新型小肽inersin抑制无乳链球菌致病性的分子机制

Lactobacillus iners is a dominant species of beneficial bacteria in female reproductive tract, while Streptococcus agalactiae is a pathogen leading to serious invasive infections and it is resistant to a wild range of antibiotics. In this study, a biosynthetic cluster for a new type of peptide (named inersin), widely present in the genomes of L. iners, is first discovered. Different from antibiotics, inersin significantly reduces the pathogenicity of S. agalactiae but barely affects its growth. This novel mode of action of inersin also hardly triggers the resistance. Therefore, this study intends to work from the biosynthesis, structure-activity relationship and mechanism of action of inersin. Functions of the key biosynthetic genes or gene cluster will be determined. Based on that, the in vitro biosynthesis system for inersin will be constructed. Combining with the technique such as site-directed mutagenesis and nuclear magnetic resonance, the structural basis for the toxicity-attenuation activity of inersin will be resolved. In addition, the molecular mechanism of inersin to regulate the two-component system against the pathogenicity of S. agalactiae will be elucidated, by investigating reduction activity, and protein-DNA interaction and so on in vivo and in vitro. This work will be contributed to revealing the probiotic mechanism of the dominated species of vaginal Lactobacillus, and offering theoretical guidances for the development of anti-infection agents or probiotics.

在女性生殖道中，惰性乳杆菌是占主导地位的有益菌之一，而无乳链球菌是能引起严重感染的致病菌且具有广谱的耐药性。本研究首次从惰性乳杆菌基因组中发现了一种广泛存在的新型小肽（将其命名为inersin）的生物合成基因簇。不同于抗生素，inersin能显著降低无乳链球菌的致病性但几乎不影响其生长。这种作用方式新颖，且不易引发菌株的抗性。因此，本研究拟从新型小肽 inersin的生物合成途径，结构-活性关系和作用机制三方面开展工作，在阐明其生物合成关键基因或基因簇功能的基础上，建立其体外生物合成系统；结合定点突变和核磁共振等技术，解析inersin发挥“减毒”活性的结构基础；此外通过诱导活性检测以及蛋白-DNA相互作用等体内外研究，揭示inersin调控无乳链球菌双组份系统而抑制其致病性的分子机制。本研究将有助于阐明生殖道主导乳杆菌的益生机制，为抗感染药物或微生态制剂的研发提供理论指导。

惰性乳杆菌和无乳链球菌是人体生殖道菌群的常见微生物，与人体的健康和疾病密切相关，然而当前对于它们之间的相互作用研究较少，特别是小肽介导的调控作用。本项目以惰性乳杆菌来源的新颖小肽inersin为主要研究对象，研究其生物合成途径，结构和生物活性，以及对无乳链球菌双组份调控系统的作用机制。结果显示：（1）inersin对于其生物合成基因的诱导活性较弱，且其生物合成可能与惰性乳杆菌肽酶IneT或生殖道其它微生物（例如普氏菌和卷曲乳杆菌）IneT-like蛋白相关。（2）inersin在模拟细胞膜环境下形成规则二级结构，且可能通过形成聚体发挥活性；inersin对无乳链球菌的生长具有抑制作用，而在亚抑制浓度下，inersin能促进无乳链球菌生物膜形成；inersin能显著影响无乳链球菌压力应答相关基因表达。（3）inersin上调无乳链球菌eDNA释放相关基因表达，且可能是通过双组份系统LiaSR介导的。此外本项目还研究了惰性乳杆菌来源的新型小肽inecin L的生物合成，结构-活性关系和作用机制，以及建立了乳杆菌高效的内源CRISPR-Cas9基因编辑系统等，这些有助于阐明惰性乳杆菌与生殖道微生物相互作用的分子机制。综上所述，本项目阐明了惰性乳杆菌-无乳链球菌互作的小分子物质和调控机制，为相关药物和益生菌的研发奠定了理论基础。