Effects of surface interaction on c-di-GMP regulation in Clostridioides difficile

Clostridioides difficile is a bacterial pathogen that causes antibiotic-associated intestinal disease. The signaling molecule cyclic diguanylate monophosphate (c-di-GMP) regulates various physiological changes such as biofilm formation, motility, and cell differentiation in many bacterial species. In C. difficile, c-di-GMP regulates motility, colonization, and virulence. In many bacteria, an increase in c-di-GMP levels is associated with the transition from a motile to a sessile lifestyle. In the presence of an abiotic surface, bacteria organize themselves in a protective manner through biofilm formation and become sessile. Prior work suggested that C. difficile in a biofilm (i.e., surface-associated) have higher intracellular c-di-GMP levels than free-living planktonic bacteria. We hypothesized that C. difficile responds to growth on a surface by increasing c-di-GMP levels. To test this hypothesis, we used a plasmid where transcription of a red fluorescent reporter, mCherry, is regulated by an upstream riboswitch that allows for transcription of mCherry under high c-di-GMP levels. With this plasmid, we quantified the percentage of the bacterial population that fluoresce red, indicative of an increase in c-di-GMP levels, at different time points. In this study, we found that the percentage of fluorescent cells on a solid surface closely resembles those in a liquid medium during a 24-hour period, suggesting that the levels of c-di-GMP remained similar in both conditions. Further, variation of fluorescence intensity of individual bacteria, indicative of variable intracellular c-di-GMP level, was also similar under the conditions tested. Additional work is needed to determine whether surface growth leads to changes in c-di-GMP at alternative time points or with different surface composition.Bachelor of Scienc