High-content assays for microbial and human cellulomics to assess sub-lethal pesticide effects.

我们小组一直在研究微生物功能与细胞表面性质之间的关系。细胞表面是任何细胞暴露于外部环境的第一个部位。随着农药等环境污染物的不断增加，评估其对人类健康和微生物环境的影响变得至关重要。我们认为，亚致死暴露于农药可以直接改变细胞的生化，物理性质和表面超微结构，最终影响细胞免疫和微生物的毒力，并发挥更大的作用，在环境功能障碍和人类疾病比目前认识。我们将继续开发选定的微生物和人类细胞膜的细胞壁模型，通过表征它们的表面生化，超微结构和物理特性。为了进一步了解表面完整性和细胞功能之间的相互作用，我们将开发体外高含量测定。将使用原子力显微镜、力谱和共聚焦显微镜开发单活细胞分析，以分别同时监测细胞表型、超微结构形态、细胞机械特性和生物化学。代谢组学将用于评估暴露于农药的细胞的分子变化，以确定指示细胞功能障碍进展的“早期预警”生物标志物。高含量分析将用于探测微生物、其细胞壁生物合成突变体和人类细胞对亚致死农药水平的反应。总之，这种多参数的生化，结构和物理数据将被用来评估细胞对农药的反应。该计划将增强我们对细胞壁和膜功能的理解，以及它们在对最终能够改变细胞功能的外部刺激的第一线反应中的作用。一旦我们充分表征农药对模型微生物和人类细胞的影响，未来的研究就可以解决它们对病原体-宿主相互作用的影响。了解环境污染物对更广泛的微生物生态系统的影响以及这些物质对人类的健康风险将最终应用于环境保护和疾病预防。

Our group has been studying the relationship between microbial function and cell surface properties. The cell surface is the first site of exposure of any cell to its external environment. With ever increasing environmental contaminants, such as pesticides, assessing their effect on human health and the microbial environment becomes paramount. We propose that sub-lethal exposure to pesticides can directly alter cell biochemical, physical properties and surface ultrastructure, ultimately affecting cellular immunity and microbial virulence and playing a greater role in environmental dysfunction and human disease than is currently appreciated. We will continue to develop models for the cell walls of select microorganisms and human cell membranes by characterizing their surface biochemical, ultrastructural and physical properties. To further understand the interplay between surface integrity and cell function, we will develop in vitro high-content assays. Single live cell analyses will be developed using atomic force microscopy, force spectroscopy and confocal microscopy to simultaneously monitor cell phenotype, ultrastructural morphology, cell mechanical properties and biochemistry, respectively. Metabolomics will be used to evaluate molecular changes to cells exposed to pesticides to identify "early warning" biomarkers indicative of progression towards cellular dysfunction. High content assays will be used to probe microbes, their cell wall biosynthetic mutants, and human cells in response to sub-lethal pesticide levels. Taken together, this multi-parameter biochemical, structural and physical data will be used to evaluate cellular responses to pesticides. This program will enhance our understanding of cell wall and membrane function, and their role in first line responses to external stimuli ultimately capable of altering cellular function. Once we fully characterize pesticide effects on model microbes and human cells, future studies can address their effects on pathogen-host interactions. Understanding the impact of environmental contaminants on the broader microbial ecosystem and the health risk of these agents to humans will have ultimate application in environmental protection and disease prevention.