Lipopolysaccharide 3-deoxy-D-manno-octulosonic acid (Kdo) core determines bacterial association of secreted toxins

Abstract

In contrast to cholera toxin (CT), which is secreted solubly by Vibrio cholerae across the outer membrane, heat-labile enterotoxin (LT) is retained on the surface of enterotoxigenic Escherichia coli (ETEC) via an interaction with lipopolysaccharide (LPS). We examined the nature of the association between LT and LPS. Soluble LT binds to the surface of LPS deep-rough biosynthesis mutants but not to lipid A, indicating that only the Kdo (3-deoxy-d-manno-octulosonic acid) core is required for binding. Although capable of binding truncated LPS and Kdo, LT has a higher affinity for longer, more complete LPS species. A putative LPS binding pocket is proposed based on the crystal structure of the toxin. The ability to bind LPS and remain associated with the bacterial surface is not unique to LT, as CT also binds to E. coli LPS. However, neither LT nor CT is capable of binding to the surface of Vibrio. The core structures of Vibrio and E. coli LPS differ in that Vibrio contains a phosphorylated single Kdo-lipid A, and E. coli LPS contains unphosphorylated Kdo2-lipid A. We determined that the phosphate group on the Kdo core of Vibrio LPS prevents CT from binding, resulting in the secretion of soluble toxin. Because LT binds E. coli LPS, it remains associated with the extracellular bacterial surface and is released in association with outer membrane vesicles. We propose that difference in the extracellular fates of LT and CT contribute to the differences in disease caused by ETEC and Vibrio cholerae.

Fig. 1. LT binds to the surface of E. coli LPS mutants

A, immunoblot of LT binding to wild type (K-12) and LPS mutant (Rd and Re) E. coli strains. Incubations of bacteria with buffer, LT, and LT preincubated with soluble O55 LPS are indicated. B, soluble Re LPS blocks LT from binding Re E. coli. Immunoblot of LT alone; and incubations of Re E. coli with buffer, LT, and LT preincubated with purified Re LPS, as indicated. C, Kdo inhibits LT binding to Re E. coli. Immunoblot of LT alone; and incubations of Re E. coli with buffer, LT, and LT preincubated with purified Re LPS or Kdo, as indicated.

Fig. 2. LT does not bind Vibrio LPS, however CT binds E. coli LPS

A, detection of surface-associated LTB and CTB expressed by Vibrio sp. 60 and secretion-competent E. coli by BODIPY-G_M1 labeling. Error bars = S.E., n ≥ 3. B, immunoblot of E9034P incubated with buffer, CT, and CT preincubated with O55 LPS, as indicated.

Fig. 3. Toxin binding is blocked by phosphorylation of the Kdo core of Vibrio LPS

Immunoblots of LT binding to E. coli strains CJB26 and Kdo1 (A), and Kdo2 and Kdo1P (B), incubated with buffer and LT, as indicated. C, Detection by BODIPY-G_M1 labeling of secreted LTB on the surface of Vibrio sp. 60 expressing LTB and Vibrio sp. 60 expressing LTB and Kdo2-Lipid A. Error bars = S.E., n ≥ 3.

Fig. 4. Proposed location of LPS binding site on the toxin

A, view from the top of an isolated LTB/CTB subunit, as if looking down into central pore of the pentamer. Star denotes putative binding site. B, oblique side view of a LTB/CTB-pentamer. Green, G_M1 pentasaccharide; blue, putative LPS binding site. Adapted from PDB file 2CHB of CT, using Swiss PDBviewer.