Institut Pasteur, Département de Microbiologie, Unité Pathogenèse de Helicobacter, CNRS UMR 2001, Paris, France.
Université de Paris, Sorbonne Paris Cité, Cellule Pasteur, Paris, France.
PLoS Pathog. 2021 Jan 14;17(1):e1009193. doi: 10.1371/journal.ppat.1009193. eCollection 2021 Jan.
Cellular metal homeostasis is a critical process for all organisms, requiring tight regulation. In the major pathogen Helicobacter pylori, the acquisition of nickel is an essential virulence determinant as this metal is a cofactor for the acid-resistance enzyme, urease. Nickel uptake relies on the NixA permease and the NiuBDE ABC transporter. Till now, bacterial metal transporters were reported to be controlled at their transcriptional level. Here we uncovered post-translational regulation of the essential Niu transporter in H. pylori. Indeed, we demonstrate that SlyD, a protein combining peptidyl-prolyl isomerase (PPIase), chaperone, and metal-binding properties, is required for the activity of the Niu transporter. Using two-hybrid assays, we found that SlyD directly interacts with the NiuD permease subunit and identified a motif critical for this contact. Mutants of the different SlyD functional domains were constructed and used to perform in vitro PPIase activity assays and four different in vivo tests measuring nickel intracellular accumulation or transport in H. pylori. In vitro, SlyD PPIase activity is down-regulated by nickel, independently of its C-terminal region reported to bind metals. In vivo, a role of SlyD PPIase function was only revealed upon exposure to high nickel concentrations. Most importantly, the IF chaperone domain of SlyD was shown to be mandatory for Niu activation under all in vivo conditions. These data suggest that SlyD is required for the active functional conformation of the Niu permease and regulates its activity through a novel mechanism implying direct protein interaction, thereby acting as a gatekeeper of nickel uptake. Finally, in agreement with a central role of SlyD, this protein is essential for the colonization of the mouse model by H. pylori.
细胞金属内稳态是所有生物体的关键过程，需要严格的调节。在主要病原体幽门螺杆菌中，镍的获取是一个必要的毒力决定因素，因为这种金属是酸抗性酶脲酶的辅因子。镍的摄取依赖于 NixA 渗透酶和 NiuBDE ABC 转运体。到目前为止，据报道细菌金属转运体在转录水平受到控制。在这里，我们揭示了幽门螺杆菌中必需的 Niu 转运体的翻译后调控。事实上，我们证明了 SlyD，一种结合肽基脯氨酰顺反异构酶（PPIase）、伴侣和金属结合特性的蛋白质，是 Niu 转运体活性所必需的。通过双杂交测定，我们发现 SlyD 与 NiuD 渗透酶亚基直接相互作用，并确定了对该接触至关重要的基序。构建了不同 SlyD 功能域的突变体，并用于进行体外 PPIase 活性测定和四种不同的体内试验，以测量幽门螺杆菌中的镍细胞内积累或转运。在体外，SlyD PPIase 活性被镍下调，而与其报道的结合金属的 C 端区域无关。在体内，只有在暴露于高镍浓度时，SlyD PPIase 功能的作用才显现出来。最重要的是，SlyD 的 IF 伴侣结构域被证明是在所有体内条件下激活 Niu 的必要条件。这些数据表明，SlyD 是 Niu 渗透酶活性功能构象所必需的，并且通过一种新的机制调节其活性，涉及直接的蛋白质相互作用，从而充当镍摄取的守门员。最后，与 SlyD 的核心作用一致，该蛋白对于幽门螺杆菌在小鼠模型中的定植是必需的。
