Department of Biological and Environmental Sciences and Technologies (DiSTeBA), University of Salento, Via Provinciale Monteroni n. 165, 73100, Lecce, Italy.
Core Research Laboratory-Siena, Institute for Cancer Research and Prevention (ISPRO), 53100, Siena, Italy.
J Biomed Sci. 2022 Jun 28;29(1):45. doi: 10.1186/s12929-022-00829-8.
In Neisseria meningitidis the HrpA/HrpB two-partner secretion system (TPS) was implicated in diverse functions including meningococcal competition, biofilm formation, adherence to epithelial cells, intracellular survival and vacuolar escape. These diverse functions could be attributed to distinct domains of secreted HrpA.
A yeast two-hybrid screening, in vitro pull-down assay and immunofluorescence microscopy experiments were used to investigate the interaction between HrpA and the dynein light-chain, Tctex-type 1 (DYNLT1). In silico modeling was used to analyze HrpA structure. Western blot analysis was used to investigate apoptotic and pyroptotic markers.
The HrpA carboxy-terminal region acts as a manganese-dependent cell lysin, while the results of a yeast two-hybrid screening demonstrated that the HrpA middle region has the ability to bind the dynein light-chain, Tctex-type 1 (DYNLT1). This interaction was confirmed by in vitro pull-down assay and immunofluorescence microscopy experiments showing co-localization of N. meningitidis with DYNLT1 in infected epithelial cells. In silico modeling revealed that the HrpA-M interface interacting with the DYNLT1 has similarity with capsid proteins of neurotropic viruses that interact with the DYNLT1. Indeed, we found that HrpA plays a key role in infection of and meningococcal trafficking within neuronal cells, and is implicated in the modulation of the balance between apoptosis and pyroptosis.
Our findings revealed that N. meningitidis is able to effectively infect and survive in neuronal cells, and that this ability is dependent on HrpA, which establishes a direct protein-protein interaction with DYNLTI in these cells, suggesting that the HrpA interaction with dynein could be fundamental for N. meningitidis spreading inside the neurons. Moreover, we found that the balance between apoptotic and pyroptotic pathways is heavily affected by HrpA.
脑膜炎奈瑟菌的 HrpA/HrpB 双组分分泌系统（TPS）被认为参与多种功能，包括脑膜炎球菌竞争、生物膜形成、上皮细胞黏附、细胞内存活和空泡逃逸。这些不同的功能可以归因于分泌 HrpA 的不同结构域。
使用酵母双杂交筛选、体外下拉测定和免疫荧光显微镜实验来研究 HrpA 与动力蛋白轻链 Tctex 型 1（DYNLT1）之间的相互作用。使用计算机模拟分析 HrpA 结构。使用 Western blot 分析来研究凋亡和细胞焦亡标志物。
HrpA 羧基末端区域作为锰依赖性细胞溶素起作用，而酵母双杂交筛选的结果表明 HrpA 中间区域具有结合动力蛋白轻链 Tctex 型 1（DYNLT1）的能力。体外下拉测定和免疫荧光显微镜实验证实了这种相互作用，结果显示感染上皮细胞中脑膜炎奈瑟菌与 DYNLT1 共定位。计算机模拟显示 HrpA-M 界面与 DYNLT1 相互作用的结构与神经毒性病毒的衣壳蛋白与 DYNLT1 相互作用的结构相似。事实上，我们发现 HrpA 在脑膜炎奈瑟菌感染和在神经元细胞内运输中发挥关键作用，并且参与调节凋亡和细胞焦亡之间的平衡。
我们的研究结果表明，脑膜炎奈瑟菌能够有效地感染和在神经元细胞中存活，并且这种能力依赖于 HrpA，它在这些细胞中与 DYNLTI 建立直接的蛋白-蛋白相互作用，这表明 HrpA 与动力蛋白的相互作用对于脑膜炎奈瑟菌在神经元内的传播可能是至关重要的。此外，我们发现凋亡和细胞焦亡途径之间的平衡受到 HrpA 的严重影响。
