Signal Transduction Architectures Underlying Population-Scale Decisions in Vibrio cholerae

Abstract

Biofilms are communities of bacterial cells engulfed in a self-produced matrix that protects resident cells from threats, including antibiotics, shear stress, and predation. Over the past two decades, the intracellular and intercellular signaling systems that control the biofilm lifecycle have been revealed. In many species of bacteria, including the global pathogen and model biofilm former Vibrio cholerae, two core signaling systems play overarching roles in driving bacterial populations to form biofilms, and these systems dictate biofilm architectural features. First, quorum sensing, a mechanism of cell density-dependent chemical communication that orchestrates collective behaviors. Second, the second messenger c-di-GMP molecule that modulates effectors to control gene expression. In V. cholerae, high levels of c-di-GMP promote biofilm formation, while high cell density quorum-sensing signaling inhibits biofilm formation. The goal of my thesis was to define how biofilm formation and dispersal are shaped by the integration of c-di-GMP and quorum-sensing signaling in V. cholerae. I developed an experimental system that enables precise modulation of the activity of a V. cholerae c-di-GMP-metabolizing enzyme and I characterized its signaling properties. I chose the NspS-MbaA system, which detects the polyamines spermidine and norspermidine. Detection of spermidine by NspS-MbaA inhibits biofilm formation via MbaA-mediated c-di-GMP degradation, while detection of norspermidine promotes biofilm formation via MbaA-mediated c-di-GMP production. I showed that the system is highly sensitive to polyamines and that the c-di-GMP produced or degraded by MbaA has an exquisitely specific effect on biofilm gene expression. Thus, although MbaA appears to transmit c-di-GMP information locally to a biofilm gene-specific effector, I also showed that alterations in the global intracellular c-di-GMP pool can elicit biofilm-specific transcriptional changes. Using the NspS-MbaA system and ligands for quorum-sensing receptors, I modulated c-di-GMP signaling and quorum-sensing signaling simultaneously. Contrary to expectation, I discovered that the high cell density quorum-sensing signaling regime enhanced polyamine-mediated biofilm formation. I found that the synergy between polyamine signaling and quorum-sensing signaling is a consequence of upregulation of the operon containing nspS and mbaA that occurs in the high cell density quorum-sensing signaling regime. Higher levels of NspS and MbaA enhance the sensitivity the signal relay to changes in polyamine concentrations, and thus, intracellular c-di-GMP levels are significantly elevated in the high cell density and high norspermidine signaling regime. I further characterized the mechanism by which elevated intracellular c-di-GMP overcomes quorum-sensing mediated repression of genes encoding biofilm structural genes. I propose that quorum sensing confers plasticity to biofilm morphogenesis by altering the levels of c-di-GMP-metabolizing enzymes as the bacterial population grows. In particular, by increasing NpsS-MbaA levels over the course of growth, the bacterial population delays commitment to norspermidine-mediated biofilm formation until members of the community detect one another’s presence via quorum sensing. Further tests of this model of quorum-sensing and c-di-GMP signal integration for other c-di-GMP-metabolizing receptors would clarify its generality. Going forward, probing the interactions between quorum-sensing and c-di-GMP signaling in species that occupy diverse niches and that have lifestyles that differ dramatically from that of V. cholerae could deliver a unified picture of how the coordination of sensory signaling systems is linked to the ecological and evolutionary roles that biofilms play across the bacterial domain.