Characterization of Agriculturally Relevant Extracellular Vesicles

The major goals of the project are to develop new analytical tools to detect and characterize Listeria extracellular vesicles (EVs) and use those tools to test the hypothesis that Listeria's EVs participate in anaerobic respiration via EET. A highly sensitive biosensor for Listeria EVs will be developed based on the Electrochemical Enzyme Immunoassay Biosensor (EEIB) platform Dr. Worden's lab recently developed with support from the National Science Foundation. An EEIB specific for Listeria EVs would allow the EV concentration to be measured rapidly and accurately and thus help elucidate the function of the EVs in Listeria growth and disease development. Results of this research could yield new commercial biosensor systems to rapidly and sensitively detect Listeria in food products, enable development of new strategies to control growth of Listeria in areas where food is produced, processed, stored, or transported. It could also help detect, prevent, or mitigate the effects of, Listeria infections in farm animals, as well as other animal disease process that involve EVs.The objectives of the project are listed below:Develop an Electrochemical Enzyme Immunoassay Biosensor (EEIB) system to measure Listeria EV concentration. Evaluate the EEIB's performance properties for measuring Listeria EV concentration in food samples and liquid samples used for Listeria research.1A: Use proteomic analysis of Listeria EVs to identify antigenic proteins present in Listeria EVs. Purchase commercially available antibodies to antigenic proteins on Listeria EVs, if possible.1B: If satisfactory antibodies to antigenic proteins on Listeria EVs are not commercially available, contract with a vendor to produce polyclonal antibodies against Listeria EVs.1C: Use EEIB-fabrication protocols developed in Dr. Worden's lab to produce EEIB to measure Listeria EV concentration in liquid samples.1D: Evaluate the performance properties of the EEIB system to detect Listeria EVs.Develop improved analytical tools for EVs needed to advance research to understand the biological role of EVs and engineer EV-enabled systems to enhance agriculture.2A: Adapt the pBLM method to study EV interactions with biomembranes. This work will be carried out using mammalian exosomes being studied in Dr. Kanada's lab and Listeria EVs being studied in Dr. Hardy's lab. 2B: Adapt the tethered BLM method study EV interactions with biomembranes. This work will be carried out using mammalian exosomes being studied in Dr. Kanada's lab and Listeria EVs being studied in Dr. Hardy's lab. Characterize EVs produced by Listeria monocytogenes. This work will be carried out in collaboration with Jonathan Hardy's lab.3A: Develop procedures to produce sufficient quantities of Listeria EVs for the studies. The approach will incorporate strategies used to scale-up fermentations in the Protein Expression Laboratory directed by Dr. Worden. The strategies involve production of multi-liter volumes of cell suspension and use of filtration and/or centrifugation to separate cells and cell debris from the EVs.3B: Measure EV properties, including size distribution and zeta potential. 3C: Characterize the EVs' electrochemical properties, including the presence of redox-active molecular species that can be oxidized and/or reduced using potential-sweep techniques including cyclic voltammetry (CV), and differential pulse voltammetry (DPV). If the EVs show evidence of redox-active species, as evidenced by faradaic peaks in the CV or DPV curves, an attempt will be made to characterize the redox-active species.3D: Characterize the EVs' interactions with BLM that mimic biomembranes. These studies will be performed both with both the pBLM method, which is well-suited for characterizing nanoparticle-induced biomembrane pore formation, and the tBLM method, which is well-suited for measuring adsorption of nanoparticles to the biomembrane and removal of lipids from the biomembrane. Test Listeria monocytogenes for the ability to exhibit energy taxis toward oxidized flavin derivatives under anaerobic conditions. Flavin derivatives will include those recently shown to be used by Listeria, as well as those Dr. Worden's lab showed could drive energy taxis for Shewanella species.4A: Evaluate Listeria monocytogenes' ability to migrate chemotactically toward insoluble electron acceptor particles submerged in dilute agar while growing under anaerobic conditions on a carbon substrate that cannot be fermented. Conduct the tests with and without the presence of flavin derivatives likely to serve as electron mediators.4B: Test Listeria monocytogenes for the ability to migrate chemotactically toward the oxidized form of flavins likely to serve as electron mediators while cells are growing on a carbon substrate that cannot be fermented.4C: Test Listeria monocytogenes for the ability to migrate chemotactically toward oxidized Listeria exosomes in dilute agar while cells are growing under anaerobic conditions on a carbon substrate that cannot be fermented.4D: If Listeria monocytogenes is found to exhibit energy taxis, test strategies to control Listeria growth in anaerobic conditions (e.g., in biofilms) by inhibiting electron transfer to extracellular electron acceptors and energy taxis by Listeria.