Vibrio cholerae c-diGMP signaling: Motile to biofilm transition and transmission

PROJECT SUMMARY The signaling nucleotide cyclic dimeric guanosine monophosphate (c-diGMP) is broadly conserved in bacteria and is a key regulator of central cellular processes including motility, biofilm formation, and virulence. Our understanding of how c-diGMP controls biofilm formation, the environmental signals modulating c-diGMP levels and biofilm formation, and the dynamics and consequences of c-diGMP signaling during infection remains limited. This proposal aims to fill critical gaps in our understanding of how c-diGMP signaling and biofilm formation control the infection cycle of Vibrio cholerae, which causes the disease cholera, an important global public health problem. We will address these information gaps through two specific aims. 1) Determine mechanisms of signal sensing and response in c-diGMP signaling networks; and 2) Analysis of c-diGMP signaling during infection. Under the first aim, the molecular mechanism(s) of activation of key c-diGMP signaling proteins will be determined using structural and ligand-binding studies. The down-stream c-diGMP signaling pathways initiated upon surface attachment will be analyzed by employing microscopy-based community tracking methods to measure motility, division, and c-diGMP levels. The mechanism by which specific key c- diGMP signaling proteins act to regulate surface attachment and biofilm matrix production will be determined using a combination of genetic and biochemical approaches. Under the second aim, we will analyze key c-diGMP signaling pathways that are activated during infection. This part of the proposal will track and examine the mechanisms underlying temporal and spatial dynamics of c-diGMP production and virulence gene regulation during infection using state-of-the-art imaging tools and novel c-diGMP sensors. The proposed work will greatly advance our understanding of how c-diGMP signaling operates, identify the inputs that influence c-diGMP production and degradation, and unveil the biological consequences of c-diGMP signaling in vivo. This research promises molecular and mechanistic insights that will allow us to devise ways to control c-diGMP signal transduction pathways governing motility, biofilm formation, and virulence, ultimately identifying potential therapeutic or preventive targets that can be exploited for preventive measures against cholera.