C. burnetii causes the zoonotic disease Q fever and resides within monocytes/macrophages manipulating innate immune response. C. burnetii requires de novo protein synthesis and a functional Type IVB Secretion System (T4BSS) to modulate host the eukaryotic transcription factor Nuclear Factor-κB (NF-κB) signaling an essential regulator of innate response. Multiple pathogens including C. burnetii deliver eukaryotic-type Ank-containing effectors to hijack NF-κB signaling. C. burnetii Nine Mile isolate encodes 5 Ank-containing effectors (AnkA, -C, -F, -G, and -K). Experiments found that 1) AnkK and AnkC significantly contributes to C. burnetii’s ability to inhibit NF-κB dependent gene expression and 2) ectopic expression of AnkK or AnkC in HEK293/hTLR4-MD2-CD14 cells blocks accumulation of NF-κB in the nucleus. The central focus of this proposal is to determine mechanistically how the C. burnetii effectors, AnkK and AnkC, modulate NF-κB to promote intracellular survival and replication. The central hypothesis by accomplishing the following specific aims: 1. Determine the contribution of AnkK and -C to C. burnetii’s virulence. The working hypothesis is that C. burnetii AnkK and AnkC promotes pathogen virulence by modulating NF-κB activation. To test this, we will generate complementation strains of C. burnetii NM II (NM II) ankK::Tn and ankC::Tn mutants and investigate in vitro growth rescue and p65 nuclear translocation for phenotype rescue in tissue culture cells. To investigate in vivo growth rescue, we will employ the SCID mice/NM II infection model. Employing CYA-translocation assays, we will then test Type 4 secretion system requirement. Furthermore, we will generate ΔdotA, ΔAnkK, and ΔAnkC gene knockouts and their complementation strains in virulent C. burnetii NM I (NM I) to infect C57Bl/6N mice and determine the relative contribution of AnkK and AnkC to bacterial virulence in vivo. 2. Determine the mechanism of AnkK/AnkC-dependent NF-κB inhibition during C. burnetii infection. The working hypothesis is C. burnetii AnkK or AnkC interacts with host targets in the canonical NF-κB pathway or potentially sequesters p65 to block its nuclear translocation. We will deplete p65 in host cells to investigate growth rescue of NM II ankK::Tn and ankC::Tn mutants. We will examine expression, phosphorylation and degradation status of individual components of the canonical NF-κB pathway in stable THP-1 cell lines expressing eGFP-tagged AnkK and AnkC. To identify host-binding partner(s), pull down assays (GFP-trap) will be performed with THP-1 stable cells expressing eGFP-AnkK and –AnkC. To determine C. burnetii AnkK or AnkC driven modulation of host p65-dependent gene expression in infected cells, we ...