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

在本提案的框架内，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)的功能和作用被命名为933Wp42和进一步推测的原噬菌体编码的同源蛋白与肠出血性大肠杆菌(EHEC)的生长和毒力有关。Neu5，9Ac2在人体肠道的粘蛋白中浓度较高。首先，将构建单个缺失突变体和所有相应基因的缺失组合(O157：H7株EDL933中的7个基因和O104：H4株LB226692中的4个基因)。这些突变株利用Neu5，9Ac2作为生长碳源的能力将被研究。此外，还将分析，在竞争模型中，这7个假定的O-乙酰酯酶的存在是否会导致生长的选择性优势。此外，933Wp42和EDL933菌株的其他6种可能的酶是否可以利用粘蛋白中除Neu5，9Ac2以外的其他碳水化合物，以及是否可以发现更多的酶活性，这将被阐明。在这方面，我们将研究933Wp42酶是否通过裂解真核细胞(Vero细胞)的表面分子来调节志贺毒素2的毒性。此外，编码可能的同源蛋白的开放阅读框将被重组表达，纯化，并在标准的酯酶分析中分析它们的功能。基于相应基因定位于噬菌体基因组的观察，这些蛋白是否对噬菌体本身具有重要作用并与噬菌体颗粒相关。这一研究结果将有助于更详细地了解O-乙酰酯酶对EHEC的生长、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.