Levanova Nadezhda, Tabakova Irina, Jank Thomas, Belyi Yury
Faculty of Medicine, Institute for Experimental and Clinical Pharmacology and Toxicology, University of Freiburg, Freiburg, Germany.
Gamaleya Research Centre, Moscow, Russia.
Methods Mol Biol. 2019;1921:277-287. doi: 10.1007/978-1-4939-9048-1_18.
Legionella pneumophila is a facultative intracellular pathogen responsible for legionellosis, a severe lung disease in humans. This bacterium uses a type 4b secretion system to deliver various effector proteins into the cytoplasm of a eukaryotic target cell. Among those is the glucosyltransferase Lgt1. This effector modifies serine-53 in eukaryotic elongation factor 1A (eEF1A) by mono-O-glucosylation. Modification of eEF1A results in inhibition of protein synthesis and death of the eukaryotic cell, processes which are thought to contribute to Legionella infection. Here we describe a protocol for isolation of the glucosyltransferase Lgt1 from L. pneumophila culture followed by assaying its enzymatic activity using C-UDP-glucose autoradiography.
嗜肺军团菌是一种兼性胞内病原体，可引发军团病，这是一种人类严重的肺部疾病。这种细菌利用4b型分泌系统将多种效应蛋白输送到真核靶细胞的细胞质中。其中包括葡糖基转移酶Lgt1。这种效应蛋白通过单O-糖基化修饰真核延伸因子1A（eEF1A）中的丝氨酸-53。eEF1A的修饰会导致蛋白质合成抑制和真核细胞死亡，这些过程被认为有助于军团菌感染。在此，我们描述了一种从嗜肺军团菌培养物中分离葡糖基转移酶Lgt1，然后使用C-UDP-葡萄糖放射自显影法测定其酶活性的方法。
