Inverse regulation of Vibrio cholerae biofilm dispersal by polyamine signals

Bacteria frequently colonize environmental habitats and infection sites by forming surface-attached multicellular communities called biofilms. Participating in the biofilm lifestyle allows bacteria to collectively acquire nutrients and resist threats (Flemming et al., 2016). By contrast, the individual free-swimming state allows bacteria to roam. The global pathogen Vibrio cholerae undergoes repeated rounds of clonal biofilm formation and disassembly, and both biofilm formation and biofilm exit are central to disease transmission as V. cholerae alternates between the marine niche and the human host (Conner et al., 2016; Gallego-Hernandez et al., 2020; Tamayo et al., 2010). The V. cholerae biofilm lifecycle occurs in three stages. First, a founder cell attaches to a substate; second, through cycles of growth, division, and extracellular matrix secretion, the biofilm matures; and finally, when the appropriate environmental conditions are detected, cells leave the biofilm through a process termed dispersal (Bridges et al., 2020). Bacterial cells liberated through dispersal can depart their current environment and repeat the lifecycle in a new niche, such as in a new host (Guilhen et al., 2017). In general, the initial stages of the biofilm lifecycle, from attachment through maturation, have been well studied in V. cholerae and other bacterial species, whereas the dispersal phase remains underexplored. Components underlying biofilm dispersal are beginning to be revealed. For example, in many bacterial species, the Lap system that is responsible for cell attachments during biofilm formation is also involved in dispersal (Boyd et al., 2012; Christensen et al., 2020; Gjermansen et al., 2005; Kitts et al., 2019; Newell et al., 2011).

To uncover mechanisms controlling V. cholerae biofilm dispersal, we recently developed a brightfield microscopy assay that allows us to follow the entire biofilm lifecycle (Bridges and Bassler, 2019). We combined this assay with mutagenesis and high-content imaging to identify mutants that failed to properly disperse (Bridges et al., 2020). Our screen revealed genes encoding proteins that fall into three functional groups: signal transduction, matrix degradation, and cell motility. Our hypothesis is that these classes of components act in sequence to drive biofilm dispersal. First, the cues that induce dispersal are detected by the signal transduction proteins. Second, activation of matrix digestion components occurs. Finally, cell motility engages and permits cells to swim away from the disassembling biofilm. The majority of the genes identified in the screen encoded signal transduction proteins. Elsewhere, we characterized one of the signaling systems identified in the screen, a new two-component phospho-relay that we named DbfS-DbfR (for Dispersal of Biofilm Sensor and Dispersal of Biofilm Regulator) that controls biofilm dispersal (Bridges et al., 2020). Of the remaining signaling proteins identified in the screen, four are proteins involved in regulating production/degradation of, or responding to, the second messenger molecule cyclic diguanylate (c-di-GMP).

c-di-GMP regulates biofilm formation in many bacteria including V. cholerae in which low c-di-GMP levels correlate with motility and high c-di-GMP levels promote surface attachment and matrix production, and thus, biofilm formation (Conner et al., 2017). c-di-GMP is synthesized by enzymes that contain catalytic GGDEF domains that have diguanylate cyclase activity (Conner et al., 2017). c-di-GMP is degraded by proteins with EAL or HD-GYP domains that possess phosphodiesterase activity. V. cholerae encodes >50 proteins harboring one or both of these domains, underscoring the global nature of c-di-GMP signaling in this pathogen. The activities of these enzymes are often regulated by environmental stimuli through ligand binding sensory domains; however, in most cases, the identities of ligands for GGDEF- and EAL-containing enzymes are unknown (Römling et al., 2013). Of the c-di-GMP regulatory proteins identified in our dispersal screen, only one, the hybrid GGDEF/EAL protein, MbaA, which has previously been shown to regulate V. cholerae biofilm formation (Bomchil et al., 2003), has a defined environmental stimulus: MbaA responds to the ubiquitous family of small molecules called polyamines, aliphatic cations containing two or more amine groups (Miller-Fleming et al., 2015). Our dispersal screen also identified PotD1, a periplasmic binding protein that mediates polyamine import and that has also been shown to control V. cholerae biofilm formation (Cockerell et al., 2014; McGinnis et al., 2009). Thus, identification of these two genes in our screen suggested that beyond roles in biofilm formation, polyamine detection could be central to biofilm dispersal. In the present work, we explore this new possibility.

