Characterization of Biofilms by Correlated Mass Spectrometric and Raman Imaging

项目摘要 大多数细菌在自然和临床环境中生长为表面附着的生物膜，这是细菌 自组装成细胞外聚合物（EPS）的包裹基质的群落。到 形成这些细菌生物膜群落并感染宿主细胞，这是一种细胞间信号传导过程， 群体感应（quorum sensing，QS）是非常重要的。对于机会致病菌铜绿假单胞菌和 金黄色葡萄球菌，QS调节许多基因的表达，这些基因对生物膜起始，EPS 生产和毒性。虽然已经了解了许多关于调节生物膜形成的选择因子， 在体外和动物模型中，多物种细胞群形成生物膜的具体机制并不清楚。 明白此外，治疗日益严重的艾滋病病毒感染所需的治疗能力， 未来几十年将需要更深入地了解多物种相互作用及其对多基因的调控， 在宿主细胞感染期间，在扩展的空间和时间尺度上表现的表达谱。 已经发展的相关质谱和拉曼化学成像方法 我们的联合研究小组规避了以前的技术所施加的限制， 检查已经从细胞中取出的少量细胞或整个细胞群， 感兴趣的条件。相反，我们的相关化学成像方法允许确定和空间 在混合细菌群落中绘制单个细菌物种及其微生物产物的图谱 在表面原位生长。 我们的长期目标之一是设计检测和诊断策略， 了解细菌相互作用和特征生物分子的产生。我们将继续努力， 通过使用铜绿假单胞菌进行单培养和共培养实验， 金黄色葡萄球菌作为模式病原体。相关的化学成像将用于定义发病和 细胞间群体感应信号在空间和时间上的范围。生物分泌组及其相互作用 共培养生物膜中的特定物种将被表征为物种内和物种间的相互作用。还有， 将进行离体肺测定以研究空间生物膜分泌物组。这项研究将利用 能够在空间上绘制由这些和其他病原细菌产生的特定化学产物， 产生对宿主定殖、感染和毒力的深入和治疗信息性的见解。

PROJECT SUMMARY Most bacteria in natural and clinical settings grow as surface-attached biofilms, which are bacterial communities that have self-assembled into an encased matrix of extracellular polymeric substances (EPS). To form these bacterial biofilm communities and infect host cells, an intercellular signaling process described as quorum sensing (QS) is very important. For the opportunistic pathogens Pseudomonas aeruginosa and Staphylococcus aureus, QS regulates the expression of many genes important to biofilm initiation, EPS production, and virulence. While much has been learned about select factors that regulate biofilm formation in vitro and in animal models, the specific mechanisms by which multispecies groups of cells form biofilms are not understood. Furthermore, the therapeutic capabilities needed to treat the increasingly virulent infections of the coming decades will require deeper insight into multi-species interactions and their regulation of multiple gene expression profiles as represented over extended spatial and temporal scales during host cell infection. The correlated mass spectrometric and Raman chemical imaging approaches that have been developed by our combined research group circumvent the limitations imposed by previous technology which allowed examination of either a small number of cells or entire cell populations that had been removed from the conditions of interest. In contrast our correlated chemical imaging methods allow the determination and spatial mapping of individual bacterial species and their microbial products within a mixed bacterial community growing in situ on surfaces. One of our long-term goals is the design of detection and diagnostic strategies informed by an understanding of bacterial interactions and signature biomolecule production. We will continue our work toward this goal by conducting mono-culture and co-culture experiments using Pseudomonas aeruginosa and Staphylococcus aureus as model pathogens. Correlated chemical imaging will be used to define the onset and range of intercellular quorum sensing signaling in space and time. The biosecretome and interactions of specific species in co-culture biofilms will be characterized for both intra- and inter-species interactions. Also, an ex vivo lung assay will be created to study the spatial biofilm secretome. This research will exploit the ability to spatially map specific chemical products produced by these and other pathogenic bacteria thereby yielding deep and therapeutically informative insights into host colonization, infection, and virulence.