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

O-乙酰酯酶 933Wp42 和同源蛋白对肠出血性大肠杆菌 (EHEC) 生长和毒力的功能和作用分析

• 批准号: 261322118
• 负责人: Professor Dr. Herbert Schmidt
• 金额: --
• 依托单位: Fachgebiet Lebensmittelmikrobiologie und -hygiene (150a)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/261322118?language=en
• 关键词: Analysis; function; role; acetyl; esterase

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.

在当前提议的框架内，滋贺毒素噬菌体编码的酶5-N-乙酰基-9-O-乙酰神经氨酸（Neu 5，9Ac 2）-酯酶（简称：O-乙酰基酯酶）命名为933 Wp 42，进一步推定的原噬菌体编码的同源蛋白质的功能和作用对肠出血性大肠杆菌（EHEC）的生长和毒力。Neu 5，9Ac 2以较高浓度存在于人肠的粘蛋白中。首先，将构建所有相应基因（O 157：H7菌株EDL 933中的7个基因和O 104：H4菌株LB 226692中的4个基因）的单缺失突变体和缺失组合。将研究这些突变体利用Neu 5，9Ac 2作为生长碳源的能力。此外，它将被分析，是否存在的7个假定的O-乙酰酯酶的竞争模型中的生长引起的选择性优势。 另外，我们将阐明933 Wp 42和菌株EDL 933的其他6种推测的酶是否可以利用除Neu 5，9Ac 2之外的粘蛋白中的其他碳水化合物，以及是否可以发现其他的酶活性此外，编码推定的同源蛋白的开放阅读框将被重组表达、纯化并在标准酯酶测定中分析以分析它们的功能。通过对O-acetyl-esterase基因定位于噬菌体基因组的观察，阐明了O-acetyl-esterase蛋白是否对噬菌体本身具有重要意义，是否与噬菌体颗粒相关，从而有助于更详细地了解O-acetyl-esterase在EHEC生长、营养竞争和毒力特性中的功能和作用。

项目成果

Escherichia coli O157:H7 Strain EDL933 Harbors Multiple Functional Prophage-Associated Genes Necessary for the Utilization of 5-N-Acetyl-9-O-Acetyl Neuraminic Acid as a Growth Substrate

• DOI: 10.1128/aem.01671-16
• 发表时间: 2016-10-01
• 期刊: APPLIED AND ENVIRONMENTAL MICROBIOLOGY
• 影响因子: 4.4
• 作者: Saile, Nadja;Voigt, Anja;Schmidt, Herbert
• 通讯作者: Schmidt, Herbert

Growth advantage of Escherichia coli O104:H4 strains on 5-N-acetyl-9-O-acetyl neuraminic acid as a carbon source is dependent on heterogeneous phage-Borne nanS-p esterases.

• DOI: 10.1016/j.ijmm.2018.03.006
• 发表时间: 2018-06
• 期刊: International journal of medical microbiology : IJMM
• 作者: Nadja Saile;L. Schwarz;Kristina Eissenberger;J. Klumpp;Florian W. Fricke;H. Schmidt