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Infection and Immunity, May 2000, p. 2470-2474, Vol. 68, No. 5
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

pH-Induced Conformational Changes in Clostridium difficile Toxin B

Maen Qa'Dan, Lea M. Spyres, and Jimmy D. Ballard*

Department of Botany and Microbiology, The University of Oklahoma, Norman, Oklahoma 73019

Received 23 November 1999/Returned for modification 7 January 2000/Accepted 17 January 2000

Toxin B from Clostridium difficile is a monoglucosylating toxin that targets substrates within the cytosol of mammalian cells. In this study, we investigated the impact of acidic pH on cytosolic entry and structural changes within toxin B. Bafilomycin A1 was used to block endosomal acidification and subsequent toxin B translocation. Cytopathic effects could be completely blocked by addition of bafilomycin A1 up to 20 min following toxin treatment. Furthermore, providing a low extracellular pH could circumvent the effect of bafilomycin A1 and other lysosomotropic agents. Acid pH-induced structural changes were monitored by using the fluorescent probe 2-(p-toluidinyl) naphthalene-6-sulfonic acid, sodium salt (TNS), inherent tryptophan fluorescence, and relative susceptibility to a specific protease. As the toxin was exposed to lower pH there was an increase in TNS fluorescence, suggesting the exposure of hydrophobic domains by toxin B. The change in hydrophobicity appeared to be reversible, since returning the pH to neutrality abrogated TNS fluorescence. Furthermore, tryptophan fluorescence was quenched at the acidic pH, indicating that domains may have been moving into more aqueous environments. Toxin B also demonstrated variable susceptibility to Staphylococcus aureus V8 protease at neutral and acidic pH, further suggesting pH-induced structural changes in this protein.

* Corresponding author. Mailing address: The University of Oklahoma, 770 Van Vleet Oval, Norman, OK 73019. Phone: (405) 325-5133. Fax: (405) 325-7619. E-mail: Jballard{at}ou.edu.

Infection and Immunity, May 2000, p. 2470-2474, Vol. 68, No. 5
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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