Bacterial peptidylarginine deiminase, a link between gums and joint disease

摘要 牙龈卟啉单胞菌，慢性牙周炎的关键致病菌，分泌多种毒力 因素，包括一种酶，肽精氨酸脱亚胺酶(PAD)，是致病物种所特有的 卟啉单胞菌属。与人类内源性Pad相似，这种细菌酶将精氨酸残基转化为 蛋白质对瓜氨酸有较强的选择性，但对C-末端Arg有很强的偏好。我们推测牙龈假单胞菌 PPAD活性不仅参与了牙周炎的发病机制，而且可能参与了牙周炎的发病过程。 免疫耐受的破坏与类风湿关节炎(RA)的发展，类风湿关节炎是由 针对瓜氨酸表位的自身抗体。这一论点得到了PPAD在 与Arg特异的牙龈痛蛋白酶(RGPS)结合，可以产生大量的瓜氨酸 源自细菌和宿主蛋白的多肽，包括纤维蛋白原和烯醇化酶-均可接受 类风湿关节炎中的自身抗原。此外，牙龈假单胞菌的瓜氨酸蛋白刺激人牙龈成纤维细胞(HGF) 产生前列腺素E2(PGE2)，它是骨吸收的最有效的介质之一，参与了 帕金森病和类风湿关节炎的发病机制。此外，我们未公布的数据显示，RESTING和P.gigivalis 来自牙周炎患者的瓜氨酸刺激的HGFs比细胞产生更多的PGE2 来自健康的捐赠者。最后，我们发现了PPAD基因在临床牙龈假单胞菌中的两种等位基因形式。 分离株，其中一株编码一种超活性的酶亚型，晶体结构表明它与之结合 底物比其他形式的PPAD具有更高的亲和力。更紧密的底物结合可能有助于 提出了PPAD作为半抗原载体蛋白的第二个重要功能(细菌和/或宿主蛋白- 衍生的瓜氨酸肽)呈递给B细胞，随后T细胞被激活和分解 半抗原/载体机制中的免疫耐受。根据这些发现，我们推测PPAD是直接 对免疫耐受和随后的抗瓜氨酸蛋白抗体(ACPA)的破坏负责 产生-RA临床发病前的关键病理事件；PD的严重性及其与RA的联系是 与牙龈假单胞菌菌株亚群表达的PPAD超活性变体的发生有关。 为了从实验上验证这些假设，我们提出了以下具体目标：(I)研究泛函 瓜氨酸化细菌蛋白在牙龈假单胞菌与成纤维细胞病理相互作用中的相关性 确定T细胞靶向PPAD是否有助于通过半抗原/载体产生ACPA 机制；及(Iii)分析携带超活性PPAD基因的牙龈假单胞菌菌株在 帕金森病严重程度的背景及其与相关类风湿关节炎的关系。 该项目的成功完成将推动该领域向前发展，为 了解帕金森病-类风湿性关节炎的发病机制，从而为开发新的策略提供场所 治疗这些令人衰弱的疾病。

Summary Porphyromonas gingivalis, a keystone pathogen in chronic periodontitis secretes a large variety of virulence factors, including an enzyme, peptidylarginine deiminase (PAD), unique for pathogenic species of the Porphyromonas genus. Alike human endogenous PADs, the bacterial enzyme converts arginine residues in proteins to citrulline but it has a strong preference for C-terminal Arg. We postulated that P. gingivalis PAD (PPAD) activity not only contributes to pathogenesis of periodontitis but also it may be involved in the breakdown of immunotolerance and the development of rheumatoid arthritis (RA), a diseases driven by autoantibodies targeting citrullinated epitopes. This contention is supported by findings that PPAD, in conjunction with Arg-specific gingipain proteases (Rgps), can generate a large spectrum of citrullinated peptides derived from bacterial and host proteins, including fibrinogen and enolase – both accepted autoantigens in RA. Also, citrullinated proteins of P. gingivalis stimulate human gingival fibroblasts (HGF) to produce prostaglandin E2 (PGE2), one of the most potent mediators of bone resorption implicated in the pathogenesis of PD and RA. Moreover, our unpublished data show that both resting and P. gingivalis citrullinome-stimulated HGFs, derived from periodontitis patients, produce significantly more PGE2 than cells from healthy donors. Finally, we discovered two allelic forms of the ppad gene among P. gingivalis clinical isolates, one encoding a superactive isoform of the enzyme, which crystal structure suggests that it binds substrates with higher affinity than the other form of PPAD. The tighter substrate binding may contribute to proposed second important function of PPAD as the carrier protein of haptens (bacteria and/or host protein- derived citrullinated peptides) presented to B cell, followed by T cell activation and the breakdown of immunotolerance in a hapten/carrier mechanism. Based on these findings we postulate that PPAD is directly responsible for the breakdown of immunotolerance and subsequent anti-citrullinated protein antibody (ACPA) production - key pathological events preceding clinical onset of RA; and the severity of PD and its link to RA is associated with the occurrence of a superactive variant of PPAD expressed by subset of P. gingivalis strains. To experimentally verify these hypotheses, we propose the following specific aims: (i) investigate the functional relevance of citrullinated bacterial proteins in pathological interactions between P. gingivalis and fibroblasts; (ii) determine if PPAD targeting by T cells provides help for the production of ACPA through a hapten/carrier mechanism; and (iii) analyse the prevalence of P. gingivalis strains carrying the superactive PPAD gene in the context of the severity of PD and relation to associated RA. Successful completion of this project will drive the field forward by providing a novel perspective to the understanding the pathogenesis of PD-RA associations, thus offering venues to develop novel strategies to treat these debilitating diseases.