Mukherjee Rukmini, Bhattacharya Anshu, Tomaskovic Ines, Mello-Vieira João, Brunstein Melinda Elaine, Başoğlu Marion, Veenendaal Tineke, Bailey Henry, Colby Thomas, Misra Mohit, Eimer Stefan, Klumperman Judith, Münch Christian, Matic Ivan, Dikic Ivan
Institute of Biochemistry II, Faculty of Medicine, Goethe University, Frankfurt, Germany.
Buchmann Institute for Molecular Life Sciences, Goethe University, Frankfurt, Germany.
EMBO J. 2025 Jun 12. doi: 10.1038/s44318-025-00483-4.
Legionella pneumophila is an intracellular pathogen that causes Legionnaires' disease. The bacteria release effector proteins, some of which remodel host autophagic-lysosomal pathways. One such effector is RavZ, which delipidates ATG8 proteins, making compromising autophagy in Legionella-infected cells. Here we show that SidE effectors also affect these pathways, by mediating phosphoribosyl-ubiquitination (PR-Ub) of the autophagic SNARE proteins STX17 and SNAP29. STX17 modification induces recruitment of STX17-positive membranes from the endoplasmic reticulum to Legionella-containing phagosomes, forming replicative vacuoles. Using proximity labeling, biochemistry and Legionella infection studies, we define a mechanism by which autophagy is hijacked by bacteria to recruit ER membranes to the bacterial vacuole, via a structure bearing autophagy markers but not fusing with lysosomes. Mass-spectrometric identification of PR-Ub sites and mutational studies show that phosphoribosyl-ubiquitination of STX17 alters its interaction with ATG14L, which causes ER membranes to be recruited to the bacterial vacuole in a PI3K-dependent manner. On the other hand, phosphoribosyl-ubiquitination of SNAP29 inhibits the formation of the autophagosomal SNARE complex (STX17-SNAP29-VAMP8) via steric hindrance, thus preventing the fusion of bacterial vacuoles with lysosomes.
嗜肺军团菌是一种胞内病原体，可引发军团病。该细菌会释放效应蛋白，其中一些效应蛋白会重塑宿主的自噬 - 溶酶体途径。其中一种效应蛋白是RavZ，它会去除ATG8蛋白的脂质，从而损害军团菌感染细胞中的自噬作用。在这里，我们表明SidE效应蛋白也会影响这些途径，通过介导自噬性SNARE蛋白STX17和SNAP29的磷酸核糖基泛素化（PR-Ub）。STX17的修饰会诱导内质网中STX17阳性膜募集到含军团菌的吞噬体，形成复制泡。通过邻近标记、生物化学和军团菌感染研究，我们确定了一种机制，即细菌劫持自噬，通过一个带有自噬标记但不与溶酶体融合的结构，将内质网膜募集到细菌液泡。对PR-Ub位点的质谱鉴定和突变研究表明，STX17的磷酸核糖基泛素化改变了其与ATG14L的相互作用，这导致内质网膜以PI3K依赖的方式被募集到细菌液泡。另一方面，SNAP29的磷酸核糖基泛素化通过空间位阻抑制自噬体SNARE复合体（STX17-SNAP29-VAMP8）的形成，从而阻止细菌液泡与溶酶体的融合。
