Characterization and Reassembly of a Regular Array in the Cell Wall of Clostridium difficile GAI 4131

Abstract

The cell wall of Clostridium difficile GAI 4131 was revealed by electron microscopy to have an outer layer composed of a nearly square array and contained the two major proteins with molecular weights of 38 kDa and 42 kDa. The properties and reassembly of the two major proteins into the regular array were investigated. When the isolated cell walls were treated with hydrophobic bond-disrupting agents or a chelating agent specific for Ca2+, the two major proteins were effectively removed and the regularly arranged outer layer disappeared. The amino acid composition of the two major proteins differed from each other. The two major proteins also gave different peptide maps from each other upon proteolysis with Staphylococcus aureus V8 protease. The major proteins solubilized from the isolated cell walls with 8 M urea or 4 M guanidine hydrochloride could be reassembled into open-ended cylinders possessing the native regular pattern by dialysis against neutral buffer containing 5 mm CaCl2. The reassembled cylinders purified by centrifugation on a Percoll density gradient were composed of almost equal amounts of the 38 kDa and 42 kDa proteins ond freed from the other proteins. These results suggest that the regular array in the outer cell wall layer is constructed from the two major cell wall proteins and requires Ca2+ for its assembly.

Molecular characterization of the surface layer proteins from Clostridium difficile

“…This is a complex motif (Lupas et al ., 1994) present at the N‐terminus of SLPs from several Gram‐positive bacteria, including B. anthracis and Clostridium thermocellum , and is also found at the C‐terminus of some cell‐associated exoenzymes (Engelhardt and Peters, 1998). However, the SLH domain is absent from the SLPs of other bacteria including Lactobacilli (Masuda et al ., 1989; Egelseer et al ., 1998). It was originally thought that the SLH domain was involved in the anchoring of the S‐layer to the underlying peptidoglycan layer, but it has now been shown to function in binding to secondary cell wall polymers (Mesnage et al ., 1999).…”

Chemical Probes of Surface Layer Biogenesis in Clostridium difficile

“…The C. difficile cell is completely coated by the surface layer (S-layer), a paracrystalline proteinacious array that mediates host-pathogen interactions (5,6). Previously we have shown that the key structural components of the S-layer are the result of proteolytic cleavage of a precursor protein, SlpA, into high-and low-molecular-weight components, namely the HMW and LMW S-layer proteins (SLPs) (5).…”

“…C lostridium difficile is a Gram-positive antibiotic-resistant anaerobe that can cause severe gastrointestinal disease in humans , and livestock and is a leading cause of hospital-acquired infection . The C. difficile cell is completely coated by the surface layer (S-layer), a paracrystalline proteinacious array that mediates host–pathogen interactions , . Previously we have shown that the key structural components of the S-layer are the result of proteolytic cleavage of a precursor protein, SlpA, into high- and low-molecular-weight components, namely, the HMW and LMW S-layer proteins (SLPs) .…”

Characteristics and Immunological Roles of Surface Layer Proteins inClostridium difficile

“…Our knowledge of SLPs has increased over the past decade; SLPs play important roles not only in growth and survival but also in the interaction with the host and its immune system. All C. difficile strains also express crystalline or paracrystalline SLPs on the outer cell surface [ 16 ]. Recent studies have shown that C. difficile SLPs are involved not only in adhesion to host intestinal cells but also in the induction of cytokine production and the recognition of C. difficile by the immune system [ 17 18 19 ].…”