Extracellular vesicles released in response to Yersinia pestis

SUMMARY Extracellular vesicles (EVs) are membrane-bound vesicles released by cells that are potent vehicles for intercellular communication. The signaling capacity of EVs is mediated by incorporation of different biomolecules (e.g. proteins, lipids, nucleic acids, and carbohydrates) within individual vesicles. Neutrophils produce EVs upon recognition of a variety of stimuli, but the biological properties of EVs change depending on the stimuli encountered. Thus, depending on stimulation, neutrophil-derived EVs have been shown to induce both pro- and anti-inflammatory responses in recipient cells. Neutrophils can also produce a subset of EVs that are directly microbiostatic to bacteria and fungi. However, the EV response by neutrophils has only been characterized for very few bacterial species, and therefore, we have a limited understanding of the full potential of neutrophil-derived EVs in response to different bacterial infections. Yersinia pestis is the causative agent of the human disease known as plague. Y. pestis evades immune cell recognition via direct interactions with innate immune cells that disrupt the normal responses by these cells. Specifically, Y. pestis uses a type three secretion system (T3SS) to directly secrete bacterial proteins (called Yops) into host cells, which disrupt specific host cell signaling pathways. The outcomes of Yop translocation into host neutrophils include: blocking phagocytosis, inhibition of the generation of reactive oxygen species, and decreased production of pro-inflammatory cytokines by neutrophils. Recently, we and others have shown that Y. pestis is able to block neutrophil granule exocytosis in a T3SS-dependent manner. Together, these data show that Y. pestis efficiently alters endocytic and exocytic activities by neutrophils. Despite its ability to disrupt endocytic and exocytic pathways, the ability of Y. pestis to alter the production of EVs by host cells has not been previously investigated. With a growing appreciation for EVs in inflammation and bacterial clearance, our lack of a proper understanding of the EVs released by innate immune cells in response to Y. pestis represents a critical knowledge gap in the immune response to Y. pestis. Based on previously published studies and our preliminary data, we hypothesize that Y. pestis actively alters the production of EVs by innate immune cells, and that this alteration has a direct impact on how EVs can influence the immune response to the bacterium. To test this hypothesis, in Aim 1 we will define the composition of the payloads packaged into EVs by neutrophils in response to Y. pestis infection and whether the T3SS and Yop effectors alter the production of EVs. In Aim 2, we will determine the impact of EVs isolated from neutrophils infected with Y. pestis or a Y. pestis strain lacking the Yop effectors on immune cell response to Y. pestis infection. Completion of these Aims will provide for the first time a comprehensive description of the EVs produced by human neutrophils in response to infection with Y. pestis.