Investigation of the Colloidal Properties of Extracellular Vesicles from Yeast and Bacteria: Implications for Environmental Transport

Abstract

When considering the complex communication patterns between diverse collections of organisms in the environment, extracellular vesicles must be considered. Extracellular vesicles (EVs) are nanoscale, colloidal particles that are secreted by all cell types. While the functions of EVs have been investigated in a variety of environmental contexts, including nutrient scavenging, immune responses, and genetic material delivery, the exact mechanisms by which they are transported in the environment have been overlooked. This prevents a deeper understanding of the intercellular or inter-organismal communication that occurs in different environmental compartments and highlights a need to investigate EV transport. To be able to evaluate or predict transport most effectively, a foundational understanding of the surface properties of EVs must be first established. Through this dissertation, factors (pH, ionic strength, organic content) influencing the surface properties of EVs are investigated to better determine their transport tendencies in the environment. Moreover, three organisms’ EVs (Gram-negative bacteria Pseudomonas fluorescens, Gram-positive bacteria Staphylococcus aureus, and yeast Saccharomyces cerevisiae) are studied to provide a kingdom-spanning perspective on the range of possible surface properties, and thus transport patterns.To evaluate their surfaces, two primary colloidal properties of EVs were studied: zeta potential and attachment efficiency. A proxy for surface charge, zeta potential provides initial characterizations for the electrostatic trends of EVs. From these data, conditions where humic acid concentration are high or ionic strength is low seem to most impact zeta potential values, while other conditions have a minimal impact on EV zeta potential. In addition, the relationship of the zeta potential of EVs to that of their corresponding parent cell is significantly different for all three organisms in this study. For attachment efficiency, the deposition potential of EVs generally seems to correlate with the electrostatic trends. For higher concentrations of humic acid and for EVs from P. fluorescens, other forces including steric or hydrophobic forces may be at play, causing deviations from expected attachment efficiencies. Beyond these two metrics, the effect of EV preparation methods is described, showing that upstream preparation methods, in particular varied size exclusion steps, influence downstream measurements. Finally, from these data, a model for EV transport is developed, showing both possible concentration profiles of EVs through a hypothetical soil column, but also the sensitivity of EVs to changes in system parameters such as flow velocity and attachment efficiency. From these findings, EVs can clearly be transported long distances, but are heavily influenced by a variety of factors, with low ionic strength, organic content, and variations in pH seeming to cause the largest changes to surface properties. For the three organisms in this study, a range of surface chemistries exists. These results have implications for future study of EVs in the environment. The diversity of EVs will likely mirror that of the cells from which they come, resulting in different capacities for transport. These trends seem to be species-dependent, calling for much more colloidal evaluation of various microbes. The conditions examined and the model developed in this dissertation can help to guide any future work for engineering EVs for specified fate outcomes as well. But, any future study of EVs in the environment must carefully consider what biases may be introduced from EV preparation methods.