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

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

Use of Translational Fusion of the MrpH Fimbrial Adhesin-Binding Domain with the Cholera Toxin A2 Domain, Coexpressed with the Cholera Toxin B Subunit, as an Intranasal Vaccine To Prevent Experimental Urinary Tract Infection by Proteus mirabilis

This is a follow-up to our previous study using an intranasal vaccine composed of MrpH, the tip adhesin of the MR/P fimbria, and cholera toxin to prevent urinary tract infection by Proteus mirabilis (X. Li, C. V. Lockatell, D. E. Johnson, M. C. Lane, J. W. Warren, and H. L. Mobley, Infect. Immun. 72:66-75, 2004). Here, we have expressed a cholera toxin-like chimera in which the MrpH adhesin-binding domain (residues 23 to 157) replaces the cholera toxin A1 ADP-ribosyltransferase domain. This chimera, when administered intranasally without additional adjuvant, is sufficient to induce protective immunity in mice