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

Abstract

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 (Neu5,9Ac 2 ) 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 Neu5,9Ac 2 . However, all tested enzymes were unable to de-O-acetylate 5-N-acetyl-4-O-acetylneuraminic acid (Neu5,4 Ac 2 ) 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 Neu5,9Ac 2 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.

Carriage of Shiga toxin phage profoundly affects Escherichia coli gene expression and carbon source utilization

“…If the results of our in vitro studies accurately predict what sugars a cell can utilize in vivo, the Stx2a phage lysogens should outcompete naive MG1655 in niches with available arabinose, fucose and N-acetyl-D-glucosamine, while be readily eliminated in the ones with N-acetylneuraminic acid and gluconate. Interestingly, certain EHEC strains carry additional functional nanS-homologues alleles, which are prophage-encoded [50][51][52]. In respect to our data, multiple nanS copies would be beneficial to compensate for potential Stx phage-dependent decrease in the ability to utilize N-acetylneuraminic acid due to downregulation of the native nan operons.…”

Mucins Inhibit Coronavirus Infection in a Glycan-Dependent Manner

“… 68 , 69 Both OC43 and the related bovine CoV are reported to bind 5,9- O -acetyl-Sia (9AcOSia, Neu5,9Ac) and 5- O -acetyl-Sia (5AcOSia, Neu5Ac) via their spike proteins. 70 − 72 Based on this knowledge, we selected bovine submaxillary mucin for our studies since the commercially available material is known to contain Neu5Ac and Neu5,9Ac and originates from a tissue of native viral targeting. 73 , 74 …”

“…OC43, like SARS-CoV, SARS-CoV-2, and MERS-CoV, is a β-CoV that binds Sia via its spike protein. OC43 is endemic in the human population, causing mild respiratory tract infections with the potential for severe complications or even fatalities in young children, the elderly, or immunocompromised individuals. , The virus originated relatively recently via zoonotic transmission from a bovine CoV. , Both OC43 and the related bovine CoV are reported to bind 5,9- O -acetyl-Sia (9AcOSia, Neu5,9Ac) and 5- O -acetyl-Sia (5AcOSia, Neu5Ac) via their spike proteins. − Based on this knowledge, we selected bovine submaxillary mucin for our studies since the commercially available material is known to contain Neu5Ac and Neu5,9Ac and originates from a tissue of native viral targeting. , …”

Early immune innate hallmarks and microbiome changes across the gut during Escherichia coli O157: H7 infection in cattle

“…However, the relationship between mucin glycosylation and susceptibility to E. coli is complex. For instance, O -acetylated sialic acids (e.g., neuraminic acid Neu5Ac) linked to glycan chains of mucin can be used by EHEC as carbon sources 54 . Further studies with multiple lectins could decipher the mucin glycan dysregulation during EHEC O157: H7 infection.…”