Polyamines are essential small molecules derived from amino acids and were likely produced by the last universal common ancestor (Michael, 2018; Miller-Fleming et al., 2015). Different organisms make distinct blends of polyamines (Michael, 2018), and they are involved in biological processes ranging from stress responses to signal transduction to protein synthesis to siderophore production (Karatan et al., 2005; McGinnis et al., 2009; Michael, 2018). In some cases, mechanisms underlying polyamine function are known. For example, the eukaryotic translation initiation factor 5A is modified with hypusine that is derived from the polyamine spermidine (Puleston et al., 2019). In most cases, however, the mechanisms by which polyamines control physiology remain mysterious (Miller-Fleming et al., 2015).

Polyamines are known to be involved in biofilm development in many bacterial species (Hobley et al., 2014; Karatan and Michael, 2013; Michael, 2018; Nesse et al., 2015; Patel et al., 2006). Intriguingly, the biofilm alterations that occur in response to a given polyamine vary among species. In V. cholerae, norspermidine, a rare polyamine produced by Vibrionaceae and select other organisms, promotes biofilm formation (Karatan et al., 2005; Lee et al., 2009). In contrast, the nearly ubiquitous polyamine spermidine, which differs from norspermidine only by a single methylene group, is not produced at substantial levels by V. cholerae and it represses V. cholerae biofilm formation (Figure 1; Karatan et al., 2005; Lee et al., 2009; McGinnis et al., 2009). Likewise, the polyamine spermine, typically made by eukaryotes, also represses V. cholerae biofilm formation (Sobe et al., 2017). Both spermidine and spermine are abundant in the human intestine (Benamouzig et al., 1997; McEvoy and Hartley, 1975; Osborne and Seidel, 1990). These results have led to the hypothesis that V. cholerae detects norspermidine as a measure of ‘self’ and spermidine and spermine as measures of ‘other’ to assess the species composition of the vicinal community (Sobe et al., 2017; Wotanis et al., 2017). V. cholerae funnels that information internally to determine whether or not to make a biofilm (Sobe et al., 2017; Wotanis et al., 2017). V. cholerae produces intracellular norspermidine; however, norspermidine has not been detected in cell-free culture fluids of laboratory-grown strains (Parker et al., 2012). Thus, if norspermidine does indeed enable V. cholerae to take a census of ‘self,’ it is apparently not via a canonical quorum-sensing-type mechanism.

Figure 1

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As mentioned above, information contained in extracellular polyamines is transduced internally by MbaA, which is embedded in the inner membrane. MbaA contains a periplasmic domain that interacts with the periplasmic binding protein NspS. nspS is located immediately upstream of mbaA in the chromosome (Cockerell et al., 2014; Karatan et al., 2005; Young et al., 2021). MbaA also possesses cytoplasmic GGDEF (SGDEF in MbaA) and EAL (EVL in MbaA) domains (Figure 1). Genetic evidence suggests that when NspS is bound to norspermidine, the complex associates with MbaA and biofilm formation is promoted (Cockerell et al., 2014). Specifically, NspS interaction with MbaA inhibits MbaA phosphodiesterase activity, leading to increased c-di-GMP levels, and in turn, elevated biofilm formation (Figure 1, left panel) (Cockerell et al., 2014). It is proposed that Apo-NspS and NspS bound to spermidine or spermine do not bind to MbaA, and thus, under this condition, biofilm formation is reduced (Cockerell et al., 2014; Sobe et al., 2017). In this case, MbaA phosphodiesterase activity is not inhibited, which leads to decreased c-di-GMP levels and repression of biofilm formation (Figure 1, right panel). Currently, it is not known whether or not MbaA has diguanylate cyclase activity. The purified MbaA cytoplasmic domain functions as a phosphodiesterase in vitro (Cockerell et al., 2014).

In a proposed second regulatory mechanism, norspermidine and spermidine are thought to control biofilm formation via import through the inner membrane ABC transporter, PotABCD1 (McGinnis et al., 2009). Following internalization, via an undefined cytoplasmic mechanism, polyamines are proposed to modulate biofilm formation (Figure 1; McGinnis et al., 2009). This hypothesis was based on the finding that elimination of PotD1, which is required for import of norspermidine and spermidine, resulted in elevated biofilm formation. Importantly, the studies reporting the MbaA and PotD1 findings used quorum-sensing-deficient V. cholerae strains (Joelsson et al., 2006) that likely cannot disperse from biofilms. Furthermore, the end-point biofilm assays used could not differentiate between enhanced biofilm formation and the failure to disperse.

