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

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.