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首页> 外文期刊>Infection and immunity >Purification and Characterization of a UDP-Glucosyltransferase Produced by Legionella pneumophila
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Purification and Characterization of a UDP-Glucosyltransferase Produced by Legionella pneumophila

机译：嗜肺军团菌产生的UDP-葡萄糖基转移酶的纯化和鉴定

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Legionella pneumophila is the agent of Legionnaires' disease. It invades and replicates within eukaryotic cells, including aquatic protozoans, mammalian macrophages, and epithelial cells. The molecular mechanisms of the Legionella interaction with target cells are not fully defined. In an attempt to discover novel virulence factors of L. pneumophila, we searched for bacterial enzymes with transferase activity. Upon screening ultrasonic extracts of virulent legionellae, we identified a uridine diphospho (UDP)-glucosyltransferase activity, which was capable of modifying a 45-kDa substrate in host cells. An approximately 60-kDa UDP-glucosyltransferase was purified from L. pneumophila and subjected to microsequencing. An N-terminal amino acid sequence, as well as the sequence of an internal peptide, allowed us to identify the gene for the enzyme within the unfinished L. pneumophila genome database. The intact gene was cloned and expressed in Escherichia coli, and the recombinant protein was purified and confirmed to possess an enzymatic activity similar to that of the native UDP-glucosyltransferase. We designated this gene ugt (UDP-glucosyltransferase). The Legionella enzyme did not exhibit significant homology with any known protein, suggesting that it is novel in structure and, perhaps, in function. Based on PCR data, an enzyme assay, and an immunoblot analysis, the glucosyltransferase appeared to be conserved in L. pneumophila strains but was absent from the other Legionella species. This study represents the first identification of a UDP-glucosyltransferase in an intracellular parasite, and therefore modification of a eukaryotic target(s) by this enzyme may influence host cell function and promote L. pneumophila proliferation.
机译：肺炎军团菌是退伍军人症的病原体。它侵入并在真核细胞内复制，包括水生原生动物，哺乳动物巨噬细胞和上皮细胞。 Legionella 与靶细胞相互作用的分子机制尚未完全阐明。为了发现 L的新型毒力因子。肺炎，我们搜索了具有转移酶活性的细菌酶。在筛选有毒军团菌的超声波提取物后，我们鉴定了尿苷二磷酸（UDP）-葡萄糖基转移酶活性，该活性能够修饰宿主细胞中的45 kDa底物。从 L中纯化出约60kDa的UDP-葡萄糖基转移酶。肺炎并进行微测序。 N末端氨基酸序列以及内部肽序列使我们能够鉴定未完成的 L中酶的基因。肺炎菌基因组数据库。完整的基因被克隆并在大肠杆菌中表达，纯化了重组蛋白并确认其具有与天然UDP-葡萄糖基转移酶相似的酶促活性。我们将该基因命名为 ugt （UDP-葡萄糖基转移酶）。 Legionella 酶与任何已知的蛋白质均未显示出显着的同源性，这表明它的结构和功能可能是新颖的。根据PCR数据，酶分析和免疫印迹分析，葡糖基转移酶似乎在 L中是保守的。肺炎链球菌菌株，但其他 Legionella 菌种均没有。该研究代表了细胞内寄生虫中UDP-葡萄糖基转移酶的首次鉴定，因此，通过该酶修饰的真核靶标可能影响宿主细胞功能并促进Lem。肺炎增殖。

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