Acute and chronic Q fever infections in humans are caused by Coxiella burnetii, which traffics to and replicates in Coxiella-containing vacuoles (CCV). Infection suppresses macrophage activation in a type 4 secretion system-dependent manner, hence our central hypothesis is characterization of T4SS-dependent effector molecules in vitro and in vivo will define “stealthy” virulence genes. This application will identify T4SS-dependent virulence determinants that modulate macrophage signaling pathways using pathogen effector mutants and host signaling mutants that are essential for restricting replication or modulating response to infection. Aim 1. Identify the range of T4SS dependent manipulation of macrophage. The working hypothesis is multiple T4SS effectors manipulate the response to infection in macrophages. Using a primary bone marrow derived macrophage (BMDM) cell culture that enables C. burnetii, RSA439 (NMII) to replicate. We will extensively characterize BMDM using transcriptomic (including single cell analysis), metabolomics and flow cytometric analysis to comprehensively map T4SS dependent pathway modulation of macrophage activation. Aim 2. Identify T4SS effectors which manipulate immune signaling. The working hypothesis is T4SS effectors target specific activation signaling pathways. To identify this broad class of T4SS effectors, tagged C. burnetii T4SS substrates will be transfected into macrophages in the context of signaling agonists. A complementary approach will use T4SS mutants to identify infection that does not modulate innate activation. Aim 3. Establish mechanistic basis for T4SS effector modulation. Current candidates derived from preliminary data include;; a) 2 Ankyrin repeat-containing proteins which dampen agonist driven NF-κb;; b) 5 T4SS effectors that are essential for replication in BMDM;; and c) 3 T4SS effectors which traffic to the nucleus (nucleomodulins). We will identify host binding partners using pull-down methods and define their role in disease using host knock-down or knock-out approaches. Each effector will be analyzed with either Tn or site-specific mutants.