铁离子调控大肠埃希菌O157:H7外膜孔蛋白OmpW抗先天免疫的分子机制

Iron is a key regulator for pathogen virulence and pathogenicity, but its regulation mechanism has not clarified clearly as yet. At present, outer membrane proteins, particular to Gram-negative bacteria, have been shown to promote pathogens to evade the host immune defense. Recently years, OmpW, an outer membrane porin protein widespread in Gram-negative bacteria, which expression regulated by iron, has found form a unique narrow hydrophobic channel, and it may be serving as transporter of iron ion. More importantly, several results of basic and clinical research have discovered that OmpW can contribute bacteria virulence and pathogenicity, and therefore cause of infectious diseases, but its mechanism has not reported to date. Our previous study on enterohemorrhagic Escherichia coli (EHEC) O157:H7 indicate that OmpW can promote bacteria resistance to bactericidal by serum complement and phagocytosis by macrophages. This finding revealed a novel pathogenic mechanism which iron modulate OmpW resistance to host innate immune. However, the resistance mechanism of OmpW and regulation mechanism by iron are still remained undefined. Therefore, on the basis of our previous studies, this project is planned firstly to determine the relations between OmpW expression and iron homeostasis, and to identify transcription factors which regulate OmpW expression by binding ferrous iron. Then, we focus on clarifying the resistance mechanism of OmpW in the processes of inhibition of bactericidal and opsonization by the complement system and affection of production of hydroxyl radicals and cytokines by macrophage cells. The project is emphasis on bacteria resistance to host innate immune defenses via its outer membrane proteins, in a new perspective for clarifying of pathogenic mechanism, and it provide new insight into the treatment of infectious diseases and a new drug target.

铁是病原菌毒力和致病性的关键调控因子，但其确切机制并未阐明。外膜蛋白是革兰氏阴性菌特有的外膜成分，可促进病原菌抵抗机体的免疫防御，是关键的致病因素。近年来，研究表明广泛存在于革兰氏阴性菌的外膜孔蛋白OmpW的表达受铁调控，且已证实其为毒力蛋白，与多种感染病的发生有关，但其机制未见报道。前期对EHEC O157:H7的研究发现，OmpW可促进细菌抗补体杀菌和抗吞噬，首次证实其抗先天免疫作用，为此我们认为铁调控OmpW抗先天免疫是病原菌致病的新机制。但OmpW抗先天免疫的分子机制及铁的调控机理尚不清楚。本项目进一步明确OmpW表达与铁平衡的关系，鉴定与铁结合并调控OmpW表达的转录因子，并着重从抑制补体系统杀菌及调理作用、对巨噬细胞产生羟自由基和细胞因子的影响等方面来阐明OmpW作用机制。本研究从病原菌抗免疫这一新角度来阐明其致病机制，并为感染病的防治提供新思路和药物作用新靶点。

细菌耐药性的产生及新致病菌株的出现，已在全球范围成为严重的公共卫生危机。目前公认铁是病原菌感染发病和宿主免疫防御这一相互作用过程中的关键调控因子，病原菌的毒力及其致病性取决于其从宿主体内获取铁的能力。因而全面揭示铁介导病原菌毒力及致病机制将有助于研发具有新型抗菌机制的抗菌药物或疫苗来应对危机。我们研究发现广泛存在于革兰氏阴性菌的外膜孔蛋白OmpW的表达受铁离子调控，且与多种感染病的发生有关。为此我们提出铁离子调控OmpW抗先天性免疫是病原菌新的致病机制，并对此开展了深入研究。首先对OmpW在细菌抗补体杀菌和抗吞噬中的作用开展了研究，并首次证实其具有抗先天免疫作用；对铁与OmpW间的关系的研究表明OmpW参与铁离子的转运，且通过结合转录因子Fur来调控OmpW的表达，揭示了铁调控OmpW的作用机制；在氧化应激下OmpW下调表达及敲除菌存活率增加则表明细菌在吞噬细胞中的存活率增加，OmpW的这一变化为细菌逃避巨噬细胞杀菌的新机制；而OmpW通过与补体旁路途径的主要抑制剂H因子的结合来抑制旁路途径的激活为细菌抗补体系统的关键机制；对OmpW的进一步研究表明该蛋白具有免疫保护性，可作为研发新型抗菌药物或疫苗的靶点。