Fic-mediated Adenylylation

描述（由申请人提供）：somni Histophilus是世界范围内的主要传染病。这种细菌产生一种叫做IbpA（免疫球蛋白结合蛋白a）的大纤维表面抗原。多杀性巴斯德菌是动物咬伤引起的最常见细菌感染的病原体，它也会产生一种称为PfhB2的大表面抗原。PfhB2与IbpA具有广泛的氨基酸序列同源性。这表明有一个小的细菌家族含有这4000个氨基酸的毒素。我们将注意力集中在IbpA上，因为有症状的动物感染H. somni的恢复期血清可识别IbpA，而无症状动物的血清不能识别IbpA。因此，IbpA的存在与H. somni毒力直接相关。IbpA的cooh末端与耶尔森氏菌III型效应蛋白YopT同源，YopT是耶尔森氏菌用来破坏宿主免疫系统的几种毒力因子之一。我们之前已经证明耶尔森菌YopT作为一种半胱氨酸蛋白酶，可以裂解和灭活Rho gtpase。我们的假设是IbpA的丝状血凝素样结构域介导对宿主细胞的附着，而其含有YopT同源序列的cooh末端在内化到宿主细胞中时充当细胞毒性效应。与我们的预期相反，我们观察到IbpA的yopt样结构域不破坏肌动蛋白细胞骨架，尽管它保留了关键的催化C/H/D三联体。相反，我们在IbpA中发现了一个毒力决定因素，该决定因素定位于被称为Fic（由c-AMP诱导的丝化）结构域的蛋白质部分。Fic结构域存在于大约1500种由细菌编码的蛋白质中，并作为单拷贝基因存在于许多真核生物基因组中。这些Fic结构域的功能是未知的。我们证明了IbpA的Fic结构域通过靶向宿主gtpase、RhoA、Rac和Cdc42诱导细胞毒性。IbpA的Fic结构域通过ATP催化GTPase开关I区酪氨酸（Tyr）残基与单磷酸腺苷（AMP）的共价加成，阻断了这些GTPase的信号传导。这种共价AMP的添加导致gtpase的下游信号传导受阻，从而导致细胞毒性。将AMP添加到GTPases的能力依赖于Fic结构域核心基序HPFxxGNGR中保守组氨酸（His）的存在。总之，我们已经确定了一类在细菌发病机制中起重要作用的新蛋白质。我们的研究结果还表明，在原核生物和真核生物中，向宿主蛋白添加AMP可能是一种被低估的翻译后修饰。该应用的具体目的是：(1)确定细菌和高等真核生物的Fic结构域是否都具有腺苷酸转移酶活性。（2A）研究fic介导的腺苷化反应的动力学性质和催化机理。（2B）阐明含Fic结构域酶的细胞底物。(3)确定IbpA如何进入哺乳动物细胞。(4)确定IbpA的Fic结构域以及与ATP非水解类似物络合的Fic结构域的x射线结构。(5)确定含有Fic结构域、非水解ATP类似物和RhoA的蛋白质复合物的x射线结构。这些研究将提供对Fic结构域进行这种新颖的翻译后修饰所使用的结构和机制的详细了解。这将共同推进我们对含有Fic结构域的蛋白质在细菌发病机制中的作用的理解。

DESCRIPTION (provided by applicant): Histophilus somni is a major infectious agent worldwide. This bacterium produces a large fibrillar surface antigen called IbpA (immunoglobulin binding protein A). Pasturella multocida, the causative agent of the most common bacterial infection due to an animal bite, also produces a large surface antigen known as PfhB2. PfhB2 shares extensive amino acid sequence identity with IbpA. This suggests that there is a small family of bacteria that harbors these 4000 amino acid toxins. We focused our attention on IbpA since convalescent serum from symptomatic animals infected with H. somni recognizes IbpA, while serum from asymptomatic animals does not. As such, the presence of IbpA directly correlates with H. somni virulence. The COOH-terminus of IbpA is homologous to the Yersinia type III effector protein, YopT, one of several virulence factors used by Yersinia to compromise the host immune system. We previously demonstrated that Yersinia YopT functions as a cysteine protease that cleaves and inactivates Rho GTPases. Our hypothesis was that IbpA's filamentous hemagglutinin-like domains mediate attachment to host cells, while its COOH-terminus containing the YopT homology sequence serves as a cytotoxic effector when internalized into host cells. Contrary to our expectations, we observed that the YopT-like domain of IbpA does not disrupt the actin cytoskeleton despite its conservation of the key catalytic C/H/D triad. Instead, we identified a virulence determinant within IbpA that is localized to a portion of the protein known as the Fic (filamentation induced by c-AMP) domain. Fic domains are found in approximately 1500 proteins encoded by bacteria and are present as single copy genes in many eukaryotic genomes. The function of these Fic domains is unknown. We demonstrated that the Fic domains of IbpA induce cytotoxicity by targeting the host GTPases, RhoA, Rac and Cdc42. The Fic domains of IbpA block signaling of these GTPases by using ATP to catalyze the covalent addition of adenosine monophosphate (AMP) to a tyrosine (Tyr) residue in the GTPase switch I region. This covalent AMP addition leads to a block in downstream signaling of the GTPases, which in turn results in cytotoxicity. The ability to add AMP to the GTPases is dependent on the presence of a conserved histidine (His) in the Fic domain's core motif, HPFxxGNGR. In summary, we have identified a new class of proteins that play an important novel role in bacterial pathogenesis. Our results also suggest that addition of AMP to host proteins may be an underappreciated post-translational modification in both prokaryotes and eukaryotes. The specific aims for this application are: (1) Determine if Fic domains from bacteria and higher eukaryotes all have adenylyl transferase activity. (2A) Study the kinetic properties and catalytic mechanisms of Fic-mediated adenylylation. (2B) Elucidate the cellular substrates of Fic domain containing enzymes. (3) Determine how IbpA enters mammalian cells. (4) Determine the X-ray structures of the IbpA's Fic domain as well as a Fic domain complexed with a non-hydrolyzed analogue of ATP. (5) Determine the X-ray structure of the protein complex containing a Fic domain, a non-hydrolyzed ATP analogue and RhoA. These studies will provide a detailed understanding of the structure and mechanism used by the Fic domain to carry out this novel post-translational modification. This will collectively advance our understanding of how the Fic domain containing proteins function in bacterial pathogenesis. PUBLIC HEALTH RELEVANCE: Prior to our work, the function of the Fic domain in bacterial pathogenesis was unknown despite its presence in over 1400 proteins from a wide variety of bacteria. We have demonstrated that the Fic domain of IbpA, a large toxin found in Histophilus somni, can disrupt the actin cytoskeleton by using ATP as a substrate to catalyze the addition of AMP to several host GTPases. This covalent addition of AMP blocks signal transduction pathways in the host that are important for combating bacterial infections.