The Pvc15 ATPase selectively associates effector proteins with the Photorhabdus virulence cassette.

IF 2.9 3区 综合性期刊 Q1 MULTIDISCIPLINARY SCIENCES Royal Society Open Science Pub Date : 2024-10-23 eCollection Date: 2024-10-01 DOI:10.1098/rsos.240948
Rhys Evans, Nicholas R Waterfield
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Abstract

The Photorhabdus virulence cassette (PVC) is an extracellular contractile injection system. In the producing bacterium, N-terminal signal peptides enable effector 'payloads' to be loaded into the PVC's hollow tube-facilitated by the 'ATPases associated with diverse cellular activities' (AAA) ATPase, Pvc15-ready for injection of the toxin or virulence factor into eukaryotic cytosols. Pvc15's function and its interaction with the signal peptide were unclear. This study describes the signal peptide diversity in extracellular contractile injection system clades and interrogates the Pvc15-signal peptide interaction using ATPase assays, cell respiratory assays and western blot quantification of Escherichia coli lysates and co-purifications of PVCs with their payloads. This study found that extracellular contractile injection system signal peptides can be grouped according to sequence alignment, owing to potentially homologous loading mechanisms. Pvc15 contains three domains, including tandem AAA domains D1 and D2. By constructing Pvc15 mutants, we found that while each domain is necessary for PVC-payload loading, domain D2 is the sole bioactive ATPase domain and rescues unstable payloads via the signal peptide. Finally, truncating the signal peptide abolishes Pvc15-dependent PVC loading and has varying effects on payload stability. This study provides crucial insights into extracellular contractile injection system effector loading mechanisms and their ATPase chaperones, and suggests that these devices could be bioengineered for injection of therapeutic proteins into human cells.

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Pvc15 ATP 酶选择性地将效应蛋白与光照杆菌毒力盒结合在一起。
光照杆菌毒力盒(PVC)是一种细胞外收缩注射系统。在生产细菌中,N端信号肽使效应 "有效载荷 "能够通过 "与多种细胞活动相关的 ATP 酶"(AAA)ATP 酶 Pvc15 装入 PVC 的空心管中,以便将毒素或毒力因子注入真核细胞溶液中。Pvc15 的功能及其与信号肽的相互作用尚不清楚。本研究描述了细胞外收缩注射系统支系中信号肽的多样性,并使用 ATPase 检测法、细胞呼吸检测法和大肠杆菌裂解物的 Western 印迹定量法以及 PVC 与其有效载荷的共纯化法来研究 Pvc15 与信号肽的相互作用。这项研究发现,由于潜在的同源负载机制,细胞外收缩注射系统信号肽可根据序列比对进行分组。Pvc15 包含三个结构域,其中包括串联 AAA 结构域 D1 和 D2。通过构建 Pvc15 突变体,我们发现虽然每个结构域都是 PVC 有效载荷装载所必需的,但结构域 D2 是唯一具有生物活性的 ATPase 结构域,可通过信号肽挽救不稳定的有效载荷。最后,截短信号肽会取消 Pvc15 依赖性 PVC 负载,并对有效载荷的稳定性产生不同的影响。这项研究为了解细胞外收缩注射系统效应物的装载机制及其 ATPase 合子提供了重要信息,并表明这些装置可以通过生物工程技术将治疗蛋白注射到人体细胞中。
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来源期刊
Royal Society Open Science
Royal Society Open Science Multidisciplinary-Multidisciplinary
CiteScore
6.00
自引率
0.00%
发文量
508
审稿时长
14 weeks
期刊介绍: Royal Society Open Science is a new open journal publishing high-quality original research across the entire range of science on the basis of objective peer-review. The journal covers the entire range of science and mathematics and will allow the Society to publish all the high-quality work it receives without the usual restrictions on scope, length or impact.
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