Here, we combine real-time biofilm lifecycle measurements, mutagenesis, a reporter of cytoplasmic c-di-GMP levels, and measurements of intra- and extracellular polyamine concentrations to define the roles that norspermidine and spermidine play in controlling the V. cholerae biofilm lifecycle. In wildtype V. cholerae, exogenous norspermidine and spermidine inversely alter cytoplasmic c-di-GMP levels and they exert their effects through the NspS-MbaA circuit. Norspermidine promotes biofilm formation and suppresses biofilm dispersal. Spermidine represses biofilm formation and promotes biofilm dispersal. Both the MbaA SGDEF and EVL domains are required for polyamine control of the biofilm lifecycle. We provide evidence that MbaA synthesizes c-di-GMP in the presence of norspermidine and degrades c-di-GMP in the presence of spermidine and that is what drives V. cholerae to form and disperse from biofilms, respectively. When MbaA is absent, V. cholerae is unable to alter the biofilm lifecycle in response to extracellular polyamines. We demonstrate that polyamine internalization via PotD1 is not required to promote V. cholerae entrance or exit from biofilms, but rather, periplasmic detection of polyamines by MbaA is the key regulatory step. Specifically, our results suggest that the ΔpotD1 mutant fails to disperse because it is unable to reimport self-secreted norspermidine. The consequence is that excess periplasmic norspermidine accumulates, is detected by MbaA, and leads to production of c-di-GMP and suppression of biofilm dispersal. Collectively, our work reveals the mechanisms by which V. cholerae detects and transduces the information contained in polyamine signals into modulation of its biofilm lifecycle. We propose that the polyamine sensing mechanisms revealed in this study allow V. cholerae to distinguish relatives from competitors and potentially the presence of predators in the vicinity and, in response, modify its biofilm lifecycle to appropriately colonize territory or disperse from an existing community.

In this study, we investigated the effects of the polyamines norspermidine and spermidine on V. cholerae biofilm dispersal. Norspermidine and spermidine, which differ in structure only by one methylene group, mediate starkly opposing effects on the V. cholerae biofilm lifecycle: norspermidine inhibits and spermidine promotes biofilm dispersal. Both function through the NspS-MbaA circuit. Thus, the polyamine binding protein NspS must harbor the exquisite capability to assess the presence or absence of a single chemical moiety as norspermidine binding drives NspS interaction with MbaA, whereas binding to spermidine prevents this interaction. Here, we speculate on the possible biological significance of our findings. We suspect that norspermidine and spermidine act as ‘self’ and ‘other’ cues, respectively. Specifically, norspermidine is a rare polyamine in the biosphere, produced only by select organisms, namely V. cholerae and closely related marine vibrios (Hamana, 1997; Michael, 2018; Yamamoto et al., 1991). The observation that laboratory-grown WT V. cholerae releases little norspermidine, at least in part due to PotD1-mediated reuptake (Figure 5C), suggests that this system does not behave like a canonical quorum-sensing pathway. However, it is possible that norspermidine is secreted by V. cholerae under some environmental conditions, or by other vibrios, and V. cholerae detects the released norspermidine via NspS-MbaA, and its dispersal from biofilms is prevented. Thus, when close relatives are nearby, as judged by detection of the presence of norspermidine, V. cholerae elects to remain in its current biofilm niche. More speculative is the notion that norspermidine functions as a cue for phage-, predator-, or toxin-induced cell lysis. Specifically, if V. cholerae can detect norspermidine released by neighboring lysed V. cholerae cells, in response to the perceived danger, V. cholerae would remain in the protective biofilm state. Conversely, detection of spermidine, a nearly ubiquitously produced polyamine (Michael, 2018), could alert V. cholerae to the presence of competing or unrelated organisms. In this case, V. cholerae would respond by dispersing from biofilms and fleeing that locale. In a seemingly parallel scenario, we previously discovered that autoinducer AI-2, a universally produced quorum-sensing signal, also drove V. cholerae biofilm repression and premature dispersal, while CAI-1, the V. cholerae ‘kin’ quorum-sensing autoinducer, did not (Bridges and Bassler, 2019). Together, our findings suggest that V. cholerae possesses two mechanisms to monitor its own numbers (norspermidine and CAI-1) and two mechanisms to take a census of unrelated organisms in the environment (spermidine and AI-2), and it responds by remaining in the biofilm state when closely related species are in the majority and by exiting the biofilm state when the density of non-related organisms is high, presumably to escape competition/danger.

