Turning on Persistence: Novel Molecular Determinants that Underpin P. gingivalis' Intracellular Survival In Epithelial Cells

家长奖摘要 越来越多的多学科证据关键地支持牙龈卟啉单胞菌，一种主要的牙周炎病原菌 成功地将口腔微生物群落重塑为病理生理状态的口腔可以生活在 与人牙龈上皮细胞(GECs)的协同作用。上皮细胞作为一个整体重要的手臂出现。 口腔粘膜中的天然防御能力，而最近的观察表明，这些细胞可以被用作 牙龈假单胞菌的优势生长环境和储存库，它们可以在细胞内繁殖并大量保留 在GEC中安然无恙。尽管如此，广泛的系统水平的分子知识仍然存在于牙龈假单胞菌和GEC上。 在这种相互作用中，关于这种中央细胞类型的有机体的细胞内生命知之甚少。我们 最近发现自噬小体的形成对牙龈假单胞菌的细胞内复制和 回避GECs中的抗微生物降解途径。我们的新的初步发现也支持 Lc3-C是以细胞内为靶点的选择性自噬途径的关键分子，它的脂化作用 病原体在抗应激分子HSP27的控制下受到牙龈假单胞菌的显著调节。 此外，谷胱甘肽过氧化物酶(GPX1)，一种主要的宿主氧化还原平衡酶和自噬通量的调节因子 在很大程度上影响了感染时GEC的全球LC3脂肪状态。HSP27或HSP27抑制 GPX1似乎严重影响微生物的细胞内运输和生存能力。中环 假说是牙龈假单胞菌诱导一种不同形式的选择性自噬，从而导致 保护细菌的生命，并最终确保牙龈假单胞菌在口腔粘膜中的持久性。至 测试这一新的假设，我们将采取双管齐下的方法，其中我们提出了选择性自噬 需要HSP27和GPX1紧密协调的作用才能形成完全发挥作用的自噬小体 牙龈假单胞菌受保护的复制生态位。目标1将定义驱动P. HSP27调控下的牙龈自噬小体组装及其破坏机制 逃避细胞降解途径的自噬通量。目标2将确立GPX1在 通过氧化还原动态平衡和抑制自噬溶酶体调节感染中的选择性自噬 机械设备。这两个目标都将采用简化主义的原代GECs培养系统，从功能上剖析 分子事件和亚细胞成分的机制和表型特征。目标3将 利用口腔上皮组织特异性基因敲除小鼠建立这两种成分的双重意义 模特们。因此，这一提议旨在填补我们基础知识中的一个重大空白，即P。 牙周炎是一种兼性的细胞内病原体，它建立了一个特权的细胞环境，并将 营养丰富的上皮细胞可能是细菌生长和持续存在的中央储存库 口腔粘膜。最终，所获得的知识可能会转化为分子策略，可以控制或 减少这种重要的机会病原体在细胞内的定植和生存方式。

SUMMARY of The PARENT AWARD A growing multidisciplinary evidence critically underpins that Porphyromonas gingivalis, a leading pathobiont of the oral cavity that successfully remodels oral microbial communities to a pathophysiological state, can live in concert with human gingival epithelial cells (GECs). Epithelial cells are emerged as an integrally important arm of innate defenses in the oral mucosa, while recent observations suggest that these cells can be exploited as privileged growth niches and a reservoir by P. gingivalis, which can intracellularly multiply and remain largely unharmed in GECs. Despite, extensive systems level molecular knowledge exists on the P. gingivalis and GEC interaction, there is considerably little known on the intracellular life of the organism in this central cell type. We recently revealed that formation of autophagosomes is critical for the P. gingivalis’ intracellular replication and evasion of the anti-microbial degradation pathways in the GECs. Our novel preliminary findings also support that lipidation of LC3-C, a key molecule in the ‘selective autophagy’ pathway, which targets intracellular pathogens is significantly modulated by P. gingivalis under the control of an anti-stress molecule, HSP27. Further, glutathione peroxidase (GpX1), a major host redox balance enzyme and a regulator of autophagic flux largely impacted on the global LC3 lipidation state of GECs upon infection. The inhibition of either HSP27 or GpX1 appears to severely affect the intracellular trafficking and viability of the microorganism. The central hypothesis is that P. gingivalis induces a distinct form of selective autophagy, which results in protection of bacterial life and ultimately securing of P. gingivalis’ persistence in the oral mucosa. To test this novel hypothesis, we will pursue two-pronged approach, where we propose the selective autophagy requires tightly coordinated actions of HSP27 and GpX1 to form autophagosomes that fully function as protected replicative niches for P. gingivalis. Aim 1 will define the selective molecular machinery that drives P. gingivalis-containing autophagosome assembly under the control of HSP27 and the mechanisms that disrupt autophagic flux for the evasion of cellular degradation pathways. Aim 2 will establish the role of GpX1 in regulating the selective autophagy in infection via redox homeostasis and suppressing autophagolysosomal machinery. Both aims will employ reductionist primary GECs culture systems to functionally dissect out the mechanisms and phenotypically characterize the molecular events and sub-cellular components. Aim 3 will establish the dual significance of these two components using oral epithelial-tissue-specific knockout mice models. Thus, this proposal aims to fill a significant gap in our fundamental knowledge that is how P. gingivalis, a facultatively intracellular pathogen, establishes a privileged cellular environment and converts nutritionally rich epithelial cells into potentially a central reservoir for bacterial growth and persistence in the oral mucosa. Ultimately, the knowledge gained may translate into molecular strategies that can control or reduce the intracellular colonization and survival methods employed by this important opportunistic pathogen.