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Cyclic di-GMP-A Newly Appreciated Bacterial Signaling Molecule

Bacteria are constantly challenged to sense, adapt, and respond to ever changing environments. Consider the pathogen Vibrio cholerae, the causative agent of the disease cholera which claims hundreds of thousands of lives every year.  V. cholerae spend the majority of its life cycle in aquatic reservoirs where it is predominantly found in biofilms.  Biofilms are surface associated communities of bacteria encased in an extracellular polymer that have increased resistance to stresses and antibiotic treatment.  Upon ingestion into the host, V. cholerae must transit down the esophagus, through the highly acidic stomach environment, and colonize the small intestine.  V. cholerae must now sense this new environment to initiate expression of the appropriate virulence factors in the correct temporal sequence.  After multiplication to high numbers, V. cholerae undergoes a specific detachment phase that releases the bacteria back into the aquatic environment reinitiating the cycle.  All bacteria, whether or not they are pathogens, face similar challenges.

Luckily for V. cholerae, but perhaps unlucky for us, bacteria have a number of mechanisms for sensing changes to their environment and processing that information to the proper control of downstream phenotypic outputs.  One of the major mechanisms by which bacteria do this is through chemical signaling.

In the Waters lab, we predominantly study the chemical signal called cyclic di-GMP (c-di-GMP) in V. cholerae.  C-di-GMP is an intracellular signal that is a cyclic dimer of two GMP molecules.  C-di-GMP is made by enzymes containing diguanylate cyclase activity (DGC) and degraded by enzymes containing phosphodiesterase activity (PDE).  These two sets of enzymes control the levels of c-di-GMP inside the cell to control phenotypic outputs at many levels of regulation (Fig. 1).

Figure 1. C-di-GMP signaling. Diguanylate cyclase enzymes (stars) synthesize c-di-GMP while phosphodiesterase enzymes (black pentagons) degrade it. These enzymes are thought to response to environmental cues. C-di-GMP controls many phenotypes though all levels of regulation. Taken from (Waters, CM. Microbe. 2012, August, p 353-59).

C-di-GMP is an essential regulator in V. cholerae of the transition from the biofilm state in the environment to the motile, virulent state in the small intestine.  C-di-GMP is a critical component of the disease lifecycle of V. cholerae and many other bacterial pathogens.  C-di-GMP signaling systems are found in 80% of all bacteria, yet surprisingly, its importance has only been appreciated for the last decade.  Therefore, a number of fundamental question about the properties underpinning c-di-GMP signaling systems remain unanswered.  The Waters laboratory seeks to answer some of these questions and also develop new technology targeting c-di-GMP to control disease.

C-di-GMP and Gene Expression

Using a combination of molecular genetics and biochemistry, we have determined that c-di-GMP controls transcription of gene necessary for biofilm formation and motility via two transcription factors. For biofilms, c-di-GMP functions as a co-activator binding to the transcription factor VpsR. C-di-GMP inhibits motility as an anti-activator by inhibiting the ability of the transcription factor FlrA to bind to DNA. We have identified additional genes that are not regulated by these transcription factors, suggesting other machinery remains to be identified!

Funded by the NSF, we are also studying the mechanism and function of a riboswitch called Vc2 in V. cholerae that binds to c-di-GMP. Riboswitches are untranslated RNA elements that fold into a specific conformation allowing binding to small molecules. We are determining how binding Vc2 binding to c-di-GMP controls behaviors in V. cholerae.

Environmental Signals Controlling c-di-GMP

The environmental signals that regulate c-di-GMP are also mostly uncharacterized.  For V. cholerae, c-di-GMP levels are thought to be high in the environment (when biofilms are formed) and low inside the host.  However, we recently determined that bile, one of the most common components of the intestine, actually increases c-di-GMP! This response is mediated through three synthesis enzymes and one degradation enzyme. Interestingly, bicarbonate, which counteracts the effects of acid from the stomach, inhibits this bile response. As the concentration of bicarbonate is higher proximal to the intestinal epithelial cells, we hypothesize that c-di-GMP in V. cholerae is high in the lumen but low as the bacteria become associated with the lumen.

Inhibiting biofilm formation

Infections of biofilm forming bacteria are a major health challenge as these bacteria resist clearance by the immune system and treatment with antibiotics. Such infections can be seen in the lungs of cystic fibrosis patients, contamination of artificial medical surfaces such as catheters, or chronic wounds such as diabetic foot ulcers. Therefore, new approaches are needed to treat these infections. We have performed multiple high throughput small molecule screens to identify novel anti-biofilm compounds. This has led to our identification of a number of new classes of compounds that inhibit biofilms including the first ever molecules that inhibit c-di-GMP synthesis enzymes. We are now focusing on identifying small molecules that enhance the ability of antibiotics to kill biofilms which we are naming Potent and Novel Anti-biofilm Compounds that Enhance Antibiotics (PaNACEAs). We are very grateful to foundation "Hunt for a Cure" for partially funding this research!

C-di-GMP in other Bacteria

Michigan State University is a Mecca of microbiology research, and the Waters lab has initiated a number of collaborations with labs at MSU to study c-di-GMP in other bacteria.  In collaboration with George Sundin, we are examining the role of c-di-GMP during infection of the plant pathogen Erwinia amylovora.  The Sundin lab has shown that E. amylovora must form biofilms in planta to disseminate and cause disease, and we hypothesize that c-di-GMP might be a part of this process.  We are also collaborating with Beronda Montgomery and her laboratory to determine the role of c-di-GMP in the adaption of cyanobacteria to red versus green light.

Evolution of cooperative Behavior

We are working with the MSU BEACON center to determine the evolutionary principles underlie the evolution and maintenance of quorum sensing in bacteria. We have found that the ability communicate is essential for stabilizing cooperative behaviors in the bioluminescent bacterium Vibrio harveyi!


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