Mechanisms and Consequences of c-di-GMP Control of Motility and Biofilm Formation

PROJECT SUMMARY Biofilms, surface-attached microbial communities encased in an extracellular matrix, enhance the environmental survival, transmission, pathogenicity, and antibiotic resistance of pathogenic microorganisms. A key regulator of biofilm formation in bacteria is the broadly conserved nucleotide-based second messenger, cyclic dimeric guanosine monophosphate (c-di-GMP). c-di-GMP is produced by diguanylate cyclases (DGCs), degraded by phosphodiesterases (PDEs), and sensed by c-di-GMP receptor proteins. This proposal examines the molecular mechanisms and consequences of biofilm formation in Vibrio cholerae, the bacterium causing the disease cholera, an important public health problem worldwide. While interfering with biofilm formation might mitigate the global health impact of diseases like cholera, how c-di-GMP controls this process remains unclear. Our objective is to address major gaps in our understanding of the interplay between the flagellum, pili, and c-di-GMP in the motile-to-sessile switch, biofilm formation, and V. cholerae infection. Specific Aim 1 focuses on determining how c-di-GMP and specific DGC and PDE proteins modulate the torque-speed relationship of the sheathed flagellum in V. cholerae using biophysical approaches and novel reporters to quantify c-di-GMP levels. We will also examine how c-di-GMP receptor proteins, particularly PilZ domain proteins, regulate motility and biofilm formation through genetic, biochemical, and structural analyses. Specific Aim 2 examines the molecular mechanism(s) through which c-di-GMP controls the production and activity of the mannose-sensitive haemagglutinin type IV pili (MSHA), the primary mediator of initial surface attachment, which leads to biofilm formation, using structural and biochemical approaches. Specific Aim 3 dissects the role of c-di-GMP signaling in V. cholerae infection using state-of-the-art imaging tools and novel c-di-GMP sensors. This research promises molecular and mechanistic insights into c-di-GMP signal transduction pathways governing motility and biofilm formation, ultimately allowing us to devise ways to inhibit V. cholerae infection cycle. Understanding these mechanisms will also lead to new strategies for disrupting biofilms and controlling motility in other pathogens, thereby improving treatments for biofilm-related bacterial infections.