Performing Department
Animal Health Research Center
Non Technical Summary
Brucellosis is a highly prevalent disease worldwide caused by the bacterium Brucella, and generates significant animal healthand production issues by causing abortion and sterility in many agriculturally important animals. Brucella survives andproliferates inside cells of the infected host to cause disease, by injecting into host cells proteins that modulate host cellfunctions to the bacterium's advantage. This project will investigate the mode of action of one of these protein, BspA, tounderstand how it contributes to the bacterium's ability to proliferate within host cells during infection. Understanding BspA function is essential to the design of future therapeutic intervention against brucellosis.
Animal Health Component
100%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
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.
Project Methods
Specific Aim 1: Determine the role ERAD plays in the Brucella intracellular cycle. Methods used to perform this specific aim include bacterial culture, primary mammalian cell culture and differentiation, siRNA methodologies, fluorescence microscopy and image analysis.Specific Aim 2: Determine whether BspA modulates ERAD via its interaction with MARCH6. Methods used to perform this specific aim include bacterial culture and genetics, mammalian culture and transfection, protein biochemistry and purification, and molecular cloning.