PhD Oral Exam - Curtis John Logan, Biology

When studying for a doctoral degree (PhD), candidates submit a thesis that provides a critical review of the current state of knowledge of the thesis subject as well as the student’s own contributions to the subject. The distinguishing criterion of doctoral graduate research is a significant and original contribution to knowledge.

Once accepted, the candidate presents the thesis orally. This oral exam is open to the public.

Abstract

Small extracellular vesicles (sEVs) mediate intercellular communication across eukaryotes and have attracted interest as biocompatible drug delivery vehicles, but the mechanisms governing their biogenesis remain incompletely defined. This gap limits both the genetic engineering of custom cargo-loading and the bioprocess design required for production at scale. Saccharomyces cerevisiae offers a genetically amenable system in which to address this issue: core trafficking machinery including the ESCRT complexes and Rab GTPases were first defined in yeast, complete subunit deletions are viable where their mammalian counterparts are lethal, while the simpler endomembrane system carries fewer parallel routes to vesicle release. This thesis establishes S. cerevisiae as a system for dissecting conserved sEV biogenesis and for identifying genetic and environmental routes to increased bioproduction.

I first show that yeast release sEVs during acute mild heat stress, and that these vesicles transfer thermotolerance to heat-naïve recipient cells. These sEVs are exosome-class particle size, carry Bro1, the yeast orthologue of the human sEV marker ALIX, require the ESCRT-I subunit Vps23 for bioactivity, and enter recipient cells by endocytosis. Proteomics identifies HSP70 family chaperones as enriched cargo and establishes Ssa2 as required for thermotolerance transfer.

Applying this defined stress to probiotic yeasts shows that Saccharomyces boulardii ATCC MYA-796 releases 9.31-fold more sEVs per donor cell than the S. cerevisiae reference, while Kluyveromyces marxianus releases fewest of the three species; elevated output was shared by every S. boulardii strain tested. Comparative proteomics of sEV preparations across three species, with soluble contaminants removed by size-exclusion chromatography, identifies a conserved core alongside large species-restricted protein pools, while differential cytokine release from recipient THP-1-derived macrophages tracks vesicle composition rather than uptake efficiency.

A complete ESCRT deletion series, both nucleotide-locked states of Ypt7, and a graded series of Nhx1 expression establish that this machinery governs sEV release in yeast, though not as a single linear route. No deletion abolished release and those preventing membrane engagement by ESCRT-I, ESCRT-II or ESCRT-III increased output up to 7.2-fold while yielding smaller, more negatively charged particles, that no described intracellular route accounts for, indicating that yeast sEVs comprise at least two populations with distinct biogenic origins whose relative bioproduction output is set in part by ESCRT function. Ypt7 is required for release irrespective of nucleotide state, while its nucleotide state together with Nhx1 activity determines whether multivesicular bodies reach the vacuole or the plasma membrane.

Screening 52 deletions in lipid metabolism, lipid-responsive signalling and calcium homeostasis then identifies membrane lipid composition as a determinant of release, spanning a 0.50- to 18.8-fold-change in sEV bioproduction relative to wild type controls across erg2Δ, opi1Δ, snf7Δ, sac1Δ, pmr1Δ, rim20Δ, and nce102Δ. Every component of the Rim101 sensing and processing arm trended toward increased release while every transcriptional target tested was null, implicating a non-transcriptional output of that pathway and offering a candidate explanation for the ESCRT phenotype through the Rim20–Snf7 interaction. Lipidomics confirms strain-specific compositional signatures, and growth at pH 3.5 abolishes the opi1Δ increased sEV bioproduction phenotype, showing that a genetic overproduction phenotype can be reversed by the growth conditions of the donor culture.

Together these chapters define conserved determinants of sEV release in yeast and identify genetic and environmental levers for increasing it. Because the highest-producing strains release vesicles of distinct lipid composition, these levers offer routes not only to greater yield for scalable bioprocess design but to sEV populations differing in properties relevant to cargo loading, stability and recipient cell uptake efficiency. Each regulatory axis identified has a counterpart dysregulated in human disease.