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.
Highlights
Clostridium difficile is a gram-positive, anaerobic, spore-forming bacterium, and causes the antibiotic-associated diarrheal disease, C. difficile infection (CDI)
SurfaceLayer Protein A (SlpA) contains two biologically distinct entities, the high-molecular weight (HMW) and the low molecular weight (LMW) subunits, which are derived via Cwp84-mediated cleavage of a single precursor protein [16], and assemble on the bacterial surface into a paracrystalline lattice [17]
We tested a group of ribotype 078 strains (REA group BK and PFGE type NAP 7,8,9) derived from different mammalian sources with symptomatic CDI, to examine adherence to host cells of the same or different species; these strains exhibited a range of adherence corresponding to their provenance
Summary
Clostridium difficile is a gram-positive, anaerobic, spore-forming bacterium, and causes the antibiotic-associated diarrheal disease, C. difficile infection (CDI). It is a leading cause of bacterial healthcare-associated infections in hospitals in the United States [1,2], having surpassed methicillin-resistant Staphylococcus aureus (MRSA) in some hospitals for this dubious distinction. SlpA contains two biologically distinct entities, the high-molecular weight (HMW) and the low molecular weight (LMW) subunits, which are derived via Cwp84-mediated cleavage of a single precursor protein [16], and assemble on the bacterial surface into a paracrystalline lattice [17]. The two subunits associate with high affinity through the N-terminus of the HMW protein and the C-terminus of the LMW protein [18]
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