Characterizing the Role of a Bacterial Consortium in a Host Reproductive Organ

所有动物都与微生物有有益的关系。许多水生动物在体外产卵，为了成功发育，必须保护这些卵不被微生物污染。虽然有益细菌被认为有助于鸡蛋的防御，但这种保护的机制在很大程度上是未知的。夏威夷短尾乌贼是研究与有益细菌关系的模范动物宿主。这种雌性和许多其他种类的鱿鱼在其生殖道的一个特殊器官中携带细菌。这些细菌的功能尚不清楚，但它们可能有助于保护这些鸡蛋免受其他微生物的污染。这项工作将使用遗传和化学分析来确定这些有益细菌及其产生的化合物是否能抑制真菌和其他微生物的生长。这些细菌诱导鱿鱼生殖器官发育的能力也将被研究。防御共生体的研究是一个日益增长的领域，在许多陆地和水生协会的兴趣。该项目将采用多学科方法，由具有共生、化学生态学和药物发现专业知识的研究人员来阐明细菌和微生物代谢物如何影响卵子的保护和发育。拟议的研究将通过促进研究生和一些本科生研究人员的培训，将研究与教育结合起来，通过几个外展计划，重点关注来自代表性不足群体的学生。研究结果可能导致新的抗真菌和抗微生物化合物的发现。初步研究鉴定了鱿鱼附属腺（ANG）中发现的细菌联盟，并表明ANG细菌沉积在卵中保护发育中的胚胎免受真菌生物膜的侵害。此外，在没有细菌的情况下饲养的雌性鱿鱼在性成熟时不会形成ANG，这表明细菌可能是器官正常发育所必需的。基于这些结果，本研究有三个主要目标。1)从ANG和鸡蛋细菌中分离并鉴定对真菌和其他微生物生物膜有活性的生物活性代谢物，并确定生物活性菌株或分离的化合物是否能在真菌攻击时拯救鸡蛋。分子网络的使用将比较这些细菌分离物产生的次生代谢物。2)利用转录组学技术表征活性细菌分离物中的细菌基因表达。这些信息，连同来自整个ANGs、卵子和活性细菌分离物基因组的序列转录组和宏基因组，将用于预测导致抗菌次级代谢物产生的生物合成途径，这可能与卵子保护有关。3)通过检测财团中分离的ANG细菌是否能够诱导ANG的形态发生，表征细菌在ANG发展中的作用。该项目将检查没有ANG的雌性卵子是否容易受到污染，以及被真菌攻击的卵子是否可以用活性细菌分离物和/或化合物来拯救。

All animals have beneficial relationships with microorganisms. Many aquatic animals lay external eggs that must be protected from fouling by microorganisms to successfully develop. Although beneficial bacteria are thought to contribute to egg defense, the mechanisms of this protection are largely unknown. The Hawaiian bobtail squid is a model animal host for studying relationships with beneficial bacteria. Females of this and many other squid species harbor bacteria in a specialized organ of their reproductive tract. The function of these bacteria is unknown, but they may assist with protecting such eggs from fouling by other microorganisms. This work will use genetic and chemical analyses to determine whether these beneficial bacteria and the compounds that they produce can inhibit the growth of fungi and other microorganisms. The ability of these bacteria to induce development of the squid's reproductive organ will also be studied. The study of defensive symbioses is a growing area of interest in a number of terrestrial and aquatic associations. This project will take a multi-disciplinary approach by investigators with expertise in symbiosis, chemical ecology and drug discovery to shed light on how bacteria and microbial metabolites affect egg protection and development. The proposed study will integrate research with education by contributing to the training of graduate students and a number of undergraduate researchers, focusing on students from underrepresented groups through several outreach programs. Results of the study may lead to the discovery of novel anti-fungal and anti-microbial compounds.Preliminary studies characterized the bacterial consortium found in the accessory nidamental gland (ANG) of the squid Euprymna scolopes, and suggest that ANG bacteria deposited into eggs protect developing embryos from fungal biofilms. Furthermore, female squid raised in the absence of bacteria do not form an ANG by sexual maturity, suggesting that bacteria may be necessary for proper development of the organ. Based on these results, the proposed research has three main objectives. 1) Isolate and identify bioactive metabolites from ANG and egg bacteria that show activity against fungal and other microbial biofilms, and determine if bioactive bacterial strains or isolated compounds can rescue eggs upon fungal challenge. The use of molecular networks would compare secondary metabolite production by these bacterial isolates. 2) Use transcriptomics to characterize bacterial gene expression in active bacterial isolates. This information, along with sequence transcriptomes and metagenomes from whole ANGs, eggs and genomes of active bacterial isolates, would be used to predict the biosynthetic pathways leading to antimicrobial secondary metabolite production that may be involved with egg protection. 3) Characterize the role of bacteria in the development of the ANG, by testing whether ANG bacterial isolates from the consortium are capable of inducing morphogenesis of the ANG. The project would examine whether eggs from females without an ANG are susceptible to fouling, and whether eggs challenged with fungi can be rescued with active bacterial isolates and/or compounds.