulators, WspR and WspF. Phosphorylated WspR catalyzes the synthesis of c-di-GMP via its GGDEF domain while phosphorylated WspF resets the Wsp system by removing methyl groups from WspA. (B) The Chp/FimS/AlgR network. A surface-associated signal sensed by PilJ activates the Pil-Chp complex and induces the production of cAMP via activation of CyaB. The cAMP then binds to and activates the transcription factor Vfr, which stimulates pilY1 gene expression along with the response regulator AlgR. Upon complete assembly of TFP apparatus, PilY1 is secreted. The external PilY1 acts as a signal to induce SadC activity. The diguanylate cyclase SadC and the phosphodiesterase BifA inversely regulate c-di-GMP levels. P, protein phosphorylation or phosphotransfer reaction. OM, outer membrane. PG, peptidoglycan. IM, inner membrane.

Figure 2.. Model of c-di-GMP modulating systems contributing to surface contact sensing and transition from the reversible to the irreversible attachment stage in P. aeruginosa.

(A) Surface contact sensing requires the Pil-Chp and Wsp system, with activation leading to a hierarchical regulatory cascade of second messengers, cAMP and c-di-GMP, to coordinate the initial surface behaviors. cAMP levels are increased prior to c-di-GMP. (B) Psl/c-di-GMP feedback loop. Psl polysaccharide deposited by P. aeruginosa migrating across a surface acts as a signal to stimulate the two diguanylate cyclases, SiaD and SadC, to produce c-di-GMP. (C) Transition to the irreversible attachment stage requires c-di-GMP modulating systems SadC/BifA, HptB/Gac, FleQ, Lap, and SagS/NicD/PA3177. C-di-GMP levels likely increase in a hierarchical or stepwise manner upon first contact with the surface to ensure P. aeruginosa remains engaged with the surface. This increase may be linked to the sequential activation of c-di-GMP modulating systems, although little is known about the timing of activation relative to each other. As a consequence of increased c-di-GMP production, flagellar driven motility ceases while matrix production is increased.

Figure 3.. Modulation of the c-di-GMP pool by HptB and intersecting regulatory systems.

Expression of RsmY/Z through the HptB system is GacA-dependent. Under planktonic conditions, SagS regulates small regulatory RNA (sRNA) levels in an HptB-dependent manner. Under biofilm growth conditions, SagS contributes via BfiSR to the suppression of the sRNA RsmZ level. Arrows and blunt-ended lines indicate stimulatory and inhibitory interactions, respectively. P, protein phosphorylation or phosphotransfer reaction. Proteins harboring diguanylate cyclase activity are shown in green.

Figure 4.. Model of c-di-GMP modulating systems contributing to biofilm development including biofilm maturation and maintenance.

The formation of biofilms is a cyclic process that occurs in a stage-specific and progressive manner. The process is initiated following surface contact by single planktonic cells. Several developmental steps are discernable as reversible attachment (step I), irreversible attachment (step II) and biofilm maturation (steps III and IV) (9, 13). During reversible attachment, bacteria sense surface contact via the Pil-Chp and Wsp system, apparent by bacteria attaching to the substratum via the cell pole or via the flagellum (step I), followed by longitudinal attachment. Additional c-di-GMP modulating enzymes likely active at this stage include diguanylate cyclases GcbA, SiaD and SadC. Transition to the irreversible attachment stage requires c-di-GMP modulating systems HptB/Gac, SadC/BifA, and SagS/NicD/PA3177 and coincides with a reduction in flagellar reversal rates, reduction in flagella gene expression and the production of biofilm matrix components (step II). This stage is also characterized by attached cells demonstrating drug tolerance, with drug tolerance having been linked to the action of diguanylate cyclase PA3177. Biofilm maturation stages are characterized by the appearance of cell clusters that are several cells thick and are embedded in the biofilm matrix (step III) which subsequently fully mature into microcolonies (step IV). Screens of c-di-GMP modulating enzymes revealed GGDEF-domain only enzymes PA0169 (SiaD), PA0290, PA0338, PA3702 (WspR), PA1120 (YfiN), PA1107 (RoeA), PA2870, PA3177, PA4332 (SadC), PA4843 (GcbA), PA5487, HDGYP-domain only enzymes PA2572, PA4781, and EAL-GGDEF domain enzymes PA4367 (BifA), PA4602 (MorA), PA5017 (DipA) to contribute to the maturation of P. aeruginosa biofilms. Maintenance of the mature biofilm architecture, although not recognized as a distinct stage, has been shown to require the phosphodiesterases MorA and RmcA, likely by controlling polysaccharide production. Dispersion (stage V) coincides with the return to the planktonic mode of growth and low c-di-GMP levels, requiring the action of several c-di-GMP modulating enzymes including GcbA (PA4843), NicD (PA4929), DipA (PA5017), RbdA (PA0861), and NbdA (PA3311). Dispersion has been reported to coincide with the decrease in and degradation of matrix components, with dispersed cells being motile and demonstrating increased drug susceptibility relative to biofilm cells. Biofilm matrix is shown in beige. Variations in the c-di-GMP level over the course of biofilm development are indicated by the thickness of the red arrows.

Figure 5.. Dispersion cue perception and relay resulting in the modulation of c-di-GMP levels.

The membrane-associated MucR (EAL-GGDEF domain), NbdA (EAL domain only) and NicD (GGDEF domain only) are involved in perceiving and relaying dispersion cues to promote the modulation of c-di-GMP levels. The diguanylate cyclase NicD and the phosphodiesterase NbdA contribute to dispersion in a cue-specific manner, with NbdA sensing NO and NicD sensing nutrient cues. MucR has been reported to perceive both NO and nutrient cues. Receptors for other dispersion cues such as heavy metals or the fatty acid cis-2-decenoinc acid (cis-DA) have not yet been elucidated (indicated by the question mark). Relay of dispersion cues resulting in overall reduced c-di-GMP levels requires the chemotaxis-like MCP homolog, BdlA, and the c-di-GMP phosphodiesterases DipA and RbdA. BdlA, DipA, and RbdA are central to the dispersion response by NO, nutrients and heavy metals. It is unclear, however, whether BdlA, DipA and RbdA form a signaling cascade with NbdA or are involved in cis-DA induced dispersion (see question mark). BdlA is activated post dispersion cue sensing. Activation of BdlA involves phosphorylation and a burst of c-di-GMP, generated by diguanylate cyclases GcbA and NicD. Active BdlA recruits phosphodiesterase RbdA and enhances the activity of phosphodiesterase DipA, resulting in an overall reduction in c-di-GMP levels and subsequently, dispersion. No domain resolution is shown. For BdlA, domains are indicated only to indicate non-processive cleavage for BdlA activation. P, phosphorylation. Arrows indicate increased enzyme activity. IM, inner membrane. ?, unknown