HMGB1-mediated host response to chronic bacterial infection

项目总结/摘要 高迁移率族蛋白1（HMGB 1）是一种由215个氨基酸组成的蛋白质，在人类中发挥多种作用。 在细胞内，HMGB 1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），主要是通过其以高亲和力和特异性结合至 不同的DNA结构在细胞外，HMGB 1是典型的损伤相关分子模式 分子（DAMP）具有强烈的促炎功能。在这里，我们已经发现，并将表明， 内源性HMGB 1在其控制引起慢性炎症的细菌的能力方面具有迄今未知的功能， 和反复感染，从而有助于宿主-病原体相互作用的微妙平衡。为 细菌要进入慢性感染状态，它们必须呈现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， 对宿主免疫系统和抗菌剂的清除具有高度抗性。我们先前已经 表明这种依赖于eDNA的结构是由细菌蛋白质的DNABII家族稳定的，当 外源性添加可以驱使自由生活的（寄生的）细菌进入生物膜。与这些蛋白质不同的是， HMGB 1破坏了eDNA结构的稳定性，并将生物膜上的细菌驱入了嗜酸性物质中， 脆弱的国家。DNABII家族和HMGB 1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，但这些蛋白质促进了匡威的反应。 此外，内源性天然HMGB 1稳态水平限制，但不能清除慢性炎症的事实， 感染表明与HMGB 1所需的促炎功能（即释放细菌）的平衡 在强烈的炎症条件下从生物膜中分离可能导致败血症。在此，我们将根据 科学前提是，eDNA结合对于HMGB 1破坏细菌生物膜是必不可少的，而且， 将有可能将其抗生物膜活性与促炎功能分开。我确已 截短的HMGB 1至97个氨基酸，一种仍然保留完全抗生物膜活性但没有前- 炎症功能，从而可能能够提示宿主-病原体相互作用有利于宿主。通过 完成3个高度集成的具体目标，我们将确定此HMGB 1派生的能力 97-在体外作为抗生物膜剂作用于由多种人类病原体形成的生物膜， 多微生物临床样品中的生物膜，离体测定（以确定活性的宽度， 支持我们的总体假设;目的1），抗生物膜的作用机制，通过DNA的过程， 结合（AIM 2），以及在两种不同的生物膜感染动物模型中的治疗功效（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).