Metagenomic discovery and optimization of novel endolysins targeting Cutibacterium acnes to treat acne vulgaris

项目摘要 寻常痤疮（痤疮）影响多达50万人在美国每年，并可有显着的负面影响。 对心理社会功能的影响，包括焦虑、自卑、抑郁和 自杀意念痤疮皮肤杆菌在痤疮发病机制中起着核心作用，并且现在理解， 降低C。痤疮痤疮类型多样性和致病性痤疮类型IA 1的同质性增加 引发先天免疫刺激和痤疮进展。局部和口服抗生素以靶向C。痤疮残留 作为痤疮一线治疗的一部分，但继续使用抗生素构成了重大挑战， 抗生素耐药性的加剧以及对肠道健康组织的附带损害， 皮肤微生物组。考虑到这些缺点，可以提供抗微生物剂的替代物的新型抗微生物剂是必要的。 抗生素和选择性靶向C.需要不破坏有益细菌的痤疮。 内溶素是噬菌体编码的酶，可以降解细菌细胞壁。外源性内溶素 可以快速溶解目标细菌，并且由于它们与目标细胞壁中非常特异的表位结合， 对单个物种甚至亚种都有裂解特异性。鉴于这些性质，内溶素保持 作为高特异性皮肤微生物组调节剂的巨大潜力。然而，内溶素的多样性 已知目标C。痤疮是低的并且这些酶具有低活性和低溶解度。最近 宏基因组学分析表明，表皮杆菌属物种在土壤中普遍存在，并且这些表皮杆菌属物种在土壤中普遍存在。 环境可以提供丰富的、未开发的Cutibacterium多样性来源。 在黄玉生物科学公司，我们已经开发了一个专有的宏基因组平台， 内溶素的优化。我们以前利用这个平台开发了内溶素， 金黄色葡萄球菌更活跃，更耐热，pH范围更广， 基准酶。在第一阶段的提案中，我们将利用这一平台扩大 已知具有抗C.痤疮，然后利用这种多样性， 具有改善的性质的酶。为了实现这一点，我们将利用 构建内溶素“部分”-酶结构域（埃兹）和细胞壁结合结构域的文库 （CBD），我们将系统地表征其抗皮肤杆菌活性、热稳定性和pH耐受性。 最初的一组“部分”将来自已测序的皮肤杆菌基因组和细菌。然后，为了显著地 为了扩大埃兹和CBD的多样性，我们将通过计算选择约200种不同的内溶素/结构域 从专有和公共宏基因组数据库预测具有抗皮肤杆菌活性， 特征化最后，我们将利用从我们的“部件”开发中获得的序列-功能见解， 设计由埃兹和CBD组成的嵌合内溶素，其具有最有希望的特性，以产生 这些酶候选物表现出改进的活性、溶解度、热稳定性和pH范围。

PROJECT SUMMARY Acne vulgaris (acne) affects up to 50 million people in the U.S. annually and can have significant negative consequences on psychosocial functioning including higher rates of anxiety, low self-esteem, depression, and suicidal ideation. Cutibacterium acnes plays a central role in acne pathogenesis and it is now understood that a decrease in C. acnes phylotype diversity and an increase in the homogeneity of the pathogenic phylotype IA1 triggers innate immune stimulation and acne progression. Topical and oral antibiotics to target C. acnes remain part of first-line treatments for acne, but continued use of antibiotics poses significant challenges including exacerbation of antibiotic resistance as well as collateral damage to the healthy commensals in the gut and skin microbiomes. Given these drawbacks, novel antimicrobial agents that can provide alternatives to antibiotics and selectively target C. acnes without damaging beneficial bacteria are needed. Endolysins are phage-encoded enzymes that can degrade bacterial cell walls. Exogenously added endolysins can quickly lyse their target bacteria and because they bind very specific epitopes in target cell walls, they can have lytic specificity down to a single species or even sub-species. Given these properties, endolysins hold enormous potential as high-specificity skin microbiome modulators. However, the diversity of endolysins known to target C. acnes is low and these enzymes suffer from low activity and low solubility. Recent metagenomic analyses demonstrate that Cutibacterium sp. are found ubiquitously in the soil and that these environments can provide a rich, untapped source of Cutibacterium diversity. At Topaz Biosciences, we have developed a proprietary metagenomic platform for the discovery and optimization of endolysins. We have previously leveraged this platform to develop endolysins against Staphylococcus aureus that are more active, more thermostable, and have a broader pH range than benchmark enzymes. In this Phase I proposal, we will leverage this platform to expand the diversity of endolysins known to have activity against C. acnes and then exploit this diversity to develop chimeric enzymes with improved properties. To accomplish this, we will take advantage of the modularity of endolysins to build a library of endolysin “parts” – enzymatic domains (EADs) and cell wall binding domains (CBDs) that we will systemically characterize for anti-Cutibacterium activity, thermostability, and pH tolerance. An initial set of “parts” will come from sequenced Cutibacterium genomes and phages. Then, to significantly expand the diversity of EADs and CBDs, we will computationally select ~200 diverse endolysins/domains predicted to have anti-Cutibacterium activity from proprietary and public metagenomic databases for additional characterization. Finally, we will leverage sequence-function insights gained from our “parts” development to design chimeric endolysins composed of EADs and CBDs with the most promising properties to generate enzyme candidates that demonstrate improved activity, solubility, thermostability, and pH range.