Project Summary Most bacterial diseases of public health significance are caused by pathogens with an intracellular lifecycle that is integral to their virulence. These microorganisms exploit a variety of host cell functions to ensure their intracellular survival and proliferation, via delivery of effector proteins or toxins that modulate specific host processes, including intracellular membrane transport by vacuolar pathogens. The Golgi apparatus plays a central role in the host secretory traffic of cellular components and is targeted by many bacteria for the purpose of vacuole biogenesis and maintenance or intracellular nutrient acquisition. Little is known, however, about the bacterial effectors that exploit this essential organelle, nor how pathogens subvert its functions for pathogenic purposes. Bacteria of the genus Brucella, which cause the global zoonosis brucellosis, generate a replication-permissive organelle derived from the host endoplasmic reticulum (ER), the Brucella-containing vacuole (rBCV), which is essential to their pathogenesis. rBCV biogenesis requires VirB Type IV secretion system (T4SS)-mediated delivery of effector proteins that modulate specific host secretory functions. We identified several of these effectors, among which BspB targets the conserved oligomeric Golgi (COG) complex, a master regulator of Golgi-associated vesicular trafficking, to promote rBCV biogenesis and bacterial replication. Additional T4SS effectors (BspD and BspF) modulate Golgi- associated trafficking functions, further supporting a previously unsuspected role of the Golgi apparatus in the Brucella intracellular cycle. Our findings also suggest that T4SS effectors (BspB/RicA and BspF/BspD) functionally cooperate in their modulation of membrane transport pathways, arguing that Brucella modulation of Golgi-associated processes results from the integration of multiple effector functions. Here we will test the hypothesis that Brucella delivers an array of T4SS effector proteins that modulate membrane transport functions at the Golgi interface to coordinately promote bacterial proliferation. We will use genetic, cellular and biochemical approaches to first characterize the Golgi transport pathways that contribute to Brucella proliferation and define the targets and functional network of Golgi-targeting T4SS effectors. Second, we will determine the molecular modes of action of the Golgi-targeting T4SS effectors BspB, BspD and BspF. Last, we will define i) how BspB and RicA coordinately modulate ER-Golgi vesicular transport to promote rBCV biogenesis and ii) how BspF and BspD co-modulate TGN-associated transport to promote Brucella replication. The successful completion of this project will uncover bacterial effector- driven mechanisms of modulation of Golgi-associated membrane transport functions and define new paradigms of effector functions and coordination in bacterial pathogens, broadly impacting the research areas of microbial pathogenesis, cell bio...