LPHI, Univ. Montpellier, CNRS, INSERM, France.
Univ. Grenoble Alpes, CEA, CNRS, IBS, Grenoble, France.
FEBS J. 2024 Jan;291(1):177-203. doi: 10.1111/febs.16969. Epub 2023 Oct 13.
Invasion of brain endothelium protein A (IbeA) is a virulence factor specific to pathogenic Escherichia coli. Originally identified in the K1 strain causing neonatal meningitis, it was more recently found in avian pathogenic Escherichia coli (APEC) and adherent invasive Escherichia coli (AIEC). In these bacteria, IbeA facilitates host cell invasion and intracellular survival, in particular, under harsh conditions like oxidative stress. Furthermore, IbeA from AIEC contributes to intramacrophage survival and replication, thus enhancing the inflammatory response within the intestine. Therefore, this factor is a promising drug target for anti-AIEC strategies in the context of Crohn's disease. Despite such an important role, the biological function of IbeA remains largely unknown. In particular, its exact nature and cellular localization, i.e., membrane-bound invasin versus cytosolic factor, are still of debate. Here, we developed an efficient protocol for recombinant expression of IbeA under native conditions and demonstrated that IbeA from AIEC is a soluble, homodimeric flavoprotein. Using mass spectrometry and tryptophan fluorescence measurements, we further showed that IbeA preferentially binds flavin adenine dinucleotide (FAD), with an affinity in the one-hundred nanomolar range and optimal binding under reducing conditions. 3D-modeling with AlphaFold revealed that IbeA shares strong structural homology with FAD-dependent oxidoreductases. Finally, we used ligand docking, mutational analyses, and molecular dynamics simulations to identify the FAD binding pocket within IbeA and characterize possible conformational changes occurring upon ligand binding. Overall, we suggest that the role of IbeA in the survival of AIEC within host cells, notably macrophages, is linked to modulation of redox processes.
脑内皮蛋白 A (IbeA) 是一种侵袭蛋白，是病原性大肠杆菌的一种特定毒力因子。最初在引起新生儿脑膜炎的 K1 株中发现，最近在禽致病性大肠杆菌 (APEC) 和黏附侵袭性大肠杆菌 (AIEC) 中也发现了该蛋白。在这些细菌中，IbeA 促进宿主细胞的侵袭和细胞内生存，特别是在氧化应激等恶劣条件下。此外，AIEC 中的 IbeA 有助于巨噬细胞内的存活和复制，从而增强肠道内的炎症反应。因此，该因子是治疗克罗恩病中抗 AIEC 策略的一个很有前途的药物靶点。尽管具有如此重要的作用，但 IbeA 的生物学功能在很大程度上仍不清楚。特别是，其确切性质和细胞定位，即膜结合侵袭蛋白与细胞质因子，仍存在争议。在这里，我们开发了一种在天然条件下高效表达 IbeA 的方法，并证明了 AIEC 中的 IbeA 是一种可溶性同源二聚体黄素蛋白。通过质谱和色氨酸荧光测量，我们进一步表明，IbeA 优先结合黄素腺嘌呤二核苷酸 (FAD)，亲和力在纳摩尔范围内，在还原条件下最佳结合。使用 AlphaFold 进行的 3D 建模表明，IbeA 与依赖黄素的氧化还原酶具有很强的结构同源性。最后，我们使用配体对接、突变分析和分子动力学模拟来确定 IbeA 中的 FAD 结合口袋，并描述配体结合时可能发生的构象变化。总的来说，我们认为 IbeA 在 AIEC 宿主细胞内，特别是巨噬细胞中的存活作用与其对氧化还原过程的调节有关。
