Using live microbes as therapeutic candidates is a strategy that has gained traction across multiple therapeutic areas. In the skin, commensal microorganisms play a crucial role in maintaining skin barrier function, homeostasis, and cutaneous immunity. Alterations of the homeostatic skin microbiome are associated with a number of skin diseases. Here, we present the design of an engineered commensal organism, Staphylococcus epidermidis, for use as a live biotherapeutic product (LBP) candidate for skin diseases. The development of novel bacterial strains whose growth can be controlled without the use of antibiotics or genetic elements conferring antibiotic resistance enables modulation of therapeutic exposure and improves safety. We therefore constructed an auxotrophic strain of S. epidermidis that requires exogenously supplied d-alanine. The S. epidermidis NRRL B-4268 Δalr1 Δalr2 Δdat strain (SE_ΔΔΔ) contains deletions of three biosynthetic genes: two alanine racemase genes, alr1 and alr2 (SE1674 and SE1079), and the d-alanine aminotransferase gene, dat (SE1423). These three deletions restricted growth in d-alanine-deficient medium, pooled human blood, and skin. In the presence of d-alanine, SE_ΔΔΔ colonized and increased expression of human β-defensin 2 in cultured human skin models in vitro. SE_ΔΔΔ showed a low propensity to revert to d-alanine prototrophy and did not form biofilms on plastic in vitro. These studies support the potential safety and utility of SE_ΔΔΔ as a live biotherapeutic strain whose growth can be controlled by d-alanine.

中文翻译：

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使用d-丙氨酸营养缺陷型控制皮肤表皮葡萄球菌的生长。

使用活微生物作为治疗候选物是一种在多个治疗领域中获得广泛关注的策略。在皮肤中，共生微生物在维持皮肤屏障功能，体内平衡和皮肤免疫方面起着至关重要的作用。稳态皮肤微生物组的改变与许多皮肤疾病有关。在这里，我们介绍一种工程化的共生生物，表皮葡萄球菌，用作皮肤疾病的活生物治疗产品（LBP）的设计。无需使用抗生素或赋予抗生素抗性的遗传元件即可控制生长的新型细菌菌株的开发，可以调节治疗暴露并提高安全性。因此，我们构建了表皮葡萄球菌的营养缺陷型菌株。需要外源提供的d-丙氨酸。在表皮葡萄球菌NRRL B-4268Δ alr1 Δ ALR2 Δ DAT应变（SE _ΔΔΔ）包含三个生物合成基因缺失：二丙氨酸消旋酶的基因，alr1和ALR2（SE1674和SE1079）和D-丙氨酸氨基转移酶基因，DAT（ SE1423）。这三个缺失限制了d-丙氨酸缺乏培养基，人血和皮肤中的生长。在d-丙氨酸存在下，_SEΔΔΔ在培养的人皮肤模型中定居并增加了人β-防御素2的表达体外。SE _ΔΔΔ显示低的倾向回复到D-丙氨酸原养型并没有形成在塑料上生物膜体外。这些研究支持_SEΔΔΔ作为活的生物治疗菌株的潜在安全性和实用性，其生长可通过d-丙氨酸来控制。