GroEL (Hsp60) of Clostridium difficile is involved in cell adherence

Previous results have demonstrated that adherence of Clostridium difficile to tissue culture cells is augmented by various stresses; this study focuss ed on whether the GroEL heat shock protein is implicated in this process. T he 1940 bp groESL operon of C. difficile was isolated by PCR. The 1623 bp g roEL gene is highly conserved between Various C. difficile isolates as dete rmined by RFLP-PCR and DNA sequencing, and the operon is present in one cop y on the bacterial chromosome. The 58 kDa GroEL protein was expressed in Es cherichia coli in fusion with glutathione S-transferase and the fusion prot ein was purified from IPTG-induced bacterial lysates by affinity chromatogr aphy on glutathione-Sepharose. A polyclonal, monospecific antiserum was obt ained for GroEL which established by immunoelectron microscopy, indirect im munofluorescence and immunoblot analysis that GroEL is released extracellul arly after heat shock and can be surface associated. Cell fractionation exp eriments suggest that GroEL is predominantly cytoplasmic and membrane bound . GroEL-specific antibodies as well as the purified protein partially inhib ited C. difficile cell attachment and expression of the protein was induced by cell contact. suggesting a role for GroEL in cell adherence.