杂合生物素连接酶 TurboID 揭示了 flexneri 志贺氏菌中 T3SS 合酶 IpgC 的近端体。

IF 3.7 2区 生物学 Q2 MICROBIOLOGY mSphere Pub Date : 2024-11-21 Epub Date: 2024-10-31 DOI:10.1128/msphere.00553-24
Nathaline Haidar-Ahmad, Kyle Tomaro, Mathieu Lavallée-Adam, François-Xavier Campbell-Valois
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引用次数: 0

摘要

源于细菌酶 BirA 的杂合生物素连接酶可用于识别诱饵蛋白附近的蛋白质,从而确定其近端体。尽管这种方法很受欢迎,但令人惊讶的是,人们对它在原核生物中的应用知之甚少。在这里,我们比较了四种广泛使用的杂合生物素连接酶在柔性志贺氏菌(大肠杆菌的一个致病亚群)细胞质中的活性。我们的数据表明,TurboID 的生物素连接活性的动力学是所测试的生物素连接活性中最高的。此外,与其祖先 BioID 相比,TurboID 与天然 BirA 结合伙伴 BccP 和生物素算子的相互作用减少了。因此,我们评估了 TurboID 探测 III 型分泌系统(T3SS)伴侣 IpgC 和转录激活剂 MxiE 的近端体的能力。当 T3SS 处于非活性(关闭状态)时,这些蛋白分别与 T3SS 底物 OspD1 和 IpaBC 形成复合物而受到抑制。相反,当 T3SS 处于活动状态(开启状态)时,OspD1 和 IpaBC 被分泌出来,使 MxiE 和 IpgC 能够相互作用并激活它们的目标基因。利用 IpgC 和 TurboID 融合体获得的结果捕捉到了这些已知相互作用的大部分。它还表明,IpgC 在开启状态下的可用性增加,导致在其附近检测到更多的蛋白质。其中包括 T3SS ATP 酶 SpaL(又称 Spa47 或 SctN),这进一步支持了伴侣蛋白护送底物到 T3SS 的观点。有趣的是,在大肠杆菌中保守的特定蛋白质亚群在导通状态下完成了 IpgC 近端体。令人吃惊的是,它们在原核生物中的应用却很少。事实上,生物素连接酶在这些生物体内的天然结合伙伴体积小且位于细胞质中,这给研究相关蛋白质的近端体带来了独特的挑战。在这里,我们评估了四种最常见的杂合生物素连接酶,发现 TurboID 最适合用于柔性志贺氏杆菌的细胞质。利用这种方法,我们扩展了 IpgC 的近端体,使其超越了参与调节 III 型分泌系统(T3SS)信号级联的已知直接结合伙伴。当 T3SS 活跃时,IpgC 周围富集的转录因子和管家蛋白尤其值得进一步研究。我们提出了一个模型,在该模型中,IpgC 在开启状态下的可用性增加可能会使 T3SS 与其他细胞过程发生交叉对话。
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The promiscuous biotin ligase TurboID reveals the proxisome of the T3SS chaperone IpgC in Shigella flexneri.

Promiscuous biotin ligases derived from the bacterial enzyme BirA are used to identify proteins vicinal to a bait protein, thereby defining its proxisome. Despite the popularity of this approach, surprisingly little is known about its use in prokaryotes. Here, we compared the activity of four widely used promiscuous biotin ligases in the cytoplasm of Shigella flexneri, a pathogenic subgroup of Escherichia coli. Our data indicate that the kinetics of TurboID's biotinylating activity is the highest of those tested. In addition, TurboID showed reduced interaction with the natural BirA binding partners, BccP and the biotin operator, when compared to its ancestor BioID. We therefore evaluated the ability of TurboID to probe the proxisome of the type III secretion system (T3SS) chaperone IpgC and the transcriptional activator MxiE. When the T3SS is inactive (off-state), these proteins are inhibited by forming complexes with the T3SS substrates OspD1 and IpaBC, respectively. In contrast, when the T3SS is active (on-state), OspD1 and IpaBC are secreted allowing MxiE and IpgC to interact together and activate their target genes. The results obtained with the IpgC and TurboID fusions capture a good fraction of these known interactions. It also suggests that the availability of IpgC increases in the on-state, resulting in a greater number of proteins detected in its vicinity. Among these is the T3SS ATPase SpaL (also known as Spa47 or SctN), further supporting the notion that chaperones escort their substrate to the T3SS. Interestingly, a specific subset of proteins conserved in E. coli completes the IpgC proxisome in the on-state.IMPORTANCEPromiscuous biotin ligases are widely used to study protein function in eukaryotes. Strikingly, their use in prokaryotes has been rare. Indeed, the small volume and the cytoplasmic location of the biotin ligase's natural binding partners in these organisms pose unique challenges that can interfere with the study of the proxisome of proteins of interest. Here, we evaluated four of the most common promiscuous biotin ligases and found TurboID was best suited for use in the cytoplasm of Shigella flexneri. Using this method, we extended the proxisome of IpgC beyond its known direct binding partners involved in the regulation of the type III secretion system (T3SS) signaling cascade. Of particular interest for further study are transcription factors and housekeeping proteins that are enriched around IpgC when the T3SS is active. We propose a model in which the increased availability of IpgC in the on-state may allow cross-talk of the T3SS with other cellular processes.

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来源期刊
mSphere
mSphere Immunology and Microbiology-Microbiology
CiteScore
8.50
自引率
2.10%
发文量
192
审稿时长
11 weeks
期刊介绍: mSphere™ is a multi-disciplinary open-access journal that will focus on rapid publication of fundamental contributions to our understanding of microbiology. Its scope will reflect the immense range of fields within the microbial sciences, creating new opportunities for researchers to share findings that are transforming our understanding of human health and disease, ecosystems, neuroscience, agriculture, energy production, climate change, evolution, biogeochemical cycling, and food and drug production. Submissions will be encouraged of all high-quality work that makes fundamental contributions to our understanding of microbiology. mSphere™ will provide streamlined decisions, while carrying on ASM''s tradition for rigorous peer review.
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