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

在美国，细菌感染每年导致超过30万人死亡。这些感染中的许多是由细菌分泌的毒力蛋白质引发的，这些毒力蛋白质存在于含脂质的颗粒中，称为细胞外囊泡。人类排泄物（粪便）是细菌细胞外囊泡（BEV）的重要来源 在环境中。然而，人们对BEV进入废水处理和处置系统时的命运知之甚少。这个CAREER项目的总体目标是调查BEV在废水系统中的命运和健康影响。为了推进这一目标，主要研究者（PI）建议将废水样品中BEV的提取和分离与先进的蛋白质基因组学分析结合联合收割机并整合，以识别和定量废水中的BEV蛋白。然后，PI将利用这种新方法来研究废水和废水处理系统中BEV的产生，命运和持久性。该项目的成功完成将通过产生关于BEV在环境中的命运及其对公共卫生的潜在影响的新的基础知识而造福社会。通过学生教育和培训，包括指导布法罗大学的一名研究生和一名本科生，将为社会带来额外的好处。细菌释放细胞外载体作为输出其细胞内容物的通用途径（即，蛋白质、核苷酸、脂质和代谢物）以富集的浓度进入周围环境。先前的研究表明，通过呼吸或口服途径引入的某些细菌细胞外囊泡（BEV）可导致体内和体外的炎症反应。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的细菌在人类废物（粪便）中脱落，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EV携带的毒力蛋白的基础知识有限。该CAREER项目将调查废水中BEV的持久性和生产，并评估其对人类健康的潜在风险，旨在改进消毒策略以灭活BEV，并推进从废水中发现与健康相关的生物标志物。该研究的具体目标是：（1）开发和应用一种新的综合分离和蛋白基因组学方法来表征BEV蛋白及其在城市污水中的生物学功能;（2）测量BEV在污水中的生产，分配和衰变的动力学;（3）评估BEV通过常用的消毒方法（包括氯化和杀菌UV）处理后的致病潜力。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。为了实施这一职业生涯项目的教育和推广活动，主要研究者（PI）将利用布法罗大学（UB）的现有项目和资源，（1）在布法罗科学博物馆为公众开发和展示模拟废水微生物组发现的互动活动，（2）设计乐高平台以扩大K的参与度-12名学生学习组学技术及其在环境监测和疾病监测中的应用。此外，PI计划利用研究结果，设计并将组学工具和分析的经验教训纳入UB的环境工程课程。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估来支持。

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.