Deployable 3D-printed Cellular Communities for Characterizing Bacterial Social Cues and Chemical Warfare

项目摘要 病原微生物群落内细胞之间的化学相互作用对 表型状态；大量的化学信号、毒力因子和氧化还原活性物种影响着 细菌成分的毒力潜力和免疫细胞的反应。详细了解如何 “微地理”通过传递社会线索、化学战和其他系统来影响细胞行为 因此，氧气和资源限制等属性将提供潜在的有价值的新的长期 对抗感染的关键策略。精确定位细小细菌聚集体的能力，具有 在感兴趣的微生物群落中确定物理和表型属性将使这项研究成为可能 细胞之间的时变空间相互作用，例如在细胞的扩散和发展过程中可能发生的那些 感染，以及已建立的微生物群内的动态重组。 在这里，我们建议开发一个研究社会微生物学和其他 病原微生物群落中的基于化学的属性，其中细菌菌落和，在一些 例如，来自哺乳动物品系的细胞具有特定的种群大小/密度、形状、排列和 社会微生物状态将在3D打印的多孔蛋白质微容器中进行组织 可操控的光纤头。该Optrode平台将提供报告和 通过微容器进行轻松的化学交换，可控制地扰乱感兴趣的微生物环境 并将通过检测荧光来获取关于微观生态系统的详细分子信息 转录记者和其他细胞探针以及一氧化氮和一氧化氮的工程化学记者 微容器壁中含有的活性氧物种。作为对平台的验证，我们将对 群体感应和人群依赖的抗生素耐药性传播的时空属性 纤维安装的3D打印PA聚集体和模型微生物群落之间的双向 作为物理上固定的聚集体维护在视觉上可访问的培养中维护在 倒置显微镜系统。作为将方法学扩展到细菌交付的概念验证 在活体环境中，我们将在大幅面成像上3D制造定义的PA种群 光纤束。将评估纤维固定种群和固定种群之间的表型相互作用 Pa/Sa的培养。如果成功，从这些高风险、高收益研究中衍生出来的能力将具有 各种体外模型和体内微弹的中长期应用，包括慢性 伤口、胃肠道和呼吸系统的感染。

Project Summary The chemical interplay between cells within pathogenic microbial communities has profound effects on phenotypic states; numerous chemical signals, virulence factors, and redox-active species influence both the virulence potential of bacterial constituents and the response of immune cells. Detailed understanding how “micro-geography” impacts cellular behavior via transmission of social cues, chemical warfare, and other system attributes such as oxygen and resource limitation would therefore offer potentially valuable new long-term strategies key for combatting infection. A capability to precisely position small, bacterial aggregates having defined physical and phenotypic attributes within microbial communities of interest would enable the study time-variant spatial interactions between cells, such as those that may occur in the spread and progression of infections, as well as dynamic reorganization within established microbiomes. Here, we propose development of a technology platform for investigating sociomicrobiology and other chemically based attributes in pathogenic microbial communities in which bacterial colonies and, in some instances, cells from mammalian lines of defined population sizes/densities, shapes, arrangements, and sociomicrobiological status will be organized in porous protein-based microcontainers 3D printed on maneuverable optical fiber tips. This optrode platform will provide capabilities both for reporting on and controllably perturbing, microbial environments of interest by facile chemical exchange through microcontainer walls and will acquire detailed molecular information on microscopic ecosystems via detection of fluorescent transcriptional reporters and other cellular probes, as well as engineered chemical reporters for nitric oxide and reactive oxygen species incorporated in microcontainer walls. As validation of the platform, we will evaluate spatiotemporal attributes for transmission of quorum sensing and population-dependent antibiotic resistance bi-directionally between fiber-mounted 3D-printed Pa aggregates and model microbiome communities maintained as a physically stationary ensemble of aggregates in visually accessible cultures maintained on an inverted microscope system. As proof-of-concept for extending methodologies to delivery of bacterial communities to in vivo environments, we will 3D fabricate defined populations of Pa on large-format imaging fiber-optic bundles. Phenotypic interplay will be evaluated between fiber-mounted populations and stationary cultures of Pa/Sa. If successful, capabilities that derive from these high-risk, high-benefit studies will have medium- and long-term applications to various in vitro models and in vivo microbomes, including chronic infections in wounds, gastrointestinal tract, and respiratory system.