Defining macrophage-induced stressors of Bacillus anthracis at early stages of anthrax disease

About This Grant

PROJECT SUMMARY Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen with significant bioterror potential. For B. anthracis to cause systemic disease, bacterial endospores must be phagocytosed by macrophages, germinate, and survive the arsenal of antibacterial stressors imposed by the host. Understanding the nature of these stressors, and the mechanisms by which B. anthracis resists them, may reveal targets for novel therapeutics to treat anthrax and other intracellular bacterial infections. Our laboratory has discovered a two- component regulatory system in B. anthracis, HitRS, which is activated in response to damage of the bacterial cell envelope. Preliminary data have shown that HitRS is activated within murine macrophages and that deletion of hitRS results in decreased bacterial growth within these cells. This led me to hypothesize that HitRS is involved in sensing cell envelope stress imparted by the host, and that HitRS activation leads to a transcriptional response that promotes bacterial growth in the intracellular environment. The role HitRS and its regulon play in B. anthracis pathogenesis, and the host factors responsible for HitRS activation, remain unknown. To fill these gaps in knowledge, our laboratory has developed a strain of B. anthracis that lacks hitRS (∆hitRS), and a fluorescent reporter strain in which HitRS activation drives the expression of mScarlet-I. Using these tools, Aim 1 will define the regulon of HitRS using differential expression analyses, discover the role of HitRS during murine anthrax infection using an in vivo infection model, and measure the activation of HitRS during murine infection through intravital imaging. To identify the host factors responsible for HitRS activation, Aim 2 experiments will employ a microscopy-based whole genome CRISPR screen. Immortalized murine macrophages in 384-well plates will be transduced with an arrayed library of CRISPR sgRNAs using a robotic liquid handler. These mutant cells will be infected with the B. anthracis HitRS reporter strain, fixed at 4-hours post-infection, and confocal images will be taken using a high-content imaging system. Orthogonal image analysis and statistical techniques will be used to discover host genes that impact the activation of HitRS and control bacterial replication. One of these gene hits will be selected for further in vitro and in vivo experiments to determine the mechanism by which a macrophage gene activates HitRS and modulates the intracellular growth of B. anthracis.