Phosphorylation of the Synthetic Hexasaccharide Repeating Unit Is Essential for the Induction of Antibodies to Clostridium difficile PSII Cell Wall Polysaccharide

Abstract

Clostridium difficile is emerging worldwide as a major cause of nosocomial infections. The negatively charged PSII polysaccharide has been found in different strains of C. difficile and, thereby, represents an important target molecule for a possible carbohydrate-based vaccine. In order to identify a synthetic fragment that after conjugation to a protein carrier could be able to induce anti-PSII antibodies, we exploited a combination of chemical synthesis with immunochemistry, confocal immunofluorescence microscopy, and solid state NMR. We demonstrate that the phosphate group is crucial in synthetic glycans to mimic the native PSII polysaccharide; both native PSII and a phosphorylated synthetic hexasaccharide repeating unit conjugated to CRM(197) elicit comparable immunogenic responses in mice. This finding can aid design and selection of carbohydrate antigens to be explored as vaccine candidates.

Multiple Factors Modulate Biofilm Formation by the Anaerobic Pathogen Clostridium difficile

“…Staining with antibodies against C. difficile suggests that this compact biofilm may comprise surface-associated or secreted bacterial components and may be impenetrable to antibodies. Furthermore, it is interesting that we observe biofilm matrix staining for a synthetic derivative of the C. difficile surface PSII polysaccharide, which was recently reported to be immunogenic (26). The presence of a complex and perhaps impermeable matrix may protect C. difficile from unfavorable agents in vivo in the gut.…”

Biofilm formation byClostridium difficile

“…19 Additionally, staining biofilm with a synthetic derivative of the C. difficile surface PSII polysaccharide ( Fig. 1E), which was reported to be immunogenic recently, 22 revealed the presence of polysaccharide components in the biofilm matrix. In all, the data clearly show that C. difficile biofilms are composed of a thick multi-component biofilm matrix.…”

Vaccines againstClostridium difficile

“…Antibodies against the conjugated PSII detected the polysaccharide at the surface of the bacterial vegetative cells, thus confirming this molecule as a target for a carbohydrate based vaccine. 139 However the sugar coating was not as thick and uniformly distributed as expected for a capsule. Therefore, it can be hypothesized that PSII is expressed by the bacterium either as cell wall-linked polysaccharide not bound to peptidoglycan or as a conjugate with lipoteichoic acids.…”

“…138 Among these 3 carbohydrates, PSII was found to be the more abundantly expressed by most of C. difficile ribotypes, including the hypervirulent strain NAP1/027 and other clinical isolates belonging to ribotypes 001, 018, 027, 078, and 126. 139,140 Following the discovery of PSII, it was not clear whether PSII was part of a capsule or a surface glycoprotein, or released to the external surface of the bacterium. Antibodies against the conjugated PSII detected the polysaccharide at the surface of the bacterial vegetative cells, thus confirming this molecule as a target for a carbohydrate based vaccine.…”