Oxidative Stress Resistance Mechanisms in Filifactor Alocis

A.项目摘要/摘要 最近的口腔微生物组研究已经认识到无数尚未培养的和挑剔的 已经显示出与牙周病严重程度有很强相关性的微生物。很可能，新兴的 与传统的“红色复合体”相比，新的病原体可能在该病中起到更重要的作用。 牙龈卟啉单胞菌、连翘坦纳氏菌和齿密螺旋体。以前就有这样的一个 未被识别的微生物Filifactor alocis是一种革兰氏阳性、无糖分解、专性厌氧杆菌。几个 最近的研究发现，这种细菌在成人或难治性牙周炎患者中的水平明显更高。 并建议将其作为牙周病的诊断指标。 目前，关于赤霉菌的生存机制和毒力的信息很少或根本没有。 这主要是由于没有一种有效的遗传系统来允许对F. Aocis基因组。在对环境胁迫的反应中，我们的初步研究表明，芦荟相对 与牙龈假单胞菌相比，它更能抵抗过氧化氢诱导的氧化应激。此外，在过氧化氢诱导的压力下 在此条件下，牙龈假单胞菌的存活率在金黄色葡萄球菌的存在下可提高4倍以上。这些 观察表明，华南星可能有能力改变/减少氧化应激环境和 稳定牙周袋中的微生物群落。在RNA-seq分析中，转录图谱 在H_2O_2诱导下，与牙龈假单胞菌共培养时(与F·alocis单独培养相比)， 胁迫，阿洛克氏酵母中最高表达的基因编码一种可能的锰ABC转运蛋白 FA0894-FA0895-FA0896-FA0897。锰已被提出用来解毒活性氧物种和 保护细菌免受氧化应激。我们的假设是假想的三磷酸腺苷转运蛋白， FA0894-FA0897可能在增强对牙龈假单胞菌的保护/存活方面发挥重要作用。 H_2O_2诱导的胁迫。我们希望了解F。alocis潜在毒力因子的调控。 对与牙龈假单胞菌的相互作用作出反应，并评估它们是否有助于保护/持久性牙周炎。 牙周炎会破坏牙周组织的氧化环境。建议的具体目标是：(1) 评价华支睾吸虫ABC转运蛋白FA0894-FA0897在保护/存活中的特异性作用(S) 在H_2O_2诱导的压力下，牙龈假单胞菌。(2)建立高效的纤溶酶原基因系统，包括 无标记的框内缺失系统和/或转座子突变系统。 总而言之，产生的数据将有助于对分子的全面评估 机制(S)和整体相互作用涉及F.alocis和‘Keystone’病原菌P.它还将 生成可促进开发新的治疗干预措施的多微生物模型系统 帮助控制和预防牙周病。此外，新的遗传操作系统 将有助于揭示金黄色葡萄球菌在牙周病病因学中的相对意义。

A. PROJECT SUMMARY/ABSTRACT Recent oral microbiome studies have recognized a myriad of as-yet-culturable and fastidious organisms that have shown a strong correlation with periodontal disease severity. It is likely that the emerging new pathogens may play a more significant role in the disease compared to the traditional “red complex” bacteria Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola. One such previously unrecognized organism, Filifactor alocis, is a Gram-positive, asaccharolytic, obligate anaerobic rod. Several recent studies have found this bacterium at significantly higher levels in adult or refractory periodontitis patients and have suggested that it could be included as a diagnostic indicator of periodontal disease. Currently, there is little or no information on survival mechanisms and virulence of F. alocis. Primarily, this is due to the unavailability of an efficient genetic system to allow genetic manipulations of the F. alocis genome. In response to environmental stress, our preliminary studies showed that F. alocis is relatively more resistant to H2O2-induced oxidative stress compared to P. gingivalis. Also, under H2O2-induced stress conditions, the survival of P. gingivalis is enhanced more than 4-fold in the presence of F. alocis. These observations suggest that F. alocis may have the ability to modify/reduce the oxidative stress environment and stabilize the microbial community of the periodontal pocket. In an RNA-seq analysis, the transcriptional profile of F. alocis showed that in coculture with P. gingivalis (compared to F. alocis monoculture) under H2O2-induced stress, the most highly upregulated genes in F. alocis encode for a putative manganese ABC transporter FA0894-FA0895-FA0896-FA0897. Manganese has been proposed to detoxify reactive oxygen species and protect bacteria from oxidative stress. It is our hypothesis that the F. alocis hypothetical ATP transporter, FA0894-FA0897, may play an important role in enhanced protection/survival of P. gingivalis against H2O2-induced stress. We wish to understand the modulation of F. alocis potential virulence factors in response to interaction with P. gingivalis and evaluate if they contribute to protection/persistence of P. gingivalis against the oxidative environment of the periodontal community. The proposed specific aims are: (1) to evaluate the specific role(s) of F. alocis putative ABC transporter FA0894-FA0897 in the protection/survival of P. gingivalis under H2O2-induced stress. (2) to develop an efficient genetic system for Filifactor that includes a markerless, in-frame deletion system and/or transposon mutagenesis system. Collectively, the data generated will facilitate a comprehensive assessment of the molecular mechanism(s) and overall interplay involving F. alocis and the ‘keystone’ pathogen P. gingivalis. It will also generate a polymicrobial model system that may facilitate the development of novel therapeutic interventions to aid in the control and prevention of periodontal disease. Furthermore, the new genetic manipulation system will help to uncover the relative significance of F. alocis in the etiology of periodontal disease.