Browsing by Subject "NanS"

Now showing 1 - 2 of 2

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.
Molekularbiologische und physiologische Untersuchungen zur Bedeutung phagenkodierter Sialinsäureesterasen von enterohämorrhagischen Escherichia coli (EHEC)
(2019) Saile, Nadja; Schmidt, Herbert
Enterohemorrhagic Escherichia coli (EHEC) are responsible for severe disease in humans such as hemorrhagic colitis or the life-threatening hemolytic uremic syndrome. The main virulence factors are Shiga toxins (Stx) and a type-III-secretion system. For colonization of the colon, they have to compete with the intestinal microbiota for limiting substrates such as 5-N-acetyl neuraminic acid (Neu5Ac). For the catabolism of Neu5Ac, E. coli possess the genes nanA, nanT, nanE, nanK, nagA, and nagB. Many sialic acids are terminally bound to mucins of mucus in the colon. E. coli is not able to use bound Neu5Ac, because it cannot express sialidases for cleavage of Neu5Ac from mucin. However, sialic acids can be cleaved by sialidases from anaerobic bacteria in the colon and are then available. Many E. coli strains encode the nanCMS operon. NanC is a porin protein, NanM is a mutarotase and NanS is an O-acetyl esterase. NanS converts 5-N-acetyl-9-O-acetyl neuraminic acid (Neu5,9Ac2) and Neu5,8Ac2 to Neu5Ac and offers a substrate niche. There are multiple prophage-located nanS-homologous open reading frames in the chromosomes of EHEC, that are designated as nanS-p (p = phage) in this study. The question of this thesis was, why EHEC possess multiple nanS-p genes, if their corresponding proteins are used for energy production and for a competition advantage of EHEC. Furthermore, the influence of the sialidase BTSA of Bacteroides thetaiotaomicron on the NanS-p dependent utilization of mucin should be investigated. The strains used in this study were the pathogenic E. coli O157:H7 strain EDL933 as well as the pathogenic O104:H4 strains LB226692 and C227-11phicu (variant of C227-11 cured in its stx-prophage), and the apathogenic E. coli strains AMC 198 (nanS+, nanS-p-) and C600deltananS (nanS-, nanS-p-). The chromosome sequences of EDL933 and LB226692 were analyzed in silico. NanS-p2 and NanS-p4 of EDL933 were expressed recombinantly and the pH- and temperature-optimum as well as potential substrates were investigated. nanS/nanS-p deletion mutants of EDL933 and C227-11phicu were generated and cultivated with Neu5,9Ac2 or mucin. The courses of the growth curves were analyzed by turbidity measurement or determination of the viable cell counts. In co-cultivation experiments the medium was supplemented with the AMC 198 strain and/or BTSA and/or NanS-p. EDL933 possess the chromosomal nanS and seven nanS-p genes, while LB226692 has no nanS, but five nanS-p genes. It was confirmed, that all nanS-p genes are located in the late regulated gene region of the prophages. The putative NanS-p proteins include the domain 303, which is known for its esterase activity in NanS, and the domain of unknown function 1737. The analyzed recombinant NanS-p proteins de-O-acetylated the substrates Neu5,9Ac2 and mucin of bovine submaxillary gland and showed a temperature- and pH-optimum of 40-50 °C and 7-9, respectively. A gene-dose effect was identified because the generation times of the deletion mutant strains increased with the number of deleted nanS-p genes in the chromosomes. After extracellular supplementation with NanS-p, the mutant strains regained the original growth kinetic of the wildtype strain. The mono-deletion of nanS in EDL933 (EDL933deltananS) caused no chances in the growth kinetic of the strain with Neu5,9Ac2. Therefore, NanS is not necessary for growth of EDL933 on Neu5,9Ac2. C600deltananS could not catabolize Neu5,9Ac2, as expected. The original course of the growth curve of a C600 cultivation was recuperated by C600deltananS if NanS-p was added to the medium. In a co-cultivation the viable cell count of C227-11phicu increased, while AMC 198 increased hardly. Only after deletion of all nanS-p genes in C227-11phicu, the viable cell count of AMC 198 increased. In a mucin-containing medium with BTSA as supplement EDL933 and EDL933deltananS could grow in contrast to EDL933deltananSdeltananS-p1a-p7. This shows, that the combination of BTSA and NanS-p supports the growth of EDL933 in a mucin-containing environment. The results of this work showed for the first time the importance of prophage-encoded O-acetyl esterases for EHEC bacteria and are an excellent basis for further work that contribute to a profound scientific understanding of the sialic acid catabolism in EHEC and other pathogenic E. coli. They identify sialic acids as substrates of a potential nutrient niche in the gut and establish a potential location for therapeutic approaches.