Recipient Organization
WASHINGTON STATE UNIVERSITY
240 FRENCH ADMINISTRATION BLDG
PULLMAN,WA 99164-0001
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, BspF, tounderstand how it contributes to the bacterium's ability to modulate immune responses during infection. Understanding BspFfunction is essential to the design of future therapeutic intervention against brucellosis.
Animal Health Component
(N/A)
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. 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. BspF is important for Brucella-induced alteration of secretory trafficking during infection, but not for rBCV biogenesis and bacterial replication. Whether BspF contributes to down-regulation of cellular immune responses, another key aspects of Brucella pathogenesis, is unknown. Here we propose to elucidate the role of BspF during infection in the context of modulation of host secretory trafficking. Our preliminary studies have identified the host protein ACAP1 as an interacting partner of BspF. ACAP1 is a GTPase activating protein (GAP) that downregulates the small GTPase ARF6. ARF6 controls vesicular trafficking at the plasma membrane, so its potential modulation by BspF may impact exocytosis of secretory vesicles. With these considerations in mind, our working hypothesis is that BspF modulates ARF6-dependent vesicular trafficking via ACAP1 to mediate its effect on host protein secretion. To begin testing this hypothesis, we will pursue the following aims:Specific Aim 1: Determine whether BspF-mediated modulation of secretory trafficking contributes to down-modulation of cellular immune responses. We will test whether deletion of bspF in Brucella i) alters surface presentation of immune molecules on infected cells and ii) alters secretion of pro-inflammatory cytokines by infected cells. These experiments will define the potential role of BspF in immune down-modulation by Brucella.Specific Aim 2: Determine whether BspF modulates ARF6 activity to impair secretory trafficking. We will examine i) whether BspF-mediated inhibition of secretory trafficking occurs via the ACAP1-ARF6 pathway; ii) whether BspF modulates ACAP1 inactivation of ARF6. These experiments will reveal the molecular mode of action of BspF that leads to defective secretory trafficking in Brucella-infected cells.
Project Methods
Specific Aim 1: Determine whether BspF-mediated modulation of secretory trafficking contributes to down-modulation of cellular immune responses. Methods used to perform this specific aim include bacterial culture, primary mammalian cell culture and differentiation, quantitative PCR, fluorescence microscopy, flow cytometry and image analysis.Specific Aim 2: Determine whether BspF modulates ARF6 activity to impair secretory trafficking. Methods used to perform this specific aim include bacterial culture, mammalian culture and transfection, protein biochemistry and purification, and molecular cloning.