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Clostridiodies difficile (C. difficile) is a bacterium that is responsible for many gastrointestinal infections and is considered by the CDC to be a pathogen of significant importance. One unusual aspect of C. difficile infections is that after antibiotic treatment there is a high chance of a reoccurrence, where the disease will reappear without additional exposure. The mechanism of the reoccurrence is currently unknown, but it is hypothesized that potential mechanisms of reoccurrence could be residual biofilm presence or resistant spores could provide a reservoir for reinfection. C. difficile produces type IV pili (T4P), extracellular appendages which provide a variety of functions for cells, such as natural competence, twitching motility, horizontal gene transfer, and biofilm formation. The function of T4P can vary between species, however the structure generally remains the same, with T4P being composed of a helical fiber of proteins. T4P are composed of a multitude of proteins, with one protein composing most of the fiber, termed the major pilin, with PilA1 being the major pilin for C. difficile. Other proteins, termed minor pilins, are incorporated sporadically throughout the pilus, with minor pilins of note for C. difficile being PilJ, PilW, and PilK. Minor pilins serve a variety of purposes in T4P systems, including DNA binding and the formation of the pilus tip complex, which is required for polymerization. Previous research by our group into the mechanism of biofilm formation has shown several results: 1) treatment of biofilm with DNase causes degradation, indicating that extracellular DNA plays a stabilizing role in the structure of biofilms. 2) removal of minor pilins PilJ and PilW cause a significant decrease in biofilm formation from the natural phenotype. 3) Minor pilins PilJ and PilW exhibit DNA binding. From this, we have developed the hypothesis that minor pilins PilJ and PilW have a stabilizing effect on biofilm by binding to extracellular DNA. To determine how these non-canonical DNA receptors recognize eDNA, we have used HADDock (High Ambiguity Driven bimolecular Docking) to predict the DNA binding site of PilJ, which has predicted two possible binding sites. The first predictive site is between α-helix 149-159 and loop 206-210, and the second predictive site is between loops 141-149, 190-197, 232-248. When modeled into a pilus, these predictive sites exhibit shape complementarity, and do not result in overlap with the pilus body, indicating that these could be potentially valid binding sites. From the HADDock predictions, we are currently working toward making PilJ mutants to perform DNA binding assays through EMSA or ITC. We predict that the mutants which correspond to the DNA binding sites will result in a deficiency in DNA binding when the affinities are determined. Additionally, we are investigating the minor pilin PilK, and hypothesize that it takes a place in the tip complex of the pilus, as it appears to resemble other known tip proteins. We are currently working to determine the structure of PilK by producing crystals through vapor diffusion and analyzing them through x-ray crystallography. We have produced diffracting crystals which contain a maltose binding protein crystallization chaperone, which will allow us to solve the structure through molecular replacement. As we have not produced a dataset suitable to solve the structure through molecular replacement, we intend to gather additional data, as well as anomalous data through soaks, which may facilitate solving the structure. Funding was received from NIH P20-GM113126, NSF 2310647 and the UNL UCARE program.
Bauer et al. (Fri,) studied this question.