PROJECT SUMMARY Acanthamoeba castellanii is a free-living amoeba (FLA) that causes usually fatal central nervous system infections like granulomatous amebic encephalitis. FLAs like A. castellanii are bacterivorous and co-exist with Pseudomonas aeruginosa. P. aeruginosa are ubiquitous environmental Gram-negative bacteria. Given that A. castellanii creates an evolutionary pressure for P. aeruginosa to defend itself against amoebal grazing, we hypothesized that P. aeruginosa may produce one or more secreted amoebicidal compounds that can be harnessed to treat amoeba infections in humans. We have since demonstrated that the cell-free supernatants of P. aeruginosa strain PA14 are lethal to A. castellanii trophozoites. Our preliminary data has established that trophocidal activity resides in a <3 kDa (“small molecule”) fraction of the cell-free supernatant, which is not toxic to mammalian cells. Importantly, the small molecule fraction prepared from ᐃrhlRᐃrhlI mutant bacteria (PA14ᐃrhlRᐃrhlI), which do not secrete rhamnolipids, retains trophocidal activity. Using a bioactivity guided fractionation pipeline developed by the Crawford lab, I have begun to narrow down fractions which retain activity. I will determine the identity and structure of small molecules present in these active fractions through mass spectrometry and nuclear magnetic resonance spectroscopy. The isolated compounds will be tested for efficacy against FLAs and toxicity against human cells. The PA14ᐃrhlRᐃrhlI small molecule fraction causes rapid A. castellanii cell rounding and detachment (within 40 minutes) and complete amoeba death between 8 and 10 hours. In the second aim of this proposal, I will determine the mechanism of cell death of small molecules produced by P. aeruginosa by assessing essential cellular processes such as cell membrane permeability, changes in mitochondrial activity, and DNA damage. My long-term goal is to understand the key chemical weapons that P. aeruginosa is secreting to kill A. castellanii trophozoites. Knowledge of these natural compounds and their mechanism of action will help us develop novel therapeutic agents for devastating human infections by A. castellanii and other free-living amoebae. This project encompasses a broad interdisciplinary training in not only microbiology and chemistry, but also molecular biology. My training plan entails immersing myself in two distinct but intertwined fields, rigorous integrative coursework, multiple opportunities for mentoring and developing scientific communication skills. This training plan will provide me with a supportive, collaborative, and gratifying environment that will allow me to develop and succeed as an emerging female Latina scientist in field.