Characterization and studies of the cellular interaction of native colonization factor CS6 purified from a clinical isolate of enterotoxigenic Escherichia coli.
CS6 is a widely expressed colonization factor of enterotoxigenic Escherichia coli (ETEC). To date, CS6 has not been well characterized in its native state. Here, we purified CS6 for the first time from an ETEC clinical isolate. Purified CS6 was composed of two structural subunits, CssA and CssB, which were present in equal amounts and tightly linked through noncovalent, detergent-stable association. The CssA subunit was poorly immunogenic, whereas CssB was highly immunogenic. Although the predicted molecular mass of CssA is 15 kDa, the purified CssA has an effective molecular mass of 18.5 kDa due to fatty acid modification. When purified CS6 was screened for its ability to bind with different extracellular matrix proteins, fibronectin (Fn) was found to interact with CS6 as well as CssA in a dose-dependent and saturable manner. This interaction was inhibited both by a synthetic peptide corresponding to the C-terminal hydrophilic, surface-exposed region of CssA (positions 112 to 126) and by the antibody derived against this region. Enzyme-linked immunosorbent assay results showed that CssA interacted with the 70-kDa N-terminal domain of Fn. The modifications on CssA probably do not play a role in Fn binding. Preincubation of INT 407 cells with CssA, but not CssB, inhibited ETEC binding to these cells. The results suggested that CS6-expressing ETEC binds to Fn of INT 407 cells through the C-terminal region of CssA. Purified CS6 was found to colocalize with Fn along the junctions of INT 407 cells. Based on the results obtained, we propose that CS6-expressing ETEC binds to the intestinal cells through Fn for colonization.
Ghosal
Abhisek
A
Division of Biochemistry, National Institute of Cholera & Enteric Diseases, P33 C.I.T. Road, Scheme XM, Beliaghata, Kolkata 700 010, India. chatterjeens@icmr.org.in
Bhowmick
Rudra
R
Banerjee
Rajat
R
Ganguly
Sandipan
S
Yamasaki
S
S
Ramamurthy
T
T
Hamabata
T
T
Chatterjee
Nabendu Sekhar
NS
D000942:Antigens, Bacterial / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D001422:Bacterial Adhesion; D002460:Cell Line; D003001:Cloning, Molecular; D054307:Enterotoxigenic Escherichia coli / Q000737:chemistry* / Q000302:isolation & purification; D004927:Escherichia coli Infections / Q000382:microbiology*; D029968:Escherichia coli Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D017422:Sequence Analysis, DNA
D000942:Antigens, Bacterial; C098406:CS6 antigen, E coli; D029968:Escherichia coli Proteins; D005353:Fibronectins; D021122:Protein Subunits