Browsing by Subject "NanS-p"

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Funktionelle Charakterisierung von 9-O-Acetylesterasen enterohämorrhagischer Escherichia coli
(2019) Feuerbaum, Stefanie; Schmidt, Herbert
Enterohemorrhagic Escherichia coli (EHEC) colonize the human colon and compete against commensal E. coli for limited nutrition. Colonic goblet cells produce glycoproteins called mucin, which are part of the mucosal barrier. This barrier is free of bacterial penetration and is important for the protection of the epithelium. The main glycoprotein in the large intestine is mucin 2 (MUC2), which is heavily O-glycosylated with terminal glycan-bounded sialic acids. These aminosugars naturally exists with up to four O-acetylgroups at position C4, C7, C8 and C9. The grade of O-acetylation acts as a protection for enzymatic degradation by glycosidases. Commensal E. coli and the pathogenic strain O157:H7 EDL933 encodes the single chromosomal 9-O-acetylesterase NanS, which is important for the metabolismus of sialic acids by de-O-acetylation of Neu5,9Ac2 to the basic structure Neu5Ac and Acetate. Both can be used as an energy source. Pathogenic E. coli O157:H7 EDL933 and O104:H4 C227-11Φcu encode further several prophage-encoded 9-O-acetylesterases (NanS-p). Recent studies demonstrated that NanS-p producing EHEC bacteria reveal a higher replication rate in Neu5,9Ac2 containing medium compared with commensal E. coli. This could be an advantage during colonization in human large intestine, where Neu5,9Ac2 is the most common sialic acid in mucins. The aim of this study was to further characterize the enzymatic function of prophage-encoded 9-O-acetylesterases in carbohydrate metabolism and during infection in vitro. To analyze the NanS-p mediated cleavage of mucin-derived O-acetylneuraminic acids, HPTLC and nanoESI MS analyses were performed. The results revealed, that recombinant expressed NanS-p cleave-off acetyl residues from up to tri-O-acetylated Neu5Ac and Neu5Gc. While NanS-p were able to de-O-acetylate glycan-bounded sialic acids at positions C7, C8 and C9, the tested enzymes were not able to hydrolyze the acetyl ester from position C4. The lower specificity of the NanS-p leads to higher availability of mucin-derived substrates for sialidases of commensal bacteria B. thetaiotaomicron. Further analyses reveal a NanS-p dependent mucin degradation in cell culture assays. Mutant strains EDL933ΔnanSΔnanS-p1-nanS-p7 and EDL933ΔnanSΔnanS-p1-nanS-p5 were not able to degrade the mucinlayer, while the wildtype strain O157:H7 EDL933 could disrupt the mucosal barrier of LS 180 cells. The NanS-p dependent adherence of O157:H7 EDL933 and O104:H4 C227-11Φcu to epithelial cells HT-29 was demonstrated by performing adherence assays. The deletion of nanS-p genes revealed less adhered bacteria compared to wildtype strains. Further performed swimming assays could show the impact of NanS-p on motility. The wildtype strains O157:H7 EDL933 and O104:H4 C227-11Φcu have shown less motility compared to nanS-p deletion mutants. Taken together, NanS-p show an important role during infection and could contribute to the preferred colonization of the large intestine due to their impact of the motility, disruption of the mucosal barrier and mediation of adherence of EHEC to the epithelium.