Brucellosis is a highly prevalent bacterial zoonosis worldwide caused by Brucella spp., inflicting significant public health burden in endemic areas and causing biodefense concerns due to Brucella high infectivity. Control and treatment of human brucellosis are limited by a lack of prophylactic measures and by the varying efficacy of long-term antibiotics treatments, highlighting the need for alternative therapies. Brucella undergoes an intracellular cycle that supports its survival, proliferation and persistence within mammalian hosts and is required for pathogenesis. The bacterium resides intracellularly within a membrane-bound compartment, the Brucella-containing vacuole (BCV), whose evolution from an initial endosomal vacuole (eBCV) to a replicative organelle (rBCV) derived from the endoplasmic reticulum (ER) is controlled by the Brucella VirB Type IV secretion system. The VirB apparatus delivers effector proteins into host cells that modulate host functions, and is required for rBCV biogenesis and bacterial replication. Following replication, rBCVs are converted into functionally distinct vacuoles (aBCVs) through an autophagy-related process associated with cell-to-cell spread, suggesting that aBCVs mediate bacterial egress and dissemination. Despite the obvious importance of bacterial egress in disseminating infections, and the established role of aBCVs in this process, little is known about this particular stage of the Brucella intracellular cycle. Here we propose to fill this knowledge gap and define the mechanisms and functional requirements of aBCV formation. Specifically, we will test the hypotheses that i) the VirB apparatus controls conversion of rBCV to aBCVs;ii) aBCVs mediate bacterial egress via secretory autophagy. First, we will take advantage of a controllable VirB expression system that circumvents VirB early requirements in the cycle to test its role in aBCV formation and define bacterial determinants of this specific stage. Second, we will use live cell imaging approaches to visualize the dynamics of aBCV formation and bacterial egress and establish whether aBCVs mediate bacterial release. Last, we will test via siRNA-mediated depletion of secretory autophagy proteins whether this particular pathway is involved to define the host determinants of aBCV formation and bacterial egress. The proposed studies will therefore advance our molecular understanding of Brucella VirB-mediated subversion of host functions and bacterial egress mechanisms, and how this pathogen may disseminate during infection, ultimately providing novel targets for therapeutic intervention against brucellosis.

Public Health Relevance

Brucellosis is a widespread disease of animals and humans inflicting significant public health burden worldwide, which is caused by the zoonotic bacterial pathogen Brucella. Brucella resides and proliferates within host cells during infection, and eventually exits from infected cells to further disseminate within the host. This proposal aims to characterize the mechanisms of bacterial dissemination, which will lead to the development of new targets for therapeutic intervention against brucellosis.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Exploratory/Developmental Grants (R21)
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Special Emphasis Panel (ZRG1)
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Mukhopadhyay, Suman
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Washington State University
Veterinary Sciences
Schools of Veterinary Medicine
United States
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Miller, Cheryl N; Smith, Erin P; Cundiff, Jennifer A et al. (2017) A Brucella Type IV Effector Targets the COG Tethering Complex to Remodel Host Secretory Traffic and Promote Intracellular Replication. Cell Host Microbe 22:317-329.e7
Miller, Cheryl; Celli, Jean (2016) Avoidance and Subversion of Eukaryotic Homeostatic Autophagy Mechanisms by Bacterial Pathogens. J Mol Biol 428:3387-98
Smith, Erin P; Miller, Cheryl N; Child, Robert et al. (2016) Postreplication Roles of the Brucella VirB Type IV Secretion System Uncovered via Conditional Expression of the VirB11 ATPase. MBio 7:
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Celli, Jean (2015) The changing nature of the Brucella-containing vacuole. Cell Microbiol 17:951-8