Project Summary Intracellular microbes with a vacuolar lifestyle share an ability to remodel host cell compartments and functions to support specific stages of their infectious cycle. The cellular and molecular details of how microbial vacuoles functionally evolve during a pathogen’s intracellular cycle to promote their virulence are not well understood. Here we aim to define cellular processes driving the functional evolution of the intracellular vacuole of the zoonotic bacterium Brucella abortus, which transitions from a replicative niche to an egress organelle. B. abortus primarily infects phagocytes and remodels its original phagosome into the replicative Brucella-containing vacuole (rBCV), an organelle derived from the host endoplasmic reticulum (ER) that supports intracellular proliferation. rBCVs subsequently convert into autophagosome- like vacuoles (aBCVs) that mediate post-replication bacterial egress. Autophagy is a conserved eukaryotic process of selective or non-selective capture of cellular content within membrane-bound autophagosomes for lysosomal degradation, including the selective degradation of organelles such as the ER via dedicated autophagy receptors. We have shown that aBCV biogenesis from rBCVs requires a subset of conventional autophagic machineries and an active bacterial VirB Type IV secretion system, but the process, selectivity and regulation of this vacuolar conversion remain enigmatic. Brucella infection triggers the Unfolded Protein Response (UPR) during the rBCV stage via the innate immune sensor STING, provoking an ER- centered stress response that promotes bacterial replication within rBCVs. Whether the UPR also contributes to aBCV biogenesis is unknown. STING-dependent UPR induces ER-phagy, whose selectivity could mechanistically drive the capture of ER-derived rBCVs by autophagosomes to form aBCVs. Based on preliminary evidence that i) Brucella infection influences ER-phagy; ii) rBCVs recruit distinct ER-phagy receptors and iii) STING is required for aBCV biogenesis, here we will test the overall hypothesis that aBCV biogenesis is mediated by selective ER-phagy of rBCVs via a STING-dependent process. Aim1 will determine i) whether Brucella modulates ER-phagy, ii) whether specific ER-phagy receptors are required for aBCV biogenesis and iii) the autophagic cascade engaged during aBCV biogenesis. Aim 2 will determine whether the role of STING in aBCV biogenesis is via induction of the UPR or its activity as an ER-phagy receptor. The successful completion of these aims will establish new concepts of functional evolution of bacterial vacuoles, a common feature of the infectious cycle of many microbial pathogens that is poorly understood.