Macrophage Killing Of Intracelluar Pathogen Fransicella Tularensis: Functional Genomic Analysis

Francisella tularensis (FT) is a highly virulent, facultative intracellular bacterium which causes tularemia, and is a potential bioweapon. Interferon‐γ has been reported to play a crucial role in inhibition of intracellular replication of FT in human monocyte‐derived macrophages and in mice, but the mechanisms for this protective effect are poorly characterized. Our hypotheses were that IFN‐g limits intracellular pathogen FT by activation of specific genes, and functional genomic screening can identify these genes. We transduced human macrophage THP‐1 cells with a genome‐wide lentiviral siRNA library targeting ~47,000 human genes and ESTs. We screened for RNA interference resulting in a phenotype that failed to show the beneficial effects of IFN‐g on intracellular FT. We sorted macrophages with highest GFP‐FT load. The siRNA inserts were amplified by PCR from genomic DNA in these cells, and siRNA targets were identified by cloning and DNA sequencing. From 5 sorting experiments, a total 4078 clones were sequenced, and 3386 genes/transcripts/ESTs were identified. We found 212 genes of interest, with 61 found in > 3 sorts, and 2 found in 4 sorts. Validation is underway using PCR arrays to confirm expression in IFN‐g‐activated primary human macrophages challenged with FT. This approach may prove useful to identify mechanisms in macrophage innate immunity against intracellular pathogens. Supported by DTRA grant (Defense Threat Reduction Agency)