C-DI-GMP SIGNALLING IN BACTERIA-NEW OPPORTUNITIES FOR THE DEVELOPMENT OF ANTI-BIOFILM DRUGS
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Bacterial infections are becoming more difficult to treat, due to the emergence of resistance strains to almost every antibiotic. Most bacteria that infect humans to cause diseases are biofilm forming bacteria and it has been shown that most antibiotics that are in clinical use today are not very effective against bacteria in a biofilm. Despite the difficulty in eradicating bacterial biofilms, there is no anti-biofilm drug in clinical use today. Cyclic diguanylate (c-di-GMP) is a second messenger in bacteria and it that is synthesized in the cytosol, in response to a changing bacterial environment to regulate bacterial physiology. Due to the central role that c-di-GMP plays in bacteria, there are interests in understanding c-di-GMP signaling with the hope that detailed understanding of c-di-GMP signaling would provide avenues to design small molecules that could be used to inhibit bacterial virulence and biofilm formation, which c-di-GMP regulates. We aim to understand the determinants of c-di-GMP binding to receptor proteins and RNA riboswitches, using c-di-GMP analogs. We designed a concise synthetic strategy to make c-di-GMP and analogs. These analogs were used to study the structure-activity-relationship (SAR) of c-di-GMP. The polymorphisms of the synthesized analogs were studied using a panel of spectroscopic techniques. It was discovered that one analog of c-di-GMP, namely 2'-F-c-di-GMP, which had a high propensity to exist in the closed conformation could potently inhibit c-di-GMP synthesis by c-di-GMP synthases. Since c-di-GMP promotes biofilm formation in bacteria, these analogs represent good starting points to design anti-biofilm agents to treat persistent bacterial infections. C-di-AMP was recently discovered as another important bacterial signaling molecule and it is mostly found in Gram-positive bacteria. C-di-AMP controls many processes in bacteria, including growth, cell wall synthesis, ion transport, and sporulation. Many of the receptor or effector proteins that mediate c-di-AMP signaling remain to be characterized and there is lack of knowledge regarding how environmental factors regulate c-di-AMP metabolism. We developed a new fluorescent probe for the "real-time" detection of c-di-AMP. This assay could be used to find inhibitors of c-di-AMP signaling as well as studying the enzymatic proficiencies of c-di-AMP metabolism proteins.