We found that periplasmic polyamine detection by NspS-MbaA, but not polyamine import, controls the biofilm dispersal program and both the MbaA EVL and SGDEF domains are required for the V. cholerae response to polyamines. Despite previous work suggesting that MbaA functions exclusively as a phosphodiesterase and that its SGDEF domain, which possesses a serine substitution in the active site relative to the canonical GGDEF active site, is inert with respect to c-di-GMP synthesis, our data suggest that when norspermidine is present, MbaA does synthesize c-di-GMP (Figure 3C, Figure 4C), as has also been shown for other SGDEF proteins (Pérez-Mendoza et al., 2011). Therefore, MbaA is likely a bifunctional protein capable of both c-di-GMP synthesis and degradation. We wonder how such an arrangement benefits V. cholerae in its response to polyamines. We speculate that the ability of MbaA to inversely alter c-di-GMP levels in response to two discrete cues coordinates polyamine signaling and enables more rapid and greater magnitude changes in c-di-GMP levels than would be achievable if two separate receptors existed, each harboring only a single catalytic activity and each responsive to only one polyamine.

An analogous bifunctional arrangement exists for the quorum-sensing two-component receptor LuxN from Vibrio harveyi. LuxN is an inner membrane receptor that harbors a periplasmic sensory domain responsible for detection of a self-made small molecule cue, a homoserine lactone agonist with a four-carbon acyl tail (Bassler et al., 1994). The ligand encodes species-specific information about cell population density. Competing bacterial species that co-occupy the niche in which V. harveyi resides produce similar acyl homoserine lactone signal molecules, and notably, those molecules differ only in the acyl tail length or decoration (Papenfort and Bassler, 2016). The tails of the molecules produced by the competitors usually possess more than four carbons, and they act as potent LuxN antagonists. With respect to mechanism, the self-made agonist drives LuxN into phosphatase mode while the antagonists induce LuxN to act as a kinase (Ke et al., 2015). Thus, as in the MbaA circuit, binding of the different ligands to LuxN propels opposing enzymatic activities in the cytoplasm. We speculate that ligand-driven alternations between opposing enzymatic activities could be an underappreciated mechanism that sensors employ to transmit species-specific and species-non-specific information into discrete changes in downstream behaviors.

In addition to conveying information about the species composition of the vicinal community, the MbaA SGDEF and EVL domains could also participate in dimerization and/or allosteric binding to c-di-GMP or other metabolites to mediate feedback regulation of the opposing MbaA activities. Parallel examples exist, here we present one: Caulobacter crescentus contains a GGDEF/EAL protein called CC3396, in which the GGDEF domain is non-catalytic but it nonetheless binds to GTP, which allosterically activates the phosphodiesterase activity in the neighboring EAL domain (Christen et al., 2005). A feedback mechanism could provide MbaA with the ability to integrate the information encoded in extracellular polyamine blends with cytoplasmic cues, such as the metabolic state of the cell or the current cytoplasmic c-di-GMP level. Taken together, the double ligand detection capability linked to the dual catalytic activities of the NspS-MbaA circuit allows V. cholerae to distinguish between remarkably similar ligands and, in response, have the versatility to convey distinct information into the cell to alter the biofilm lifecycle.

In summary, four signaling pathways have now been defined that feed into the regulation of V. cholerae biofilm dispersal: starvation (RpoS) (Singh et al., 2017), quorum sensing (via CAI-1 and AI-2) (Bridges and Bassler, 2019; Singh et al., 2017), the recently identified DbfS/DbfR cascade (ligand unknown) (Bridges et al., 2020), and through the current work, polyamine signaling via NspS-MbaA. We propose that integrating the information contained in these different stimuli into the control of biofilm dispersal endows V. cholerae with the ability to successfully evaluate multiple features of its fluctuating environment prior to committing to the launch of this key lifestyle transition that, ultimately, impinges on its overall survival. Because V. cholerae biofilm formation and biofilm dispersal are intimately connected to cholera disease and its transmission, we suggest that deliberately controlling the biofilm lifecycle, possibly via synthetic strategies that target polyamine signal transduction via NspS-MbaA, could be a viable therapeutic strategy to ameliorate disease.

All data generated and analyzed in this study are included in the manuscript and supporting files. Source data files have been provided in Zenodo (https://doi.org/10.5281/zenodo.4651348).

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Author details

1. Andrew A Bridges

   1. Department of Molecular Biology, Princeton University, Princeton, United States
   2. The Howard Hughes Medical Institute, Chevy Chase, United States

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - reviewing and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8132-751X

2. Bonnie L Bassler

   1. Department of Molecular Biology, Princeton University, Princeton, United States
   2. The Howard Hughes Medical Institute, Chevy Chase, United States

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   bbassler@princeton.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0043-746X

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