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

项目总结 寻常痤疮(粉刺)在美国每年影响多达5000万人，并可能有显著的负面影响 对心理社会功能的影响，包括更高的焦虑率、低自尊、抑郁和 自杀念头。痤疮皮肤杆菌在痤疮发病机制中起着核心作用，现在人们了解到一种 痤疮念珠菌系统型多样性降低，致病系统型IA1同质性增加 触发先天免疫刺激和粉刺进展。针对痤疮念珠菌的局部和口服抗生素仍然存在 是痤疮一线治疗的一部分，但继续使用抗生素带来了巨大的挑战，包括 抗生素耐药性的加剧以及对肠道和肠道健康共生的附带损害 皮肤微生物群。鉴于这些缺点，可以提供替代药物的新型抗菌剂 抗生素和选择性地针对痤疮假单胞菌而不破坏有益细菌是必要的。 内毒素是一种噬菌体编码的酶，可以降解细菌细胞壁。外源性添加的内毒素 可以快速裂解它们的目标细菌，因为它们在目标细胞壁上结合了非常特定的表位，所以它们可以 具有裂解专一性，可细化到单一物种甚至亚种。鉴于这些特性，内毒素可以 作为高特异性皮肤微生物组调节剂的巨大潜力。然而，内毒素的多样性 已知的靶标是痤疮假单胞菌，这些酶活性低，溶解度低。近期 元基因组学分析表明，该菌为枯草杆菌属。在土壤中无处不在，这些 环境可以提供丰富的、尚未开发的枯草杆菌多样性来源。 在黄玉生物科学公司，我们已经开发了一个专有的元基因组平台来发现和 内毒素的优化。我们以前曾利用这一平台开发内毒素来对抗 金黄色葡萄球菌比金黄色葡萄球菌更活跃，更耐热，pH范围更宽 基准酶。在这个第一阶段的提案中，我们将利用这个平台来扩大 已知的内溶素对痤疮假单胞菌有活性，然后利用这种多样性开发嵌合体 具有改进性能的酶。要实现这一点，我们将利用 内毒素构建内毒素“部分”--酶结构域(EADS)和细胞壁结合结构域的文库 (CBDS)，我们将系统地表征其抗皮肤细菌活性、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噬菌体。然后，值得注意的是 扩大EADS和CBD的多样性，我们将通过计算选择约200个不同的内毒素/结构域 从专利和公共元基因组数据库预测具有抗皮肤杆菌活性，用于其他 人物刻画。最后，我们将利用从我们的“部件”开发中获得的序列功能洞察来 设计由EADS和CBD组成的最有希望产生的嵌合内毒素 表现出更好的活性、溶解性、热稳定性和pH范围的候选酶。

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.