The Bioinformatic and In Vitro Studies of Clostridioides Difficile Aminopeptidase M24 Revealed the Immunoreactive KKGIK Peptide.

Clostridioides difficile (CD) is a Gram-positive pathogen responsible for CD-associated disease (CDAD), which is characterized by symptoms ranging from mild diarrhea to pseudomembranous colitis. This work is an attempt to respond to the need of novel methods for CD infection (CDI) prevention, since the number of CDI cases is still rising. A bioinformatics approach was applied to design twenty-one peptides consisting of in silico predicted linear B-cell and T-cell epitopes of aminopeptidase M24 from CD. These peptides were mapped for epitopes exploiting PEPSCAN procedure and using sera obtained from CD infected patients, umbilical cord blood, and healthy volunteers. Two new CD epitopes, (131)KKGIK(135) and (184)KGTSTHVIT(192), were identified and characterized. Immunoreactivity of the synthetic biotinylated (131)KKGIK(135) epitope was significantly higher compared to (184)KGTSTHVIT(192) epitope in Enzyme-Linked Immunosorbent Assay (ELISA) with umbilical cord blood and CDI patients' sera. Hereafter, the conjugate of bovine serum albumin and epitope (131)KKGIK(135) was evaluated in vitro on lung epithelial cell line. In vitro, a significant induction of IL-6 by conjugate was observed, thereby we postulate that this new (131)KKGIK(135) epitope possesses immunostimulating properties suggesting possibility of its use in a vaccine against Clostridioides difficile.