Characterization of Biofilms by Correlated Mass Spectrometric and Raman Imaging

项目总结 在自然和临床环境中，大多数细菌以附着在表面的生物膜的形式生长，这种生物膜是细菌 自组装成被包裹的胞外聚合物(EPS)基质的群落。至 形成这些细菌生物膜群落并感染宿主细胞，这种细胞间信号传递过程被描述为 群体感知(QS)是非常重要的。对于条件致病菌铜绿假单胞菌和 金黄色葡萄球菌，QS调节许多对生物被膜启动重要的基因的表达 生产和毒力。虽然已经了解了许多关于调节生物膜形成的选定因素 在体外和动物模型中，多物种细胞组形成生物膜的具体机制尚不清楚 明白了。此外，治疗日益严重的猪流感病毒感染所需的治疗能力 未来几十年将需要对多物种相互作用及其对多基因的调控有更深入的了解 在宿主细胞感染期间，表达谱表现在扩展的空间和时间尺度上。 已开发的相关质谱学和拉曼化学成像方法 通过我们的联合研究小组，绕过了以前技术施加的限制，这使得 对已从细胞中移除的少数细胞或整个细胞群体进行检查 利息的条件。相比之下，我们相关的化学成像方法允许确定和空间 混合细菌群落中单个细菌物种及其微生物产物的测绘 在表面上原位生长。 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标志性生物分子的产生。我们将继续努力，争取 通过使用铜绿假单胞菌和铜绿假单胞菌进行单一培养和共培养实验来实现这一目标 金黄色葡萄球菌作为模式致病菌。相关的化学成像将被用来定义起始和 细胞间群体感应信号在空间和时间上的范围。细菌的生物基因组及其相互作用 共培养生物膜中的特定物种将被描述为物种内和物种间的相互作用。另外， 将建立一种体外肺试验来研究空间生物膜分泌组。这项研究将利用 能够对这些和其他致病细菌产生的特定化学产品进行空间测绘，从而 对寄主定植、感染和毒力有深刻的和有治疗意义的见解。

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.