A Single Native Ganglioside GM(1)-Binding Site Is Sufficient for Cholera Toxin To Bind to Cells and Complete the Intoxication Pathway

Jobling, Michael G.; Yang, ZhiJie; Kam, Wendy R.; Lencer, Wayne I.; Holmes, Randall K.

Bacterial strains and construction of expression clones. All chimeric toxins in this study were produced by expression from recombinant plasmids in Escherichia coli BW27784 (araFGH pCP18-araE [37] ). This strain does not metabolize arabinose and constitutively expresses the arabinose transporter, and therefore a uniform degree of induction occurs in all cells of a culture at any arabinose concentration. To produce the expression strains, genes encoding native-length CTB or carboxy-terminally extended CTB tagged with a glycosylation-sulfation signal (24) followed by a hexahistidine peptide were cloned on compatible plasmids with different selectable markers and inducible promoters. These encoded resistance to ampicillin (Ap) with the IPTG-inducible lacUV5 promoter or resistance to chloramphenicol (Cm) and the arabinose-inducible araBAD promoter, along with the regulator araC. Variants of these CTB clones were also made with G33D substitutions that eliminate GM 1 binding. A separate plasmid (pSlacbadCTA) selectable with spectinomycin (Sp) and compatible (with a pSC101 origin) with both ctxB plasmids was used to independently express the ctxA gene under control of both the lac and araBAD promoters. Each expression strain thus contained three plasmids, encoding a native CTB subunit (wt or G33D variant, Ap, and lac promoter), a GSH6-tagged CTB subunit (G33D or wt, Cm, and ara promoter), and the wt CTA subunit (Sp and ara and lac promoters). Details of construction, including full DNA sequences, are available on request. Plasmids created or used in this study are listed in Table 1 . Plasmids pAlacCTB and pAlacCTB G33D encode Ap resistance and the wt and G33D native-length variants of CTB, respectively, under control of the lac promoter and have previously been published as pLMP1 and pLMP148, respectively (38) . Carboxy-terminally linker-hexahistidine-tagged variants of CTB and CTB-G33D were made in a compatible Cm r arabinoseinducible vector, pAR3 (39), using the hexa-His tag from pT7sh6 (40) and a glycine-rich repeat from the M13 phage PIII protein, encoding XX (EX) 4 XDPRVPSS (where X is GGGS) inserted between residues 102 and 103 of CTB. Initial experiments to make wt and CTB-G33D mixed pentamers were done using these plasmids, but we saw significant proteolysis of the polyglycine linker-tagged variants, and subsequent experiments were done with variants that had the linker replaced with the GSH6 tag-pCbadCTBGSH6 and pCbadCTB G33D GSH6.