Abstract
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, 131KKGIK135 and 184KGTSTHVIT192, were identified and characterized. Immunoreactivity of the synthetic biotinylated 131KKGIK135 epitope was significantly higher compared to 184KGTSTHVIT192 epitope in Enzyme-Linked Immunosorbent Assay (ELISA) with umbilical cord blood and CDI patients’ sera. Hereafter, the conjugate of bovine serum albumin and epitope 131KKGIK135 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 131KKGIK135 epitope possesses immunostimulating properties suggesting possibility of its use in a vaccine against Clostridioides difficile.
Highlights
Clostridioides difficile (CD) is a Gram-positive, spore-forming bacterium that grows under anaerobic conditions
CD proteins which were isolated using gentle washing-off methoddesigned designedfor forthe theisolation isolationof of surface proteins were separated using electrophoresis and subjected to Western blotsurface proteins were separated using 1D SDS-PAGE electrophoresis and subjected to Western blotting ting analysis umbilical blood serum as primary antibody
B-cellB-cell arrows) in the protein sequence. (C) Peptides selected for PEPSCAN synthesis with underlined
Summary
CD is a Gram-positive, spore-forming bacterium that grows under anaerobic conditions. It is commonly associated with CDAD occurring in hospitals and nursing homes [1,2,3,4]. CDI spreads by the fecal-oral route, mainly through spores resistant to high temperature, disinfectants, and antibiotics [5]. These spores can be found in small amounts in the soil, food, and water. They occur in the hospital environment, covering floors and hospital equipment, like beds and cabinets [5,6]. CD may be a constituent of a healthy human gut flora [7], but it starts to overgrow when an imbalance in
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