Anhui Province Key Laboratory of Veterinary Pathobiology and Disease Control, College of Animal Science and Technology, Anhui Agricultural University, Hefei, 230036, China.
Anhui Province Engineering Laboratory for Animal Food Quality and Bio-Safety, College of Animal Science and Technology, Anhui Agricultural University, Hefei, 230036, China.
Vet Res. 2023 Jan 30;54(1):6. doi: 10.1186/s13567-023-01138-0.
The type VI secretion system (T6SS) is a secretion apparatus widely found in pathogenic Gram-negative bacteria and is important for competition among various bacteria and host cell pathogenesis. Hcp is a core component of functional T6SS and transports toxic effectors into target cells by assembling to form tube-like structures. Studies have shown that Hcp simultaneously acts as an effector to influence cellular physiological activities; however, the mechanism of its activity in host cells remains unclear. To investigate the target of effector protein Hcp2a in a chicken fibroblast cell line, we first detected the subcellular localization of Hcp2a in DF-1 cells by indirect immunofluorescence assay. The results showed that Hcp2a protein was localized in the endoplasmic reticulum of DF-1 cells. We also used a streptavidin-biotin affinity pull-down assay combined with LC-MS/MS to screen DF-1 cell lysates for proteins that interact with Hcp2a and analyze the cellular functional pathways affected by them. The results showed that Hcp2a interacted with 52 DF-1 cellular proteins that are involved in multiple intracellular pathways. To further explore the mechanism of Hcp2a protein targeting the endoplasmic reticulum of DF-1 cells, we screened three endoplasmic reticulum-associated proteins (RSL1D1, RPS3A, and RPL23) from 52 prey proteins of Hcp2a for protein-protein molecular docking analysis. The docking analysis showed that the effector protein Hcp2a and the RPL23 protein had good complementarity. Overall, we propose that Hcp2a has strong binding activity to the RPL23 protein in DF-1 cells and this may help Hcp2a anchor to the endoplasmic reticulum in DF-1 cells.
VI 型分泌系统（T6SS）是一种广泛存在于致病性革兰氏阴性菌中的分泌装置，对于各种细菌之间的竞争和宿主细胞发病机制都很重要。Hcp 是功能 T6SS 的核心组件，通过组装形成管状结构将毒性效应器输送到靶细胞。研究表明，Hcp 同时作为一种效应物来影响细胞的生理活动；然而，其在宿主细胞中的活性机制尚不清楚。为了研究效应蛋白 Hcp2a 在鸡成纤维细胞系中的靶标，我们首先通过间接免疫荧光法检测 Hcp2a 在 DF-1 细胞中的亚细胞定位。结果表明，Hcp2a 蛋白定位于 DF-1 细胞的内质网中。我们还使用链霉亲和素-生物素亲和下拉法结合 LC-MS/MS 从 DF-1 细胞裂解物中筛选与 Hcp2a 相互作用的蛋白质，并分析受其影响的细胞功能途径。结果表明，Hcp2a 与 52 种参与多种细胞内途径的 DF-1 细胞蛋白相互作用。为了进一步探讨 Hcp2a 蛋白靶向 DF-1 细胞内质网的机制，我们从 Hcp2a 的 52 种宿主蛋白中筛选了三种内质网相关蛋白（RSL1D1、RPS3A 和 RPL23）进行蛋白-蛋白分子对接分析。对接分析表明，效应蛋白 Hcp2a 与 RPL23 蛋白具有良好的互补性。总体而言，我们提出 Hcp2a 与 DF-1 细胞中的 RPL23 蛋白具有很强的结合活性，这可能有助于 Hcp2a 锚定在 DF-1 细胞的内质网上。
