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.Pathogenic bacteria produce toxins that cause the symptoms of the diseases associated with infection. Several of these, including shiga toxin, pertussis toxin, cholera toxin (CT), 1 and heat-labile enterotoxin (LT), belong to a family related by structural homology. These AB 5 toxins consist of a catalytic A subunit (e.g. LTA, CTA) and a pentamer of receptor-binding B subunits (e.g. LTB, CTB) (1, 2). In addition to structural homology, the enterotoxigenic Escherichia coli (ETEC) toxin, LT, shares 80% nucleotide sequence identity with the Vibrio cholerae toxin, CT (3, 4). The ring-shaped B pentamers of both LT and CT mediate binding to the host cell receptor, G M1 (5-7). LTB is more promiscuous than CTB in that it can also bind other receptors containing a terminal galactose (6). After binding, the receptor/toxin complex is internalized by the host cell and the A subunit undergoes retrograde trafficking to the cytosol via the endoplasmic reticulum (ER) (6,8,9). Upon entry into the cytosol, the catalytic subunit constitutively activates adenylate cyclase, resulting in water and electrolyte efflux from the host cells (10).Despite the equivalent activity CT and LT exhibit in cell culture, disease caused by ETEC is much less severe than that caused by V. cholerae (11). Several factors, including the toxins themselves, are likely to contribute to the severity of these diseases. Volunteers treated with 25 g of CT lost an average of 20 liters of fluid, compared with only a 6 liter loss caused by the same amount of LT (12, 13). Hypothes...

Chemical validation of molecular mimicry: interaction of cholera toxin with Campylobacter lipooligosaccharides

“…Recently, it has been reported that Kdo2-lipid A also possesses binding affinity to Ctxb to form a complex with LOS via a GM1-independent binding region [44]. The authors demonstrated that the phosphate group on the Kdo core of LOS from Vibrio cholerae as preventing Ctxb from binding to the LOS, resulting in the secretion of soluble toxin across the outer membrane.…”

Bacterial outer membrane vesicles and the host–pathogen interaction

“…2). The minimal structure recognized by LT is the Kdo (3-deoxy-Dmanno-octulosonic acid) portion of LPS, and intriguingly, phosphorylation of Kdo inhibits binding by LT as well as its close homolog, cholera toxin (CT) (Horstman et al 2004). The difference in soluble secretion of CT by V. cholerae, compared with the vesicle-associated secretion of LT by ETEC, is therefore explained by the expression of phosphorylated Kdo on the LPS of V. cholerae.…”

Infection strategies of enteric pathogenic Escherichia coli

“…6 LT toxins are AB 5 toxins (one A subunit linked to a pentameric B subunit) and are transported across the bacterial outer membrane by the type 2 secretion system. 42 LT remains membrane-associated by binding lipopolysaccharide (LPS) 43 and is secreted in outer membrane vesicles (OMVs) that bind to ganglioside receptors on the mammalian cell (GM1a for LT-I or GD1 a/b for LT-II) via the LT-B subunit. The OMVs are then actively endocytosed and the LT transported via the Golgi and endoplasmic reticulum (ER) to the cytosol 44 where the A1 subunit then ADP-ribosylates mammalian guanine nucleotide binding protein a-subunit (G sa ).…”