Orientia tsutsugamushi Strain Ikeda Ankyrin Repeat-Containing Proteins Recruit SCF1 Ubiquitin Ligase Machinery via Poxvirus-Like F-Box Motifs
Orientia tsutsugamushi Strain Ikeda Ankyrin Repeat-Containing Proteins Recruit SCF1 Ubiquitin Ligase Machinery via Poxvirus-Like F-Box Motifs
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DOI：
10.1128/jb.00276-15
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发表时间：
2015-10-01
影响因子：
3.2
通讯作者：
Carlyon, Jason A.
中科院分区：
文献类型：
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作者：
Beyer, Andrea R.;VieBrock, Lauren;Carlyon, Jason A.
在细胞内微生物中，一个日益增长的主题是传递含有锚蛋白重复序列的效应器(ANK)，它与目标蛋白相互作用，以选择宿主细胞的功能。东方体是一种专性胞内细菌，也是森林斑疹伤寒的病原体，它编码了所有已测序微生物中最大的ANK谱系之一。它们以前被鉴定为1型分泌系统底物。在这里，在计算机和人工序列分析显示，很大比例的恙虫病原虫Ikeda anks株携带一个真核/痘病毒样的F-box基序，该基序被认为是招募宿主细胞SCF1泛素连接酶的机械。我们评估了ANK作为F-box蛋白的能力。免疫共沉淀分析表明，含有F-box的ANK与高表达和/或内源性SCF1组分相互作用。当与FLAGAnk4_01或FLAG-Ank9共表达时，谷胱甘肽S转移酶(GST)标记的SCF1组分Skp1定位于FLAG-Ank积累的亚细胞位置。重组ANK与Skp1相互作用和共定位的能力依赖于F-box。GST-Skp1从受感染的宿主细胞中沉淀出来自恙虫病原虫的Ank9，验证了病原体在感染过程中表达Ank9，以及该蛋白与Skp1结合的能力。比对恙虫病原虫、痘病毒和真核生物的F-box序列，描绘了三个高度保守且可能具有重要功能的F-box残基。这些残基的取代使GFP-Ank9与GST-Skp1相互作用的能力减弱。这些结果表明，钩端螺旋体Ikeda anks株能够参与宿主细胞的多泛素化机制，首次提供了在感染过程中具有这种作用的证据，并促进了对微生物F-box蛋白的全面了解。重要的是，含Ankyrin重复序列的蛋白(ANKs)是胞内细菌的重要毒力因子，介导蛋白质与宿主细胞靶标的相互作用。恙虫病东方体在世界上人口最稠密的地区之一会引起一种名为丛林斑疹伤寒的令人衰弱的感染，它编码了所有已测序细菌中最大的Ank武器之一。这项研究表明，恙虫病原虫Ikeda anks株也携带与宿主细胞多泛素化机制相互作用的F-box基序。通过证明东方体来源的Ank与感染细胞中的Skp1相互作用，这证明了第一个真正的东方体效应器，也是第一个含有内源性F-box的Ank-哺乳动物-宿主配体相互作用的细胞内细菌。此外，重要的是，这项工作确定了对微生物F-box功能至关重要的关键残基。
A rising theme among intracellular microbes is the delivery of ankyrin repeat-containing effectors (Anks) that interact with target proteins to co-opt host cell functions. Orientia tsutsugamushi, an obligate intracellular bacterium and the etiologic agent of scrub typhus, encodes one of the largest Ank repertoires of any sequenced microorganism. They have been previously identified as type 1 secretion system substrates. Here, in silico and manual sequence analyses revealed that a large proportion of O. tsutsugamushi strain Ikeda Anks bear a eukaryotic/poxvirus-like F-box motif, which is known to recruit host cell SCF1 ubiquitin ligase machinery. We assessed the Anks for the ability to serve as F-box proteins. Coimmunoprecipitation assays demonstrated that F-box-containing Anks interact with overexpressed and/or endogenous SCF1 components. When coexpressed with FLAGAnk4_ 01 or FLAG-Ank9, a glutathione S-transferase (GST)-tagged version of the SCF1 component SKP1 localized to subcellular sites of FLAG-Ank accumulation. The abilities of recombinant Anks to interact and colocalize with SKP1 were F-box dependent. GST-SKP1 precipitated O. tsutsugamushi-derived Ank9 from infected host cells, verifying both that the pathogen expresses Ank9 during infection and the protein's capability to bind SKP1. Aligning O. tsutsugamushi, poxviral, and eukaryotic F-box sequences delineated three F-box residues that are highly conserved and likely to be functionally important. Substitution of these residues ablated the ability of GFP-Ank9 to interact with GST-SKP1. These results demonstrate that O. tsutsugamushi strain Ikeda Anks can co-opt host cell polyubiquitination machinery, provide the first evidence that an O. tsutsugamushi Ank does so during infection, and advance overall understanding of microbial F-box proteins.IMPORTANCEAnkyrin repeat-containing proteins (Anks) are important virulence factors of intracellular bacteria that mediate protein-protein interactions with host cell targets. Orientia tsutsugamushi, which causes a debilitating infection called scrub typhus in one of the most densely populated regions of the world, encodes one of the largest Ank armamentariums of any sequenced bacterium. This study demonstrates that O. tsutsugamushi strain Ikeda Anks also bear F-box motifs that interact with host cell polyubiquitination machinery. By proving that an Orientia-derived Ank interacts with SKP1 in infected cells, this evidences the first bona fide Orientia effector and the first example of an endogenous F-box-containing Ank-mammalian-host ligand interaction for any intracellular bacterium. Also, importantly, this work identifies key residues that are essential for microbial F-box function.