CAREER: Bacterial extracellular vesicles in wastewater systems: Persistence and production to disseminate virulence proteins

在美国，细菌感染每年导致30多万人死亡。其中许多感染是由细菌分泌的含有脂质的颗粒(称为细胞外小泡)中分泌的毒力蛋白触发的。人类排泄物是环境中细菌胞外小泡(BEV)的重要来源。然而，当BEV进入废水处理和处置系统时，人们对它们的命运知之甚少。这个职业项目的首要目标是调查废水系统中BEV的命运和对健康的影响。为了推进这一目标，首席调查员(PI)建议将从废水样本中提取和分离BEV与先进的蛋白质基因组分析相结合，以鉴定和定量废水中的BEV蛋白。然后，PI将利用这一新方法来调查BEV在废水和废水处理系统中的生成、去向和持久性。该项目的成功完成将使社会受益，因为它将产生关于电动汽车在环境中的命运及其对公众健康的潜在影响的新的基本知识。通过学生教育和培训将获得更多的社会利益，包括指导布法罗大学的一名研究生和一名本科生。细菌释放细胞外载体作为一种通用途径，将其细胞内容(即蛋白质、核苷酸、脂类和代谢物)以丰富的浓度输出到周围环境中。以往的研究表明，某些细菌胞外囊泡(BEV)通过呼吸道或口服途径进入体内和体外都能导致炎症反应。这些致病功能和与宿主细胞的相互作用主要归因于BEV释放的毒力蛋白。高浓度的BEV和产生EV的细菌被排放到人类排泄物(粪便)中，并可能进入废水收集、处理和处置系统。然而，由于复杂废水和环境基质中蛋白质分析的挑战，关于废水中细菌EVS携带的毒力蛋白的基础知识有限。这个职业项目将调查BEV在废水中的持久性和产生，并评估它们对人类健康的潜在风险，目的是改进消毒战略，使BEV失活，并推进从废水中发现与健康相关的生物标志物。这项研究的具体目标是：(1)开发和应用一种新的集成分离和蛋白质组学方法来表征城市污水中的BeV蛋白及其生物学功能；(2)测量废水中BeV的产生、分配和腐烂的动力学；以及(3)评估常用的消毒方法包括氯化和杀菌紫外线处理后BEV的致病潜力。该项目的成功完成有可能通过产生新的数据和基础知识来促进对BEV在废水和环境介质中释放的毒力蛋白的检测、发现和监测，从而产生革命性的影响。为了实施这一职业项目的教育和推广活动，首席调查员(PI)将利用布法罗大学(UB)的现有计划和资源，(1)在布法罗科学博物馆为普通公众开发和展示模拟废水微生物群发现的互动活动，(2)设计乐高平台，以扩大K-12学生学习组学技术及其在环境监测和疾病监测中的应用的参与度。此外，PI计划利用研究结果来设计组学工具和分析方面的课程，并将其纳入亚利桑那州大学的环境工程课程。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Bacterial infections cause more than 300,000 deaths annually in the United States. Many of these infections are triggered by virulence proteins secreted from bacteria in lipid-containing particles, called extracellular vesicles. Human waste (feces) is an important source of bacterial extracellular vesicles (BEVs) in the environment. However, little is known about the fate of BEVs when they enter wastewater treatment and disposal systems. The overarching goal of this CAREER project is to investigate the fate and health impacts of BEVs in wastewater systems. To advance this goal, the Principal Investigator (PI) proposes to combine and integrate the extraction and separation of BEVs from wastewater samples with advanced proteogenomic analyses to identify and quantify BEV proteins in wastewater. The PI will then utilize this new approach to investigate the generation, fate, and persistence of BEVs in wastewater and wastewater treatment systems. The successful completion of this project will benefit society through the generation of new fundamental knowledge on the fate of BEVs in the environment and their potential impacts on public health. Additional benefits to society will be achieved through student education and training including the mentoring of one graduate student and one undergraduate student at the University at Buffalo.Bacteria release extracellular vehicles as a universal pathway to export their cellular contents (i.e., proteins, nucleotides, lipids, and metabolites) with enriched concentrations into the surrounding environment. Previous studies have shown that certain bacterial extracellular vesicles (BEVs) introduced through a respiratory or oral route can lead to inflammatory reactions in vivo and in vitro. Those disease-causing functions and interactions with host cells are primarily attributed to virulence proteins released by BEVs. High concentrations of BEVs and EV-producing bacteria are shed in human waste (feces) and are likely to enter wastewater collection, treatment, and disposal systems. However, limited fundamental knowledge is available about the virulence proteins carried by bacterial EVs in wastewater due to the challenges of protein analysis in complex wastewater and environmental matrices. This CAREER project will investigate the persistence and production of BEVs in wastewater and assess their potential risks to human health with the goal of improving disinfection strategies to deactivate BEVs and advancing the discovery of health-relevant biomarkers from wastewater. The specific objectives of the research are to (1) develop and apply a novel integrated separation and proteogenomic approach to characterize BEV proteins and their biological functions in municipal wastewater; (2) measure the kinetics of BEV production, partitioning, and decay in wastewater; and (3) assess the disease-causing potentials of BEVs after treatment by commonly used disinfection methods including chlorination and germicidal UV. The successful completion of this project has the potential for transformative impact through the generation of new data and fundamental knowledge to advance the detection, discovery, and monitoring of virulence proteins released by BEVs in wastewater and environmental media. To implement the educational and outreach activities of this CAREER project, the Principal Investigator (PI) will leverage existing programs and resources at the University at Buffalo (UB) to (1) develop and display interactive activities simulating wastewater microbiome discovery for the general public at the Buffalo Museum of Science and (2) design LEGO platforms to broaden the engagement of K-12 students in learning about omics technologies and their utilization in environmental monitoring and disease surveillance. In addition, the PI plans to leverage the research findings to design and incorporate lessons in omics tools and analyses into the Environmental Engineering Curriculum at UB.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.