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。许多水生动物产下的外部卵必须受到微生物的保护才能成功发育。虽然有益的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型动物宿主。这种和许多其他种类的雌性鱿鱼在其生殖道的一个专门器官中藏匿着细菌。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的细菌及其产生的化合物是否可以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。防御性共生的研究在一些陆生和水生生物协会中引起了越来越大的兴趣。该项目将由在共生、化学生态学和药物发现方面具有专业知识的研究人员采取多学科方法，以阐明细菌和微生物代谢物如何影响鸡蛋的保护和发育。这项拟议的研究将把研究与教育结合起来，通过几个外联计划，帮助培训研究生和一些本科生研究人员，重点是来自代表性不足群体的学生。本研究的结果可能导致新的抗真菌和抗微生物化合物的发现。初步研究表明，在鱿鱼头节的附属网状腺(Ang)中发现的细菌联合体，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，提出的研究有三个主要目标。1)分离和鉴定抗真菌和其他微生物生物膜的ANG和鸡蛋细菌的生物活性代谢物，并确定生物活性细菌菌株或分离出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株产生的次生代谢物。2)用转录组学方法研究活性细菌分离株中细菌基因的表达。这些信息，连同来自整个ANG、鸡蛋和活性细菌分离株基因组的序列转录本和元基因组，将被用于预测导致抗菌素次生代谢产物产生的生物合成途径，这可能与鸡蛋保护有关。3)通过检测来自该联合体的ANG细菌分离株是否能够诱导ANG的形态发生，来表征细菌在ANG发展中的作用。该项目将研究没有ANG的雌性卵子是否容易受到污染，以及是否可以用活性细菌分离和/或化合物拯救受到真菌挑战的卵子。

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.