Surface-Layer Protein A (SlpA) Is a Major Contributor to Host-Cell Adherence of Clostridium difficile

Abstract

Clostridium difficile is a leading cause of antibiotic-associated diarrhea, and a significant etiologic agent of healthcare-associated infections. The mechanisms of attachment and host colonization of C. difficile are not well defined. We hypothesize that non-toxin bacterial factors, especially those facilitating the interaction of C. difficile with the host gut, contribute to the initiation of C. difficile infection. In this work, we optimized a completely anaerobic, quantitative, epithelial-cell adherence assay for vegetative C. difficile cells, determined adherence proficiency under multiple conditions, and investigated C. difficile surface protein variation via immunological and DNA sequencing approaches focused on Surface-Layer Protein A (SlpA). In total, thirty-six epidemic-associated and non-epidemic associated C. difficile clinical isolates were tested in this study, and displayed intra- and inter-clade differences in attachment that were unrelated to toxin production. SlpA was a major contributor to bacterial adherence, and individual subunits of the protein (varying in sequence between strains) mediated host-cell attachment to different extents. Pre-treatment of host cells with crude or purified SlpA subunits, or incubation of vegetative bacteria with anti-SlpA antisera significantly reduced C. difficile attachment. SlpA-mediated adherence-interference correlated with the attachment efficiency of the strain from which the protein was derived, with maximal blockage observed when SlpA was derived from highly adherent strains. In addition, SlpA-containing preparations from a non-toxigenic strain effectively blocked adherence of a phylogenetically distant, epidemic-associated strain, and vice-versa. Taken together, these results suggest that SlpA plays a major role in C. difficile infection, and that it may represent an attractive target for interventions aimed at abrogating gut colonization by this pathogen.

New class of precision antimicrobials redefines role of Clostridium difficile S-layer in virulence and viability

“…What did come as a surprise, however, was the ability of the S-layer mutant to stably colonize and persist in the hamster gut for the 14-day duration of the experiment. Previous reports have pointed to a role for the S-layer in epithelial cell adhesion; however, these earlier studies were performed without access to an slpA -defective strain (34). The S-layer defective mutants and isogenic controls expressing the SLCT-specific slpA alleles provide the ideal controls to test these conflicting findings and better define the effect SlpA type has on these functions.…”

Cwp2 from Clostridium difficile exhibits an extended three domain fold and cell adhesion in vitro

“…The former of these results suggests Cwp2 may play a role in host cell adhesion. This reflects the results previously seen for Cwp66 and LMW SLP .…”

Clostridium difficile infection

“…44), are secreted by the bacterium and degrade the colonic mucosa. Bacterial cell surface-associated proteins have been shown to affect the adhesion of the bacterium to colon epithelial cells in vitro 45–48 ; mutations in genes encoding these proteins, or in the genes encoding proteins for their processing, generally attenuate virulence 49,50 . The expression of at least a subset of colonization factors by the bacterium, such as cell surface protein Cwp84 and surface layer protein A (SlpA), is stimulated in the presence of the antibiotics ampicillin and clindamycin 51 .…”