Bacterial peptidylarginine deiminase, a link between gums and joint disease

总结 牙龈卟啉单胞菌（Porphyromonasgingivalis）是慢性牙周炎的重要致病菌，其毒力种类繁多 因子，包括一种酶，肽基精氨酸脱亚胺酶（PAD），独特的致病物种的 卟啉单胞菌属。与人类内源性PAD类似，细菌酶将精氨酸残基转化为精氨酸。 蛋白质对瓜氨酸有很强的偏好，但对C-末端Arg有很强的偏好。我们推测牙龈卟啉单胞菌PAD （PPAD）活性不仅参与牙周炎的发病，而且可能参与牙周炎的发生发展。 免疫耐受的破坏和类风湿性关节炎（RA）的发展， 针对瓜氨酸化表位的自身抗体。这一论点得到了调查结果的支持， 与Arg特异性牙龈菌蛋白酶（Rgps）结合，可以产生大量的瓜氨酸化的 来源于细菌和宿主蛋白质的肽，包括纤维蛋白原和烯醇化酶-两者都被接受 自身抗原此外，牙龈卟啉单胞菌的瓜氨酸化蛋白刺激人牙龈成纤维细胞（HGF）， 产生前列腺素E2（PGE 2），这是骨吸收最有效的介质之一，与骨吸收有关。 PD和RA的发病机制。此外，我们未发表的数据表明，静息和牙龈卟啉单胞菌 来自牙周炎患者的瓜氨酸酶刺激的HGFs产生的PGE 2显著高于细胞 健康的捐赠者最后，我们在临床牙龈卟啉单胞菌中发现了两种ppad等位基因 分离物，其中一种编码酶的超活性同种型，其晶体结构表明它结合 与其他形式的PPAD相比具有更高亲和力的底物。更紧密的底物结合可能有助于 提出PPAD的第二个重要功能是作为半抗原（细菌和/或宿主蛋白- 来源于瓜氨酸化肽）呈递给B细胞，随后T细胞活化， 半抗原/载体机制中的免疫耐受性。基于这些发现，我们假设PPAD直接 负责免疫耐受性的破坏和随后的抗瓜氨酸化蛋白抗体（ACPA） 产生-RA临床发作前的关键病理事件; PD的严重程度及其与RA的联系是 与牙龈卟啉单胞菌菌株亚群表达的PPAD超活性变体的发生相关。 为了实验验证这些假设，我们提出了以下具体目标：（i）研究功能 瓜氨酸化细菌蛋白在牙龈卟啉单胞菌和成纤维细胞之间的病理相互作用中的相关性;（ii） 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;和（iii）分析携带超活性PPAD基因的牙龈卟啉单胞菌菌株在 PD严重程度的背景以及与相关RA的关系。 该项目的成功完成将通过提供一个新的视角来推动该领域的发展。 了解PD-RA相关性的发病机制，从而为开发新的策略提供场所， 治疗这些使人衰弱的疾病。

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 Moreover, our unpublished data show that both resting and P. gingivalis citrullinome-stimulated HGFs, derived from periodontitis patients, produce significantly more PGE2 than cells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.