Synergistic killing of bacterial pathogens by histones

项目摘要/摘要 有效治疗皮肤、深部软组织和伤口的细菌感染仍然是一项 医疗保健环境中尚未满足的主要挑战，特别是在慢性糖尿病患者中。葡萄球菌 金黄色葡萄球菌和铜绿假单胞菌是最常见的细菌，分离自慢性、非 愈合伤口。在这些特殊的细菌中出现了抗生素耐药性，导致这些感染 变得越来越难以治疗，并产生了包括甲氧西林耐药在内的多重耐药菌株 金黄色葡萄球菌(MRSA)。 这项拟议工作的目标是开发下一代抗菌剂，为其设计 其灵感来自于对哺乳动物抗菌防御途径的更好的机械理解。我们专注于我们的 关注中性粒细胞胞外陷阱(Net)的抗菌活性，它使用组蛋白杀死或 抑制微生物增殖。组蛋白的抗菌机制尚不清楚。这个 Siryaporn和Gross实验室最近报告称，组蛋白与在 NETS-抗菌肽(AMP)LL-37(中草药)-产生强大的抗菌协同作用。LL-37 在细菌膜上形成孔，使组蛋白能够进入细菌并干扰基因 表情。这对细菌有不可逆转的杀菌作用。这里提出的这项工作将利用 这一发现是通过鉴定人组蛋白和膜/细胞壁靶向抗菌剂的组合来实现的 (MTAS)产生强大的抗菌活性和协同作用。该项目的总体目标是更好地 了解组蛋白和MTA之间的抗菌协同作用机制，并利用它来建立 一类治疗皮肤感染和伤口的新疗法。 我们将通过鉴定人类组蛋白与LL-37和其他 产生最大抗菌活性和协同增效作用的MTA。我们将测试这些菌株与金黄色葡萄球菌、P. 铜绿假单胞菌和体外皮肤细菌群落(目标1)。我们将尝试增加抗菌剂 在影响Net中组蛋白功能的因素中的工程学活性，特别是化学修饰 通过瓜氨酸化和将组蛋白拴在DNA纤维上(目标2)。为了验证我们的方法，我们将测试 标准化小鼠皮肤感染模型中AIMS 1和2中确定的组蛋白和MTA的组合 (目标3)。此外，为了解决治疗糖尿病患者皮肤感染和伤口这一尚未解决的挑战， 我们将在糖尿病小鼠模型上进行测试。 这项工作的结果将提供对抗菌协同作用的机理理解，并开发 对抗抗生素耐药性上升的战略。这项研究的结果可能会创造一个新的班级 用于治疗糖尿病和非糖尿病患者皮肤感染和伤口的抗微生物疗法。 这将代表着抗菌治疗方法的游戏规则改变者。

Project Summary/Abstract The effective treatment of bacterial infections of skin, deep soft tissues and wounds continues to be a major unmet challenge in healthcare settings, especially among patients with chronic diabetes. Staphylococcus aureus and Pseudomonas aeruginosa are the most common bacteria that are isolated from chronic, non- healing wounds. Antibiotic resistance has arisen in these particular bacteria, causing these infections to become increasingly difficult to treat and giving rise to multi-drug resistant strains, including Methicillin-resistant Staphylococcus aureus (MRSA). The goal of the proposed work is to develop the next generation of antimicrobials for which the design is inspired by a better mechanistic understanding of mammalian antimicrobial defense pathways. We focus our attention on the antimicrobial activities of neutrophil extracellular traps (NETs), which use histones to kill or suppress microbial proliferation. The antimicrobial mechanism of histones has not been understood. The Siryaporn and Gross labs recently reported that the pairing of histones with an additional component found in NETs – the antimicrobial peptide (AMP) LL-37 (cathelicidin) – produces potent antimicrobial synergy. LL-37 forms pores in the bacterial membrane, which enable histones to enter the bacterium and interfere with gene expression. This has an irreversible bactericidal (killing) effect on bacteria. The work proposed here will exploit this discovery by identifying combinations of human histones and membrane-/cell wall-targeting antimicrobials (MTAs) that produce potent antimicrobial activity and synergy. The overall objective of the project is to better understand the mechanism of antimicrobial synergy between histones and MTAs, and to harness it to establish a class of new therapeutics for the treatment of skin infections and wounds. We will accomplish this objective by identifying combinations of human histones with LL-37 and other MTAs that produce the greatest antimicrobial activities and synergies. We will test these against S. aureus, P. aeruginosa, and communities of skin bacteria in vitro (Aim 1). We will attempt to augment the antimicrobial activity by engineering in factors that impact histone function in NETs, specifically chemical modification through citrullination and tethering histones to DNA fibers (Aim 2). To validate our approach, we will test the combinations of histones and MTAs identified in Aims 1 and 2 in a standardized mouse skin infection model (Aim 3). To additionally address the unmet challenge of treating skin infection and wounds in diabetes patients, we will perform the tests in a diabetic mouse model. The results of this work will provide a mechanistic understanding of antimicrobial synergy and develop a strategy to combat the rise of antibiotic resistance. The results of the study could create a new class of antimicrobial therapeutics for the treatment of skin infections and wounds in diabetic and non-diabetic patients. This would represent a game-changer in the approach to antimicrobial treatments.