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Infection and Immunity, May 2009, p. 2125-2135, Vol. 77, No. 5
0019-9567/09/$08.00+0     doi:10.1128/IAI.01397-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Characterization and Studies of the Cellular Interaction of Native Colonization Factor CS6 Purified from a Clinical Isolate of Enterotoxigenic Escherichia coli{triangledown}

Abhisek Ghosal,1 Rudra Bhowmick,1 Rajat Banerjee,2 Sandipan Ganguly,1 S. Yamasaki,3 T. Ramamurthy,1 T. Hamabata,4 and Nabendu Sekhar Chatterjee1*

National Institute of Cholera and Enteric Diseases, Kolkata, India,1 BC Guha Centre for Biotechnology and Genetic Engineering, University of Calcutta, Kolkata, India,2 Osaka Prefecture University, Osaka, Japan,3 International Medical Center of Japan, Tokyo, Japan4

Received 14 November 2008/ Returned for modification 19 December 2008/ Accepted 18 February 2009

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.

* Corresponding author. Mailing address: Division of Biochemistry, National Institute of Cholera & Enteric Diseases, P33 C.I.T. Road, Scheme XM, Beliaghata, Kolkata 700 010, India. Phone: 91(33) 2363-3372. Fax: 91(33) 2370-5066. E-mail: chatterjeens{at}icmr.org.in

{triangledown} Published ahead of print on 23 February 2009.

Editor: A. J. Bäumler

Infection and Immunity, May 2009, p. 2125-2135, Vol. 77, No. 5
0019-9567/09/$08.00+0     doi:10.1128/IAI.01397-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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