Characterization of Fluorescent Chimeras of Cholera Toxin and Escherichia coli Heat-Labile Enterotoxins Produced by Use of the Twin Arginine Translocation System

Authors

Juliette K. Tinker, Boise State UniversityFollow
Jarrod L. Erbe, Wisconsin Lutheran College
Randall K. Holmes, Boise State University

Document Type

Article

Publication Date

6-1-2005

DOI

http://dx.doi.org/10.1128/IAI.73.6.3627-3635.2005

Abstract

Cholera toxin (CT) is an AB₅ toxin responsible for the profusesecretory diarrhea resulting from Vibrio cholerae infection.CT consists of a pentameric, receptor-binding B subunit (CTB)and a monomeric A subunit (CTA) that has latent enzymatic activity.In addition to its enterotoxicity, CT has potent mucosal adjuvantactivity and can also function as a carrier molecule with manypotential applications in cell biology. In earlier studies,the toxic CTA₁ domain was replaced by several other antigenicprotein domains to produce holotoxin-like chimeras for use aspotential mucosal vaccines. In the present study we utilizedthe twin arginine translocation (tat) system to produce fluorescentCT chimeras, as well as fluorescent chimeras of Escherichiacoli heat-labile toxins LTI and LTIIb. Fusion proteins containingeither green fluorescent protein (GFP) or monomeric red fluorescentprotein (mRFP) and the A₂ domain of CT, LTI, or LTIIb were transportedto the periplasm of E. coli by the tat system, and the correspondingB polypeptides of CT, LTI, and LTIIb were transported to theperiplasm by the sec system. The fluorescent fusion proteinswere shown to assemble spontaneously and efficiently with thecorresponding B polypeptides in the periplasm to form chimericholotoxin-like molecules, and these chimeras bound to and enteredcultured cells in a manner similar to native CT, LTI, or LTIIb.The GFP and mRFP derivatives of CT, LT, and LTIIb developedhere are useful tools for studies on the cell biology of traffickingof the CT/LT family of bacterial enterotoxins. In addition,these constructs provide proof in principle for the developmentof novel chimeric CT-like or LT-like vaccine candidates containingCTA₂ fusion proteins that cannot be delivered to the periplasmof E. coli by use of the sec secretion pathway.

Publication Information

Tinker, Juliette K.; Erbe, Jarrod L.; and Holmes, Randall K.. (2005). "Characterization of Fluorescent Chimeras of Cholera Toxin and Escherichia coli Heat-Labile Enterotoxins Produced by Use of the Twin Arginine Translocation System". Infection and Immunity, 73(6), 3627-3635.