Evans Rhys, Waterfield Nicholas R
Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK.
R Soc Open Sci. 2024 Oct 23;11(10):240948. doi: 10.1098/rsos.240948. eCollection 2024 Oct.
The 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 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.
毒力盒（PVC）是一种细胞外收缩注射系统。在产生该系统的细菌中，N端信号肽可使效应“有效载荷”加载到PVC的空心管中，这一过程由“与多种细胞活动相关的ATP酶”（AAA）ATP酶Pvc15辅助，从而为将毒素或毒力因子注射到真核细胞溶质中做好准备。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进化枝中的信号肽多样性，并使用ATP酶测定、细胞呼吸测定以及对裂解物进行蛋白质免疫印迹定量分析，以及对PVC与其有效载荷的共纯化来研究Pvc15-信号肽相互作用。本研究发现，由于潜在的同源加载机制，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，包括串联的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对于PVC-有效载荷加载都是必需的，但结构域D2是唯一具有生物活性的ATP酶结构域，并通过信号肽拯救不稳定的有效载荷。最后，截短信号肽会消除Pvc15依赖性的PVC加载，并对有效载荷稳定性产生不同影响。本研究为细胞外收缩注射系统效应器加载机制及其ATP酶分子伴侣提供了关键见解，并表明这些装置可进行生物工程改造，用于将治疗性蛋白质注射到人类细胞中。
