Heat-stable enterotoxin of enterotoxigenic Escherichia coli as a vaccine target

Abstract

Enterotoxigenic Escherichia coli (ETEC) is responsible for 280 million to 400 million episodes of diarrhea and about 380,000 deaths annually. Epidemiological data suggest that ETEC strains which secrete heat-stable toxin (ST), alone or in combination with heat-labile toxin (LT), induce the most severe disease among children in developing countries. This makes ST an attractive target for inclusion in an ETEC vaccine. ST is released upon colonization of the small intestine and activates the guanylate cyclase C receptor, causing profuse diarrhea. To generate a successful toxoid, ST must be made immunogenic and nontoxic. Due to its small size, ST is nonimmunogenic in its natural form but becomes immunogenic when coupled to an appropriate large-molecular-weight carrier. This has been successfully achieved with several carriers, using either chemical conjugation or recombinant fusion techniques. Coupling of ST to a carrier may reduce toxicity, but further reduction by mutagenesis is desired to obtain a safe vaccine. More than 30 ST mutants with effects on toxicity have been reported. Some of these mutants, however, have lost the ability to elicit neutralizing immune responses to the native toxin. Due to the small size of ST, separating toxicity from antigenicity is a particular challenge that must be met. Another obstacle to vaccine development is possible cross-reactivity between anti-ST antibodies and the endogenous ligands guanylin and uroguanylin, caused by structural similarity to ST. Here we review the molecular and biological properties of ST and discuss strategies for developing an ETEC vaccine that incorporates immunogenic and nontoxic derivatives of the ST toxin.

FIG. 1.

Structures and sequence alignment of guanylate cyclase C receptor ligands. (A) Structures of the A form of human uroguanylin (left, PDB:1UYA) and the toxic domain (residues 6 to 18) of STp (right, PDB:1ETN). The N and C termini are marked. The two structures have a similar fold, and the part of the uroguanylin structure that corresponds to the ST structure is shown in cyan. The structures share two disulfide bridges (Cys7-Cys15 and Cys10-Cys19, shown as yellow sticks), and ST has an additional one (Cys6-Cys11). Note that Cys6 in ST is replaced with 5-beta-mercaptopropionate (shown as blue/yellow stick). The proposed GC-C receptor-interacting residues of ST (STp, Asn11-Pro12-Ala13) are shown as sticks. (B) Sequence alignment of the human GC-C ligands (top) and bacterial GC-C ligands (bottom). Disulfide bonds are marked with lines. Residue numbering is according to STh. The species abbreviations are as follows: Hs, Homo sapiens; Ec, Escherichia coli; Yk, Yersinia kristensenii; Ye, Yersinia enterocolitica; Vc, Vibrio cholerae.

FIG. 2.

ST mutants and effects on toxicity reported in peer-reviewed publications. The top of the figure shows an alignment of the STh and STp sequences, with the positions of the disulfide bonds marked with lines (residue numbering is according to the STh sequence). Below the alignment is an amino acid matrix, where the residues are grouped according to physicochemical properties (leftmost column). Only residues that have been mutated and tested for effect on toxicity are shown, and the following scheme is used: no shading, no effect on toxicity; gray shading, reduced toxicity; black shading, nontoxic. All mutations affect single amino acid residues, except one double mutation, which is marked with a dashed line. Two additional double mutants are not shown in the figure: STh (Pro13Gly Ala14Leu) (2) and STh (Asn12Tyr Tyr19Asn) (20, 72). The first was reported to be nontoxic but was not included since it is unclear whether the effect of toxicity was due to the change in residues or a result of the fusion construct. The latter was not included since it gave an effect similar to that of the Asn12Tyr single mutant. The ST variants used in the different reports are as follows (numbers correspond to superscript numbers in the matrix, and references are given in parentheses): 1, STp (31);, 2, STp (32); 3, STh (60); 4, STp (71); 5, STp (69); 6, STp (70); 7, STp (73). Notes: the Glu8Ala mutant (*) had an effect on DsbA-dependent disulfide bond formation; both the Phe4Gly and Tyr19Gly mutants (#) affected translocation of the ST across the outer membrane.