Chemical profiling of bacterial-fungal interactions to elucidate mechanisms of growth and survival in multi-domain microbial communities

最近，随着微生物组的特定成员越来越多地被发现，对微生物组的兴趣已经爆发。 被认为在人类和许多其他宿主的健康和疾病中发挥关键作用。[1]虽然许多研究将 微生物组成的生物学结果，决定这些结果的分子机制是 不太了解。这部分是由于难以处理大量的变量， 复杂的多域微生物群落。发酵食品微生物组提供了一个优势， 就存在的微生物数量以及对人类健康的潜在应用而言， 通过饮食。具体来说，奶酪皮是可食用的生物膜，最近被描述为一个简化的模型 具有高度可重复的微生物群落演替模式的系统。[2]这些果皮通常是 由10-12种微生物组成，特定的奶酪，如洗过的或天然的外皮，具有相似的微生物属 无论世界各地的果皮是在哪里老化的。因此，干酪皮生物膜模型可以是 用于建立驱动细菌-真菌与食物相互作用的潜在生化过程的模式 安全和饮食健康相关性。在这项研究中，我将研究细菌产生的分子， 真菌对不同生长伙伴的反应，以评估不断变化的生长伙伴的化学产出。 基于合作者达顿和沃尔夫的表型筛选，我选择了不同的丝状真菌 与细菌配对进行代谢组学分析。这些菌株在干酪凝乳琼脂上分离生长， 食品污染物E.大肠杆菌，并与一个自然的增长伙伴，假单胞菌psychrophila。我用矩阵 辅助激光解吸/电离飞行时间（MALDI-TOF）成像质谱（IMS）， 在所有条件下分子的存在和空间分布的差异。IMS数据将用于 结合真菌和细菌代谢物的化学提取物的串联质谱数据 以鉴定介导与细菌相互作用的分子和分子类别。其中三种真菌 菌株对E.大肠杆菌，推测是通过生产抗菌分子。我在 纯化和鉴定负责抑制的分子的过程。以后我会用高效液相色谱 细菌和真菌提取物的纯化和NMR，以确认分子的疑似身份并阐明 生物活性分子的新结构。这项研究的成功完成将加深我们的理解 专门的代谢物是如何参与发酵食品微生物组的，也使我们能够将 研究来检测发酵食品本身的这些分子。

Recently, interest in microbiomes has exploded as specific members of the microbiome are increasingly recognized to play key roles in health and disease in humans and many other hosts.[1] While many studies link microbial composition to biological outcomes, the molecular mechanisms that determine these outcomes are poorly understood. This is partially due to the difficulty of dealing with the large number of variables present in complex multi-domain microbial communities. Fermented food microbiomes offer an advantage of being relatively simple in terms of numbers of microbes present as well as the potential application to human health through diet. Specifically, cheese rinds are edible biofilms have recently been described as a simplified model system with highly reproducible patterns of microbial community succession.[2] These rinds are typically comprised of 10-12 microbes and specific cheeses, like washed or natural rind, have similar microbial genera present regardless of where the rind is aged across the world. As such, the cheese rind biofilm model can be used to establish patterns of underlying biochemical processes that drive bacterial-fungal interactions with food safety and dietary health relevance. For this study I will investigate the molecules produced by bacteria and fungus in response to different growing partners to assess the chemical output of changing growth partners. Based on phenotypic screening from collaborators Dutton and Wolfe, I have selected different filamentous fungi to pair with bacteria for metabolomic analysis. These strains were grown on cheese curd agar in isolation, with a food contaminant, E. coli, and with a natural growing partner, Pseudomonas psychrophila. I used matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) imaging mass spectrometry (IMS) to visualize differences in the presence and spatial distribution of molecules across all conditions. IMS data will be used in conjunction with tandem mass spectrometry data from chemical extractions of fungal and bacterial metabolites in order to identify molecules and molecular classes that mediate interactions with bacteria. Three of these fungal strains exhibit growth inhibition of E. coli, presumably by production of antimicrobial molecules. I am in the process of purifying and identifying the molecules responsible for inhibition. In the future, I will use HPLC purification and NMR of bacterial and fungal extracts to confirm suspected identity of molecules and elucidate novel structures of bioactive molecules. Successful completion of this research will deepen our understanding of how specialized metabolites are involved in fermented food microbiomes as well allow us to translate the research to detection of these molecules on the fermented foods themselves.