Clostridioides difficile (C. difficile) are ubiquitous Gram-positive spore-forming bacteria. They are relevant nosocomial pathogens causing antibiotic associated diarrhea. Furthermore, C. difficile infection may lead to severe complications, such as pseudomembranous colitis, sepsis, multiorgan dysfunction syndrome and even death. Surface of C. difficile is decorated with a proteinaceous layer known as surface (S-) layer. It functions as a protective barrier, helps in cell adhesion and sporulation and is an important virulence factor. S-layer is anchored to the cell wall through a non-covalent interaction with a secondary polysaccharide PSII. PSII is a C. difficile-specific anionic polymer composed of hexaglycosyl phosphate repeating units. Proteins involved in PSII biosynthesis are poorly understood, yet they are potential new targets for developing new C. difficile treatments.
In this master’s thesis, we focused on the CD630_27760 protein, which is a glycosyltransferase involved in the biosynthesis of PSII hexaglycosyl phosphate unit. It catalyses the transfer of sugar moiety from the sugar donor, which is either UDP-glucose (UDP-Glc) or UDP-N-acetylgalactosamine (UDP-NAcGal), to the acceptor, which is a forming sugar unit bound to undecaprenyl pyrophosphate (UndPP). Colleagues have previously successfully produced the protein in Escherichia coli (E. coli) and determined its three-dimensional structure. They had observed a spontaneous cleavage of the C-terminal part of the protein during its production. Using mass spectrometry (MALDI-TOF), we determined that the protein is cleaved in three separate sites, that give us protein fragments: 27,1 kDa, 26,6 kDa and 24,3 kDa. Position of these cleavage sites coincide with the predicted cleavage site.
Next we attempted to co-crystallize our protein with predicted sugar donors (UDP-Glc or UDP-NAcGal) and potential inhibitors. However, we failed to do so. By comparing the sequence and structure of CD630_27760 protein with homologous proteins, we determined in silico which amino acid residues are potentially involved in binding of the nucleotide part of the sugar donor: P12, L14, R16, N42, K69, R75, D91, D92, D93 in H205. The comparison with homologous proteins also showed that the C-terminal part of the protein, which is spontaneously cleaved during isolation, contributes to the bonding of the donor sugar unit and acceptor. Therefore, in order to obtain s structure with a bonded sugar donor and/or inhibitors, a full-length protein including the C-terminal part would have to be prepared.
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