Defined O-acetylated Neu5Ac substrates reveal position-specific preferences of gut bacterial sialidases.

Sialic acid on intestinal glycans is modified by O-acetylation, a modification that can protect glycans from bacterial degradation and modulate immune recognition. However, the specificity of gut bacterial sialidases toward O-acetylated sialic acid remains poorly defined. Here, we characterized six GH33 sialidases from Bacteroides spp. and Akkermansia muciniphila using chemically defined 4-methylumbelliferyl substrates bearing 4-O-, 7-O-, or 8/9-O-acetylated N-acetylneuraminic acid (Neu5Ac). All enzymes adopted the canonical β-propeller fold and displayed distinct substrate preferences. While 8/9-O-acetylated Neu5Ac was broadly tolerated and 7-O-acetylation variably accommodated, all commensal sialidases were unable to cleave 4-O-acetylated Neu5Ac. Endogenous murine caecal sialidase activity showed the same pattern. Removal of the 4-O-acetyl group by a viral sialate O-acetylesterase restored cleavage, demonstrating that the C4 acetyl group directly prevents hydrolysis. On primary colonic and splenic lymphocytes, the bacterial sialidases produced enzyme-, linkage-, cell-type-, and tissue-dependent changes in sialic acid-dependent lectin and Siglec-E-Fc binding. In colon tissue, however, only the broad-spectrum pathogen-derived NanI sialidase consistently reduced SNA, MAL2, and Siglec-E-Fc binding, while the commensal enzymes caused more restricted changes. These findings demonstrate pronounced position-dependent effects of Neu5Ac O-acetylation on the activity of the sialidases tested and reveal substantial enzyme-specific differences in the processing of defined O-acetylated substrates.