During inhalational anthrax, considerable evidence indicates that alveolar macrophages (m?s) contribute to the development of disseminated infection and disease by transporting dormant B. anthracis (Ba) spores, previously inhaled into the alveolar spaces of the lungs, to the bloodstream. Although the initial steps of spore uptake into m?s have been extensively studied, a major gap in knowledge, which is the focus of this exploratory R21 application, is the mechanism by which intracellular Ba escape infected m?s as a requisite step prior to extracellular dissemination. Here, we propose studies that challenge an existing model that predicts, analogous to several different intracellular pathogens, m? death must precede Ba release to the external environment. Instead, our preliminary data indicate that subsequent to phagocytic uptake of dormant spores into Bacillus-containing vacuoles (BCVs), a substantial fraction of intracellular Ba escape in the absence of m? killing. Because we find no evidence that Ba depart BCVs into the cytosol of infected m?s, we propose that these intracellular vacuoles represent specialized niches that are important for Ba escape. In support of this idea, infection with Ba enhances transport of lysosomal contents to the cell surface, and, induces the time-dependent enrichment of BCVs with the membrane vesicle fusion protein, synaptotagmin-7, consistent with an exocytic process. Based on these preliminary data, we will evaluate the overall hypothesis that egress of intracellular B. anthracis from infected macrophages requires remodeling of Bacillus-containing vacuoles (BCVs) from degradative compartments to exocytic-like vesicles.
In Aim 1, we will characterize the maturation of BCVs during the course of m? infection with dormant Ba spores.
In Aim 2, we will evaluate the importance of BCV remodeling for Ba egress and the outcome of infection. These studies will provide the first detailed characterization of the intracellular niche occupied by Ba spores, and contribute to our understanding of the fundamental biology underlying inhalational anthrax.
Inhalational anthrax, which is caused by the bacterium Bacillus anthracis, is generally a fatal disease in humans. The research proposed within this application seeks to understand how B. anthracis subvert the killing potential of host immune defenses. The eventual goal is to leverage these proposed studies by translating the discovery of newly identified pathways and pathogen/host determinants as potential targets for the development of effective countermeasures to prevent or treat disease in humans.
