EST‐based gene silencing for identification of macrophage resistance mechanisms against Francisella tularensis

Abstract

Francisella tularensis (FT) is a gram‐negative, highly infectious, facultative intracellular pathogen that causes tularemia, and is a potential bioterrorism agent. Macrophages represent the main site of initial FT replication but the mechanism of survival and bacteriostasis upon activation remains poorly characterized. We used genome‐wide genetic screen to identify host genes responsible for survival of infection. We used macrophages (RAW 264.7) transduced with an EST‐lentiviral "knock‐down" library targeting 40,000 sequences. The macrophages were infected with attenuated Live Vaccine Strain (LVS) FT expressing GFP. We screened for EST interference resulting in a phenotype that allows survival of infection and intracellular bacterial proliferation. Flow cytometric sorting of cells with highest FT load (i.e. top 1% by green fluorescence) was performed. The EST inserts from sorted cells were amplified from genomic DNA by PCR, and corresponding EST inserts were identified by cloning and DNA sequencing. Initial screening has analyzed 1400 clones from 6 different sorts and has identified 7 genes of interest based on repeated detection. Validation of potential targets is underway to confirm gene expression and to assess effects of directed lentiviral knockdown of candidate genes. This approach may prove useful to identify mechanisms in macrophage innate immunity against intracellular pathogens.