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 focussed on whether the GroEL heat shock protein is implicated in this process. The 1940 bp groESL operon of C. difficile was isolated by PCR. The 1623 bp groEL gene is highly conserved between various C. difficile isolates as determined by RFLP-PCR and DNA sequencing, and the operon is present in one copy on the bacterial chromosome. The 58 kDa GroEL protein was expressed in Escherichia coli in fusion with glutathione S:-transferase and the fusion protein was purified from IPTG-induced bacterial lysates by affinity chromatography on glutathione-Sepharose. A polyclonal, monospecific antiserum was obtained for GroEL which established by immunoelectron microscopy, indirect immunofluorescence and immunoblot analysis that GroEL is released extracellularly after heat shock and can be surface associated. Cell fractionation experiments suggest that GroEL is predominantly cytoplasmic and membrane bound. GroEL-specific antibodies as well as the purified protein partially inhibited C. difficile cell attachment and expression of the protein was induced by cell contact, suggesting a role for GroEL in cell adherence.
Hennequin
C
C
Université de Paris-Sud, Faculté de Pharmacie, Département de Microbiologie, 5 rue JB Clément, F-92296 Châtenay-Malabry cedex, France.
Porcheray
F
F
Waligora-Dupriet
A
A
Collignon
A
A
Barc
M
M
Bourlioux
P
P
Karjalainen
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001422:Bacterial Adhesion / Q000502:physiology*; D001483:Base Sequence; D018834:Chaperonin 60 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002522:Chlorocebus aethiops; D016360:Clostridioides difficile / Q000472:pathogenicity* / Q000502:physiology; D004761:Enterocolitis, Pseudomembranous / Q000382:microbiology; D018628:Gene Dosage; D006801:Humans; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009876:Operon; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D017422:Sequence Analysis, DNA; D014709:Vero Cells
D018834:Chaperonin 60