女性生殖道益生卷曲乳杆菌抑制白色念珠菌的分子机制

Candida albicans, a main opportunistic pathogenic fungus, is involved in the increasing prevalence of female reproductive tract infection. With the emergence of fungal resistance to antibiotics, live lactobacillus preparation has been used to inhibit C. albicans growth in human reproductive tract for keeping the balance of dominant microbes and curing reproductive tract infection diseases. However, little is known about the molecular mechanism of Lactobacilli against C. albicans, which greatly hindering the development of efficient microbial inoculums against C. albicans. Lactobacillus crispatus L49, the object of this research, is a probiotic bacteria isolated from the vagina of health female. Our study revealed that it could directly adhere to C. albicans and inhibit its growth. In this application, we planned to study the active substances against C. albicans produced by L49, their biosynthesis process, inhibition mechanism and stress response to C. albicans will also be resolved. In addition, the adhesion proteins associated with direct interaction between L. crispatus and C. albicans and their function mechanism will be studied. Moreover, the key factors and signal pathways of L. crispatus responding to the quorum sensing molecules of C. albicans such as farnesol and so on will be characterized. Revealing molecular mechanism of L. crispatus against C. albicanswill not only provide the theoretical basis for the development of new anti-candidiasis lactobacillus preparation, but also present a model for the research on the interaction between fungi and bacteria.

白色念珠菌是目前日益增多的女性生殖道真菌感染疾病的主要条件致病菌。随着耐药真菌的出现，采用乳杆菌活菌制剂来恢复生殖道优势菌群、抑制白念珠菌、防治生殖道感染疾病已成为方向及共识。但对乳杆菌抑制白念珠菌分子机制了解的缺乏限制了高效菌剂的研发。本项目的研究对象是一株来源于健康女性生殖道的益生细菌-卷曲乳杆菌，其显示了抑制白念珠菌生长及直接黏附白念珠菌等益生功能，本项目即拟在对其进行组学研究的基础上，深入解析其抑制白念珠菌的物质基础，对其产生的生物活性小肽类代谢产物的合成途径、对白念珠菌的抑制机制及对白念珠菌的胁迫响应开展研究；对卷曲乳杆菌与白念珠菌直接发生黏附作用的黏附蛋白及其功能开展研究；对卷曲乳杆菌响应白念珠菌群感效应分子法尼醇的关键因子和信号通路开展研究。通过阐明卷曲乳杆菌抑制白念珠菌的分子机制，不仅可为研发新型抗念珠菌的乳杆菌制剂提供理论基础，也将为真菌与细菌相互作用研究提供一个范例。

白念珠菌过度增殖是导致女性生殖道感染的主要原因之一，而主导健康生殖道微生物菌群的卷曲乳杆菌能抑制白念珠菌的生长，但具体的分子机制一直未得到解析。本项目在解析卷曲乳杆菌基因组特征的基础上，分析了其特有的表面蛋白Slp对白念珠菌的粘附作用，及其主要的活性代谢产物-乳酸对白念珠菌生长和形态转变的影响，并深入研究了乳酸的主要生理环境来源-卷曲乳杆菌代谢上皮细胞源的糖原-的分子机制。结果显示：（1）卷曲乳杆菌基因组上具有较多的移动元件，它们与碳源利用和压力应答等相关，这与其能在多种碳源条件下生长的表型一致，表明其具有较强的环境适应能力。（2）表面蛋白SlpA1的N端和C端分别具有较强的结合产生菌和白念珠菌的能力，表明其可能是卷曲乳杆菌直接作用于白念珠菌的物质基础。（3）乳酸具有较强抑制白念珠菌生长的能力，而在低温条件下，它却能促进白念珠菌opaque细胞长菌丝，表明乳酸在卷曲乳杆菌-白念珠菌复杂的互作中发挥重要作用。（4）卷曲乳杆菌通过一个新颖的糖苷水解酶GlgU降解胞外的糖原，最终代谢产生乳酸；GlgU在生理环境下比人源α-淀粉酶具有更高的糖原水解活性；处于可转移单元上的glgU基因仅存在于卷曲乳杆菌生殖道来源的菌株中，而不存在于肠道来源的菌株，表明卷曲乳杆菌可能通过获得glgU而定殖于富含糖原的生殖道环境，进而代谢产酸以抑制白念珠菌等病原菌的过度生长。此外本项目还研究了乳杆菌对其它碳源（例如甘露醇）的代谢调控机制，以及羊毛硫细菌素等次级代谢产物的生物合成调控机制等，这些有利于深入解析生殖道乳杆菌抑制病原菌的分子机制。综上所述，本项目解析了乳杆菌发挥益生功能或适应不同环境的分子机制，为相关药物和益生菌剂的研发奠定了理论基础。