Beyer Andrea R, Truchan Hilary K, May Levi J, Walker Naomi J, Borjesson Dori L, Carlyon Jason A
Department of Microbiology and Immunology, Virginia Commonwealth University School of Medicine, Richmond, VA, 23298, USA.
Cell Microbiol. 2015 Apr;17(4):504-19. doi: 10.1111/cmi.12380. Epub 2014 Nov 22.
SUMOylation, the covalent attachment of a member of the small ubiquitin-like modifier (SUMO) family of proteins to lysines in target substrates, is an essential post-translational modification in eukaryotes. Microbial manipulation of SUMOylation recently emerged as a key virulence strategy for viruses and facultative intracellular bacteria, the latter of which have only been shown to deploy effectors that negatively regulate SUMOylation. Here, we demonstrate that the obligate intracellular bacterium, Anaplasma phagocytophilum, utilizes an effector, AmpA (A. phagocytophilum post-translationally modified protein A) that becomes SUMOylated in host cells and this is important for the pathogen's survival. We previously discovered that AmpA (formerly APH1387) localizes to the A. phagocytophilum-occupied vacuolar membrane (AVM). Algorithmic prediction analyses denoted AmpA as a candidate for SUMOylation. We verified this phenomenon using a SUMO affinity matrix to precipitate both native AmpA and ectopically expressed green fluorescent protein (GFP)-tagged AmpA. SUMOylation of AmpA was lysine dependent, as SUMO affinity beads failed to precipitate a GFP-AmpA protein when its lysine residues were substituted with arginine. Ectopically expressed and endogenous AmpA were poly-SUMOylated, which was consistent with the observation that AmpA colocalizes with SUMO2/3 at the AVM. Only late during the infection cycle did AmpA colocalize with SUMO1, which terminally caps poly-SUMO2/3 chains. AmpA was also detected in the cytosol of infected host cells, further supporting its secretion and likely participation in interactions that aid pathogen survival. Indeed, whereas siRNA-mediated knockdown of Ubc9 - a necessary enzyme for SUMOylation - slightly bolstered A. phagocytophilum infection, pharmacologically inhibiting SUMOylation in infected cells significantly reduced the bacterial load. Ectopically expressed GFP-AmpA served as a competitive agonist against native AmpA in infected cells, while lysine-deficient GFP-AmpA was less effective, implying that modification of AmpA lysines is important for infection. Collectively, these data show that AmpA becomes directly SUMOylated during infection, representing a novel tactic for A. phagocytophilum survival.
小泛素样修饰物（SUMO）蛋白家族成员与靶底物中的赖氨酸共价连接的SUMO化是真核生物中一种重要的翻译后修饰。SUMO化的微生物调控最近成为病毒和兼性胞内细菌的一种关键毒力策略，后者仅被证明会部署负向调节SUMO化的效应蛋白。在此，我们证明专性胞内细菌嗜吞噬细胞无形体利用一种效应蛋白AmpA（嗜吞噬细胞无形体翻译后修饰蛋白A），该蛋白在宿主细胞中会发生SUMO化，这对病原体的存活很重要。我们之前发现AmpA（原APH1387）定位于嗜吞噬细胞无形体占据的液泡膜（AVM）。算法预测分析表明AmpA是SUMO化的候选蛋白。我们使用SUMO亲和矩阵沉淀天然AmpA和异位表达的绿色荧光蛋白（GFP）标记的AmpA，验证了这一现象。AmpA的SUMO化依赖于赖氨酸，因为当GFP - AmpA蛋白的赖氨酸残基被精氨酸取代时，SUMO亲和珠无法沉淀该蛋白。异位表达的和内源性的AmpA都发生了多聚SUMO化，这与AmpA在AVM处与SUMO2/3共定位的观察结果一致。仅在感染周期后期，AmpA才与SUMO1共定位，SUMO1会终止多聚SUMO2/3链。在受感染宿主细胞的胞质溶胶中也检测到了AmpA，进一步支持了其分泌以及可能参与有助于病原体存活的相互作用。事实上，虽然小干扰RNA介导的Ubc9（SUMO化所需的一种必需酶）敲低略微增强了嗜吞噬细胞无形体的感染，但在受感染细胞中进行药理学抑制SUMO化显著降低了细菌载量。异位表达的GFP - AmpA在受感染细胞中作为天然AmpA的竞争性激动剂发挥作用，而赖氨酸缺陷型GFP - AmpA的效果较差，这意味着AmpA赖氨酸的修饰对感染很重要。总的来说，这些数据表明AmpA在感染期间直接发生SUMO化，这代表了嗜吞噬细胞无形体存活的一种新策略。
