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

在当前提案的框架中，志贺毒素噬菌体编码酶 5-N-乙酰基-9-O-乙酰神经氨酸 (Neu5,9Ac2)-酯酶（简称：O-乙酰基酯酶）的功能和作用指定为 933Wp42，并进一步推定原噬菌体编码同源蛋白，用于肠出血性大肠杆菌 (EHEC) 的生长和毒力。 Neu5,9Ac2 在人肠道粘蛋白中的浓度较高。最初，将构建所有相应基因的单一缺失突变体和缺失组合（O157:H7菌株EDL933中的7个基因和O104:H4菌株LB226692中的4个基因）。将研究这些突变体利用 Neu5,9Ac2 作为生长碳源的能力。此外，还将分析 7 种假定的 O-乙酰酯酶的存在是否会在竞争模型中导致生长的选择性优势。 此外，还将阐明933Wp42和菌株EDL933的其他6种推定酶是否可以利用除Neu5,9Ac2之外的粘蛋白中的其他碳水化合物，以及是否可以发现进一步的酶活性。在此背景下，我们将研究酶933Wp42是否通过切割真核细胞（Vero细胞）的表面分子来调节志贺毒素2的毒性。此外，开放阅读编码假定的同源蛋白的框架将被重组表达、纯化并在标准酯酶测定中进行分析以分析其功能。基于对原噬菌体基因组中相应基因定位的观察，将阐明这些蛋白质是否对噬菌体本身很重要以及与噬菌体颗粒相关。这项研究的结果将有助于更详细地了解 O-乙酰酯酶对 EHEC 生长、营养竞争和毒力特性的功能和作用。

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.