Genetics and molecular specificity of sialylation of Histophilus somni lipooligosaccharide (LOS) and the effect of LOS sialylation on Toll-like receptor-4 signaling

Abstract Histophilus somni is an etiologic agent of bovine respiratory and systemic diseases. Most pathogenic strains of H. somni that have been tested (36 of 42) are able to utilize N -acetyl-5-neuraminic acid (Neu5Ac) to sialylate their lipooligosaccharide (LOS). Homologs of all the genes required for transport, metabolism, and regulation of Neu5Ac in Haemophilus influenzae were identified in the sequenced genomes of H. somni . Three open reading frames (ORFs) in H. somni strain 2336 were identified that contained homology to genes required for LOS sialylation in related bacteria. ORF-1 ( hssT-I ), ORF-2 ( hssT-II ), and ORF-3 ( neuA Hs ) were predicted to encode for putative proteins with 37% amino acid homology to an α-(2–3)-sialyltransferase in H. influenzae , 43% amino acid homology to an Haemophilus ducreyi sialyltransferase, and 72% amino acid homology to an H. influenzae CMP-Neu5Ac synthetase, respectively. The specific enzyme activity of each ORF was determined using synthetic acceptor substrates. The HssT-I sialyltransferase primarily sialylated N -acetyllactosamine (LacNAc, Gal-β-[1–4]-GlcNAc-R), which is expressed on strain 2336, whereas HssT-II preferentially sialylated lacto- N -biose (LNB, Gal-β-[1–3]-GlcNAc-R), which is expressed on a phase variant of strain 2336: strain 738. Phase variation of the terminal galactose linkage in strain 738 from β-(1–3)-(LNB) to β-(1–4)-(LacNAc) was confirmed using monoclonal antibody reactivity and nuclear magnetic resonance spectroscopy. Sialylated LOS induced significantly less chemokine response from macrophages derived from Toll-like receptor (TLR)-4 knockout mice than from de-sialylated LOS. Furthermore, sialylated LOS induced significantly less NF-κB activity from mouse-derived bone marrow macrophages than de-sialylated LOS. Therefore, sialylation inhibited LOS signaling through TLR-4. In conclusion, H. somni utilizes linkage-specific sialyltransferases to sialylate its LOS to avoid innate host defense mechanisms despite simultaneous epitope phase variation.