PROJECT SUMMARY / ABSTRACT Intraocular bacterial infections (endophthalmitis) cause a significant number of cases of blindness worldwide. Efforts to prevent damage to delicate ocular tissues during infection rely on swift and proper use of therapeutics to rapidly kill organisms and arrest potentially damaging inflammation. Currently-used antibiotics can kill organisms, but the effectiveness of anti-inflammatory drugs is controversial. No current therapies neutralize toxins which damage nonregenerative tissues in the eye. Our overarching goal is to develop more effective therapeutics which target and inhibit these events, preserving vision. Bacillus cereus (Bc) causes one of the most inflammatory and rapidly blinding forms of endophthalmitis. This common environmental organism enters the eye during trauma or bloodstream infection, and is also a reported surgical contaminant. Eyes infected with Bc can be rendered sightless in a short period of time, so effective treatment is critical to saving vision. This proposal focuses on mechanisms related to the organism and its behavior in the eye, and highlights events during the infection course prior to significant retinal damage and inflammation—a time when therapeutic intervention would be most valuable. We propose four aims that address Bc pathogenesis during important early events in endophthalmitis: Aim 1 will analyze elements of the intraocular environment that trigger germination of Bc spores. The mechanisms involved in transformation from spore to vegetative state in the eye will be examined. Aim 2 will further explore innate immune pathway recognition of potential Bc agonists. For TLR2, we will address the role of peptidoglycan deacetylation and teichoic acid as agonists. For TLR4, we will address the role of cereolysin and heat-labile surface components as agonists. Aim 3 will examine whether intraocular inflammation prompts migration of Bc throughout the eye. RNASeq data suggest that flagellar and motility systems of Bc are highly expressed in vivo. Aim 4 will test the hypothesis that during inflammation, products secreted by Bc disarm infiltrating neutrophils. Candidates include superoxide dismutase, immune inhibitor A, and cereolysin, and mutants deficient in each will be tested for potential anti-neutrophil activity. These independent but related aims are a logical extension of our ongoing research into the pathogenic mechanisms underlying bacterial endophthalmitis. For Bc endophthalmitis patients, ineffective treatment often results in significant vision loss or loss of the globe itself. Therefore, identifying factors and pathways important to early disease events disease is critical to developing novel and effective therapeutic options for this blinding infection.