The role of 9-O-acetylated glycan receptor moieties in the typhoid toxin binding and intoxication.

Typhoid toxin is an A(2)B(5) toxin secreted from Salmonella Typhi-infected cells during human infection and is suggested to contribute to typhoid disease progression and the establishment of chronic infection. To deliver the enzymatic 'A' subunits of the toxin to the site of action in host cells, the receptor-binding 'B' subunit PltB binds to the trisaccharide glycan receptor moieties terminated in N-acetylneuraminic acid (Neu5Ac) that is alpha 2-3 or alpha 2-6 linked to the underlying disaccharide, galactose (Gal) and N-acetylglucosamine (GlcNAc). Neu5Ac is present in both unmodified and modified forms, with 9-O-acetylated Neu5Ac being the most common modification in humans. Here we show that host cells associated with typhoid toxin-mediated clinical signs express both unmodified and 9-O-acetylated glycan receptor moieties. We found that PltB binds to 9-O-acetylated alpha 2-3 glycan receptor moieties with a markedly increased affinity, while the binding affinity to 9-O-acetylated alpha 2-6 glycans is only slightly higher, as compared to the affinities of PltB to the unmodified counterparts, respectively. We also present X-ray co-crystal structures of PltB bound to related glycan moieties, which supports the different effects of 9-O-acetylated alpha 2-3 and alpha 2-6 glycan receptor moieties on the toxin binding. Lastly, we demonstrate that the cells exclusively expressing unmodified glycan receptor moieties are less susceptible to typhoid toxin than the cells expressing 9-O-acetylated counterparts, although typhoid toxin intoxicates both cells. These results reveal a fine-tuning mechanism of a bacterial toxin that exploits specific chemical modifications of its glycan receptor moieties for virulence and provide useful insights into the development of therapeutics against typhoid fever. Author summary The Gram-negative rod-shaped bacteria Salmonella enterica serovar Typhi (S. enterica serovar Typhi or S. Typhi) is the cause of the life-threatening disease typhoid fever. Many millions of people including children under the age of five are affected by this infectious disease. Molecular mechanisms underlying typhoid disease progression and the establishment of chronic infection important for the transmission of this human-adapted pathogen are incompletely understood, but typhoid toxin, one of the virulence factors of S. Typhi, is suggested to contribute to these processes. Typhoid toxin consists of three functionally distinct subunits: two enzymatic 'A' subunits important for intoxicating host cells after the delivery into host cells and one homopentamer of receptor-binding 'B' subunit important for the delivery of the toxin into host cells. Typhoid toxin 'B' subunit recognizes specific three-sugar structures decorating the host cell surface, whose terminal sugar called N-acetylneuraminic acid (Neu5Ac) can be present in unmodified or modified forms. The modified Neu5Ac possesses additional chemical groups, with 9-O-acetylation being the most frequently found modification in humans. This study analyzes glycan expression profiles of primary tissues and cells associated with typhoid clinical signs, the interface between the toxin and glycan receptor moieties through biochemical and structural approaches, and typhoid toxin intoxication profiles of host cells that are genetically engineered to alter the expression of related glycans. This study reveals a fine-tuning mechanism of a bacterial toxin in exploiting a chemical modification of glycan receptor moieties for virulence and thus provides useful insights into the development of efficacious therapeutics against typhoid intoxication.