Identification of signaling pathways, matrix-digestion enzymes, and motility components controllingVibrio choleraebiofilm dispersal

Abstract

Bacteria alternate between being free-swimming and existing as members of sessile multicellular communities called biofilms. The biofilm lifecycle occurs in three stages: cell attachment, biofilm maturation, and biofilm dispersal. Vibrio cholerae biofilms are hyper-infectious and biofilm formation and dispersal are considered central to disease transmission. While biofilm formation is well-studied, almost nothing is known about biofilm dispersal. Here, we conduct an imaging screen for V. cholerae mutants that fail to disperse, revealing three classes of dispersal components: signal transduction proteins, matrix-degradation enzymes, and motility factors. Signaling proteins dominated the screen and among them, we focused on an uncharacterized two-component sensory system that we name DbfS/DbfR for Dispersal of Biofilm Sensor/Regulator. Phospho-DbfR represses biofilm dispersal. DbfS dephosphorylates and thereby inactivates DbfR, which permits dispersal. Matrix degradation requires two enzymes: LapG, which cleaves adhesins, and RbmB, which digests matrix polysaccharide. Reorientations in swimming direction, mediated by CheY3, are necessary for cells to escape from the porous biofilm matrix. We suggest that these components act sequentially: signaling launches dispersal by terminating matrix production and triggering matrix digestion and, subsequently, cell motility permits escape from biofilms. This study lays the groundwork for interventions that modulate V. cholerae biofilm dispersal to ameliorate disease.Significance statementThe pathogen Vibrio cholerae alternates between the free-swimming state and existing in sessile multicellular communities known as biofilms. Transitioning between these lifestyles is key for disease transmission. V. cholerae biofilm formation is well studied, however, almost nothing is known about how V. cholerae cells disperse from biofilms, precluding understanding of a central pathogenicity step. Here, we conducted a high-content imaging screen for V. cholerae mutants that failed to disperse. Our screen revealed three classes of components required for dispersal: signal transduction, matrix degradation, and motility factors. We characterized these components to reveal the sequence of molecular events that choreograph V. cholerae biofilm dispersal. Our report provides a framework for developing strategies to modulate biofilm dispersal to prevent or treat disease.

Inverse regulation of Vibrio cholerae biofilm dispersal by polyamine signals

“…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 NpsS-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.…”

“…The biofilm lifecycle was measured using time-lapse microscopy as described previously (Bridges and Bassler, 2019). All plots were generated using ggplot2 in R. Light production driven by the vpsL promoter was monitored as described previously (Bridges et al, 2020). Results from replicates were averaged and plotted using ggplot2 in R.…”

“…The V. cholerae biofilm lifecycle occurs in three stages. First, a founder cell attaches to a substate, second, through cell 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).…”

“…To uncover the 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). To identify the components that orchestrate V. cholerae biofilm dispersal, 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.…”

“…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).…”