HMGB1-mediated host response to chronic bacterial infection

项目概要/摘要 高迁移率组盒 1 (HMGB1) 是一种 215 个氨基酸的蛋白质，在人类中发挥多种作用。 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 DNA 代谢）。 复制、修复、重组），主要是通过其以高亲和力和特异性结合的能力 各种DNA结构。在细胞外，HMGB1 是典型的损伤相关分子模式 分子（DAMP）具有很强的促炎功能。在这里，我们已经发现并将证明， 内源性 HMGB1 具有迄今为止未知的功能，能够控制导致慢性病的细菌。 和反复感染，从而有助于宿主与病原体相互作用的微妙平衡。对于 细菌要进入慢性感染状态，它们必须呈现出一种称为生物膜的群落结构， 充满了自制的细胞外基质，通常由支架式细胞外 DNA (eDNA) 组成 它对宿主免疫系统和抗菌药物的清除具有高度抵抗力。我们之前有过 表明这种依赖于 eDNA 的结构被细菌蛋白 DNABII 家族稳定，当 外源添加可以驱动自由生活（浮游）细菌进入生物膜。与这些蛋白质不同，我们显示 HMGB1 破坏 eDNA 结构的稳定性并驱使生物膜驻留细菌进入浮游生物， 脆弱状态。 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一性和二级结构。因此我们假设 尽管它们具有相似的 DNA 结构结合偏好，但这些蛋白质促进相反的反应。 此外，内源性天然 HMGB1 稳态水平限制但未能清除慢性 感染表明与 HMGB1 所需的促炎功能（即细菌释放）的平衡 强烈炎症条件下生物膜中的细菌可能导致败血症。在此，我们将在 科学前提是，eDNA 结合对于 HMGB1 破坏细菌生物膜至关重要，而且，它 将有可能将其抗生物膜活性与促炎功能分开。确实，我们有 将 HMGB1 截短为 97 个氨基酸，这种形式仍保留完整的抗生物膜活性，但不具有亲 炎症功能，从而可能使宿主与病原体的相互作用有利于宿主。通过 完成3个高度集成的具体目标后，我们将确定这个HMGB1的衍生能力 97-mer 在体外和多种人类病原体形成的生物膜上充当抗生物膜剂 多种微生物临床样本中的生物膜，进行离体分析（以确定活性的广度和 支持我们的总体假设； AIM 1)，抗生物膜作用机制，通过 DNA 过程 结合（AIM 2），以及两种不同的生物膜感染动物模型的治疗效果（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).