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Modulation of Er-associated Degradation By Brucella Abortus

Celli, JE
Washington State University
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Intracellular bacterial pathogens modulate host cell functions to promote their survival, proliferation and persistence, traits that contribute to their pathogenic potential and ability to cause disease. Bacteria of the genus Brucella are the causative agents of the worldwide zoonosis brucellosis, a widespread disease that affects the reproduction and health of many agriculturally important animals, and inflicts significant economic losses in endemic areas. Essential to Brucella's pathogenesis is its ability to undergo an intracellular cycle within host phagocytes, during which it resides within a membrane-bound vacuole, the Brucella-containing vacuole (BCV). BCV maturation occurs along the host endocytic and secretory pathways to generate an organelle (rBCV) derived from the host cell endoplasmic reticulum (ER) that supports bacterial proliferation. rBCV biogenesis is an essential step in the infectious cycle that requires bacterial subversion of the host early secretory pathway via functions mediated by the VirB Type IV secretion system (T4SS). The VirB apparatus delivers effector proteins into infected cells that presumably modulate various cellular pathways to promote Brucella intracellular pathogenesis, yet only a few of these proteins have been identified and none of their functions are known. This knowledge gap limits our understanding of Brucella pathogenesis and the development of new, effective therapies against brucellosis.Our long-term goal is to decipher the molecular mechanisms of intracellular survival and replication of Brucella abortus, which are prominently driven by activities of the VirB T4SS and exploitation of the secretory pathway and ER functions. We have recently identified a series of proteins (named Bsp, for Brucella secreted proteins) that are delivered into host cells by the VirB T4SS during infection, among which three (BspA, BspB and BspF) impair host cell secretory trafficking. BspA is important for rBCV biogenesis and Brucella intracellular replication and binds the host E3 ubiquitin ligase MARCH6, a key component of the ER-associated degradation (ERAD) pathway. Interestingly, pharmacological interference with ERAD enhances Brucella intracellular replication, suggesting that ERAD exerts a suppressive effect on the bacterium's infectious cycle. Based on this preliminary data, our working hypothesis is that Brucella modulates ERAD via delivery of the T4SS effector BspA to promote biogenesis of its replicative vacuole and proliferation. To begin testing this hypothesis, we will pursue the following aims:Specific Aim 1: Determine the role ERAD plays in the Brucella intracellular cycle. We will use pharmacological ERAD inhibitors and siRNA-mediated depletions of key ERAD components in macrophages to define i) whether ERAD impairs rBCV biogenesis or bacterial replication and ii) which step(s) of the ERAD pathway influence(s) the bacterium's infectious cycle. These experiments will establish the role ERAD plays in Brucella intracellular pathogenesis.Specific Aim 2: Determine whether BspA modulates ERAD via its interaction with MARCH6. We will determine using both ectopic expression and infection models i) whether BspA modulates MARCH6 activity and stability, and ii) whether BspA alters ERAD. These experiments will define whether BspA targets ERAD via its interaction with MARCH6 and may reveal the molecular mode of action of BspA in promoting rBCV biogenesis and bacterial replication.
Funding Source
Nat'l. Inst. of Food and Agriculture
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Bacterial Pathogens