HMGB1-mediated host response to chronic bacterial infection

项目摘要/摘要 高迁移率族蛋白1(HMGB1)是一种由215个氨基酸组成的蛋白质，在人体内发挥多种作用。 在细胞内，HMGB1与染色质相关，并参与几乎所有类型的DNA代谢(例如， 复制、修复、重组)，主要是通过高亲和力和特异性结合到 各种DNA结构。在细胞外，HMGB1是典型的损伤相关分子模式 具有很强的促炎功能的分子(湿)。在这里，我们已经发现，并将展示， 内源性HMGB1在控制引起慢性疾病的细菌方面具有迄今未知的功能 以及反复感染，从而有助于宿主与病原体相互作用的微妙平衡。为 细菌要进入慢性感染状态，它们必须采用一种称为生物膜的群落结构， 充满了自制的细胞外基质，通常由支架细胞外DNA(EDNA)组成 这对宿主免疫系统和抗菌剂的清除都具有高度的抵抗力。我们之前已经 表明这种依赖于Edna的结构由细菌蛋白的DNABII家族稳定，当 外源添加会将自由生活的(浮游)细菌驱入生物膜。与这些蛋白质不同的是，我们发现 HMGB1破坏了Edna结构的稳定，并驱使生物膜驻留的细菌进入浮游生物， 脆弱的国家。DNABII家族和HMGB1在体外具有相似的DNA结构结合偏好 尽管缺乏一级氨基酸序列同源性和二级结构。因此，我们假设 尽管这些蛋白质具有相似的DNA结构结合偏好，但它们促进了反向反应。 此外，内源性HMGB1稳定状态水平限制了慢性疾病，但未能清除 感染提示与HMGB1的平衡S需要促炎功能，即释放细菌 在强烈的炎症条件下从生物膜中释放出来可能会导致败血症。在此，我们将在 科学前提是Edna结合对于HMGB1破坏细菌生物膜是必不可少的，而且，它 将有可能将其抗生物被膜活性与促炎功能分开。事实上，我们有 截短的HMGB1到97个氨基酸，这种形式仍然保留了充分的抗生物被膜活性，但没有促进 炎症功能，从而可能使宿主-病原体的相互作用有利于宿主。穿过 在完成3个高度集成的具体目标后，我们将确定此HMGB1派生的能力 97-mer在多种人体病原体体外形成的生物膜上作为抗生物膜制剂，以及 多菌临床样本中的生物膜，体外分析(以确定活性和 支持我们的主要假设；目标1)，通过DNA过程的抗生物被膜的作用机制 结合(AIM 2)，以及在两种不同的生物被膜感染动物模型中的治疗效果(AIM 3)。

PROJECT SUMMARY/ABSTRACT High Mobility Group Box 1 (HMGB1) is a 215-amino acid protein that plays multiple roles in humans. Intracellularly, HMGB1 is chromatin-associated and involved in virtually all types of DNA metabolism (e.g. replication, repair, recombination), primarily through its ability to bind with high affinity and specificity to various DNA structures. Extracellularly, HMGB1 is the prototypical damage-associated molecular pattern molecule (DAMP) with strong pro-inflammatory functions. Here, we have discovered, and will show, that endogenous HMGB1 has a heretofore unknown function in its ability to control bacteria that cause chronic and recurrent infections, which thereby contributes to the delicate balance of host-pathogen interactions. For bacteria to enter a chronic infection state, they must assume a community architecture called a biofilm, replete with a self-made extracellular matrix commonly composed of scaffolded extracellular DNA (eDNA) that is highly resistant to clearance by both the host immune system and antimicrobials. We have previously shown that this eDNA-dependent structure is stabilized by the DNABII family of bacterial proteins, that when added exogenously can drive free-living (planktonic) bacteria into a biofilm. Unlike these proteins, we show that HMGB1 destabilizes the eDNA structure and drives biofilm-resident bacteria into the planktonic, vulnerable state. The DNABII family and HMGB1 have similar DNA structure binding preferences in vitro despite a lack of primary amino acid sequence identity and secondary structure. We therefore hypothesize that despite their similar DNA structure binding preferences, these proteins facilitate converse reactions. Further, the fact that endogenous native HMGB1 steady state levels restrict, but fail to clear, chronic infections suggests a balance with HMGB1’s needed pro-inflammatory functions, i.e. release of bacteria from biofilms under strong inflammatory conditions could lead to sepsis. Herein, we will work under the scientific premise that eDNA-binding is essential for HMGB1 to disrupt bacterial biofilms and further, that it will be possible to separate its anti-biofilm activity from pro-inflammatory functions. Indeed, we have truncated HMGB1 to 97 amino acids, a form which still retains full anti-biofilm activity but without pro- inflammatory functions, thereby likely able to tip the host-pathogen interaction in favor of the host. Through the completion of 3 highly integrated specific aims, we will determine the capacity of this HMGB1 derived 97-mer to act as an anti-biofilm agent on biofilms formed by diverse human pathogens in vitro as well as biofilms within polymicrobial clinical samples, assayed ex-vivo (to determine the breadth of activity and support our overarching hypothesis; AIM 1), the anti-biofilm mechanism of action, through a process of DNA binding (AIM 2), and the therapeutic efficacy in two distinct animal models of biofilm infections (AIM 3).