Bacillus anthracis is a ubiquitous soil bacterium and NIAID category A pathogen that causes anthrax. Although research has revealed important insights into this deadly bacterium (e.g., the activity of the tripartite toxins), the pathogenesis of anthrax has yet to be satisfactorily explained and there is much we do not understand. Chromosomal factors important for pathogenicity have been understudied, and we know very little about how the innate immune system acts to protect against anthrax infection. We have demonstrated that B. anthracis can produce a lethal infection in the soil nematode and model invertebrate C. elegans. This discovery opens up the possibility to study anthrax infection using forward and reverse genetics on an unprecedented scale. B. anthracis can infect and proliferate inside the nematode, eventually devouring it from the inside out. C. elegans, however, has a potent innate immune system that shares many pathways and effector mechanisms with that of mammals. The nematode appears capable in some cases of mounting an effective immune response against B. anthracis. This proposal will exploit this novel C. elegans-B. anthracis system to identify, on an unprecedented scale, (i) host genes involved in innate defense against B. anthracis infection and (ii) B. anthracis genes involved in establishing infection and circumventing innate defenses in an intact animal. Affymetrix microarrays will be used to identify potential host immunity genes up-regulated in response to B. anthracis infection. These genes will be systematically knocked down in C. elegans using RNAi. Treated animals will be screened for hypersensitivity to B. anthracis infection in order to identify novel genes involved in innate defense. A large-scale genetic screen for C. elegans mutants hypersensitive to B. anthracis infection will also be performed. On the pathogen side of the equation, random transposon and/or plasmid integrational mutant libraries of B. anthracis will be generated and screened for virulence attenuation in a high-throughput C. elegans infection assay. Avirulent mutants will be screened for growth within murine macrophages as a preliminary assessment of potential reduced pathogenicity in mammals. Results from this R21 study will generate novel reagents and hypotheses to be explored in a future R-01 or P-01 proposal on B. anthracis virulence mechanisms and relevant host innate defenses, including direct extension into mammalian model systems. [unreadable] [unreadable] [unreadable] [unreadable]