De-O-Acetylation of mucin-derived sialic acids by recombinant NanS-p esterases of Escherichia coli O157:H7 strain EDL933.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain EDL933 encodes the single chromosomal 9-O-acetylesterase NanS, and several copies of prophage-encoded 9-O-acetylesterases (NanS-p). These enzymes have recently been shown to cleave 5-N-acetyl-9-O-acetyl neuraminic acid (Neu5,9Ac₂) to yield de-O-acetylated Neu5Ac, the latter of which may serve as a carbon and/or nitrogen source. In the current study, we investigated the NanS- and NanS-p-mediated digestion of synthetic O-acetylated neuraminic acids and bovine submaxillary glands mucin (BSM)-derived O-acetylneuraminic acids by high-performance thin-layer chromatography (HPTLC) and nano electrospray ionization mass spectrometry (nanoESI MS). Initial HPTLC analyses showed the expected activity of NanS and NanS-p variants for Neu5,9Ac₂. However, all tested enzymes were unable to de-O-acetylate 5-N-acetyl-4-O-acetylneuraminic acid (Neu5,4 Ac₂) in our test system. The nanoESI MS analysis of neuraminic acids after treatment of BSM with NanS-p gave evidence that NanS-p variants of EHEC O157:H7 strain EDL933 cleave off O-acetyl groups from mono-, di-, and tri-O-acetylated Neu5Ac and N-glycolylneuraminic acid (Neu5Gc), regardless of the carbon positions C7, C8 or C9 of the acetate esters. This enzyme activity leads to neuraminidase-accessible Neu5Ac and Neu5Gc on mucin glycans. Moreover, we could demonstrate by HPTLC analyses that recombinant Bacteroides thetaiotaomicron sialidase (BTSA-His) was able to cleave Neu5Ac and Neu5,9Ac₂ from BSM and that the combination of BTSA-His with both NanS-His and NanS-p-His derivatives enhanced the release of de-O-acetylated core Neu5Ac and Neu5Gc from mammalian mucin O-glycans. Growth experiments with EHEC wildtype strain EDL933, its nanS and nanS/nanS-p1a-p7 mutant and exogenous BTSA-His in BSM demonstrated that the presence of BTSA-His enhanced growth of EDL933 and the nanS deletion mutant but not the nanS/nanS-p1a-p7 mutant. Thus, we hypothesize that the expression of sialic acid O-acetylesterases with a broad specificity could be an advantage in competition with the gut microbiota for nutrients and facilitate EHEC colonization in the human large intestine.

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