Isolation of recombinant antibodies directed against surface proteins of Clostridium difficile

Abstract

Clostridium difficile has emerged as an increasingly important nosocomial pathogen and the prime causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis in humans. In addition to toxins A and B, immunological studies using antisera from patients infected with C. difficile have shown that a number of other bacterial factors contribute to the pathogenesis, including surface proteins, which are responsible for adhesion, motility and other interactions with the human host. In this study, various clostridial targets, including FliC, FliD and cell wall protein 66, were expressed and purified. Phage antibody display yielded a large panel of specific recombinant antibodies, which were expressed, purified and characterised. Reactions of the recombinant antibodies with their targets were detected by enzyme-linked immunosorbent assay; and Western blotting suggested that linear rather than conformational epitopes were recognised. Binding of the recombinant antibodies to surface-layer proteins and their components showed strain specificity, with good recognition of proteins from C. difficile 630. However, no reaction was observed for strain R20291—a representative of the 027 ribotype. Binding of the recombinant antibodies to C. difficile M120 extracts indicated that a component of a surface-layer protein of this strain might possess immunoglobulin-binding activities. The recombinant antibodies against FliC and FliD proteins were able to inhibit bacterial motility.

Surface layer proteins from virulent Clostridium difficile ribotypes exhibit signatures of positive selection with consequences for innate immune response

“…These strains exhibit increased antibiotic and disinfectant resistance [19, 20] increased sporulation rates [21] and other possible modes of action for virulence [22]. Another recent study has shown antibody raised against slpA from C difficile strain 630 (PCR ribotype 012) does not cross react with slpA from ribotype 027 [23]. Despite slpA being examined as a vaccine candidate [24], problems may still arrive due to high sequence variability of the protein coding sequences for SLPs between strains.…”

Host Immune Responses to Surface S-Layer Proteins (SLPs) of Clostridioides difficile

“…In another study, Shirvan et al generated and expressed specific recombinant antibodies against SLPs such as Cwp66 and SlpA from C. difficile 630 proteins using phage display and showed that these recombinant antibodies reacted to SLPs and their components in a strain-specific manner with high specificity [ 68 ].…”

Rapid-format recombinant antibody-based methods for the diagnosis of Clostridioides difficile infection: Recent advances and perspectives

“…Some of the major and common C. difficile antigens are its surface proteins, including surface-layer proteins (SLPs), cell wall protein 66 (Cwp66) and 84 (CWP84), flagellin FliC, flagellar cap protein FliD, which play key role in attachment to intestinal mucus and have been used as target biomolecules in previous studies ( Merrigan et al, 2013 ; Kandalaft et al, 2015 ; Shirvan and Aitken, 2016 ). Additionally, glutamate dehydrogenase (GDH) is a constitutive enzyme produced in large amounts by all toxigenic and non-toxigenic strains of C. difficile and can be easily detected in stool samples ( Arimoto et al, 2016 ; Kelly et al, 2021 ).…”