Anaplasma phagocytophilum hijacking of host cell monoubiquitination

描述（由申请人提供）：嗜吞噬细胞无形体是一种专性细胞内细菌，侵入中性粒细胞，引起新出现的和潜在致命的感染，人粒细胞无形体病。嗜吞噬芽胞杆菌所在的宿主细胞衍生的液泡劫持了循环内体，这一过程对细菌的生存至关重要。这种独特的病原体占据的细胞器如何支配内吞分选尚不清楚。单泛素化是最近出现的一种翻译后修饰，调节内噬分选，特别是循环内体途径。单泛素本身是一种分选信号，用该片段修饰的蛋白质与内吞机制进行广泛的接触。我们发现嗜吞噬芽胞杆菌液泡膜积累单泛素，而P100，一种定位于液泡膜的效应物，携带三个f -box。F-box基序首先在真核生物中被发现，并与SCF泛素连接酶复合物相互作用，催化蛋白质的泛素化。当GFP-P100在真核细胞中异位表达时，单泛素与GFP-P100和在嗜吞噬细胞空泡膜上的内源性P100共定位。含有F-box基序的P100区域暴露在嗜吞噬芽胞杆菌液泡膜的细胞质表面，当它在感染细胞中异位表达时，竞争性地抑制细菌生长。P100是一种新的细菌效应物，因为当许多微生物F-box蛋白利用宿主细胞多泛素化时，P100是F-box效应物的第一个例子，它利用单泛素化。我们的研究将验证P100 f -box招募SCF泛素连接酶复合物来指导嗜吞噬细胞芽胞杆菌液泡膜上宿主蛋白的单泛素化，这对细菌生长很重要的假设。在此过程中，我们将破译一种新发现的细菌致病机制，并将推进微生物在多种甚至是杀微生物的宿主细胞中选择泛素途径茁壮成长的策略。

DESCRIPTION (provided by applicant): Anaplasma phagocytophilum is an obligate intracellular bacterium that invades neutrophils to cause the emerging and potentially fatal infection, human granulocytic anaplasmosis. The host cell-derived vacuole in which A. phagocytophilum resides hijacks recycling endosomes, a process that is essential for the bacterium's survival. How this unique pathogen-occupied organelle commandeers endocytic sorting is poorly understood. Monoubiquitination has recently emerged as a posttranslational modification that regulates endocytic sorting, particularly the recycling endosome pathway. Monoubiquitin is itself a sorting signal, and proteins decorated with the moiety make extensive contacts with endocytic machinery. We discovered that the A. phagocytophilum vacuolar membrane accumulates monoubiquitin and that P100, an effector that localizes to the vacuolar membrane, carries three F-boxes. The F-box motif was first identified in eukaryotes and interacts with the SCF ubiquitin ligase complex, which catalyzes ubiquitination of proteins. Monoubiquitin colocalizes with GFP-P100 when it is ectopically expressed in eukaryotic cells and with endogenous P100 on the A. phagocytophilum vacuolar membrane. The P100 region that contains the F-box motif is exposed on the cytosolic face of the A. phagocytophilum vacuolar membrane and competitively inhibits bacterial growth when it is ectopically expressed in infected cells. P100 is a novel bacterial effector because, while many microbial F-box proteins exploit host cell polyubiquitination, P100 is the first example of an F-box effector that co-opts monoubiquitination. Our investigations will test the hypothesis that the P100 F-boxes recruit the SCF ubiquitin ligase complex to direct monoubiquitination of host proteins on the A. phagocytophilum vacuolar membrane and that doing so is important for bacterial growth. In doing so, we will decipher a newly discovered bacterial pathogenic mechanism and will advance knowledge of the strategies by which microbes co-opt ubiquitin pathways to thrive in diverse, even microbiocidal, host cells.