Analysis of the function and role of the O-acetyl-esterase 933Wp42 and homologues proteins for growth and virulence of enterohemorrhagic Escherichia coli (EHEC)

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In the framework of the current proposal, the function and role of the Shiga toxin-phage-encoded enzyme 5-N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2)-esterase (shortly: O-acetyl-esterase) designated 933Wp42 and further putative prophage encoded homologues proteins for growth and virulence of enterohemorrhagic Escherichia coli (EHEC). Neu5,9Ac2 occurs in higher concentrations in mucin of the human intestine. Initially, single deletion mutants and combinations of deletions of all corresponding genes (7 genes in O157:H7 strain EDL933 and 4 genes in O104:H4 strain LB226692) will be constructed. The ability of these mutants to utilize Neu5,9Ac2 as a carbon source for growth will be investigated. Furthermore, it will be analyzed, whether the presence of the 7 putative O-acetyl-esterases cause a selective advantage for growth in a competition model. In addition, it will be clarified, whether 933Wp42 and the other 6 putative enzymes of strain EDL933 can utilize, besides Neu5,9Ac2, further carbohydrate compounds in mucin, and whether further enzyme activities can be found.In this context, we will investigate whether the enzyme 933Wp42 modulates the toxicity of Shiga toxin 2 by cleaving surface molecules of eukaryotic cells (Vero cells).Moreover, the open reading frames encoding the putative, homologues proteins will be expressed recombinantly, purified and analyzed in standard esterase enzyme assays to analyze their function. Based on the observation that the corresponding genes are localized in prophage genomes it will be clarified whether the proteins may be important for the phage itself and are associated with the phage particles.The results of this study will help to get a more detailed insight in function and role of the O-acetyl-esterase for growth, competition for nutrients, and virulence properties of EHEC.