# Determining the mechanism of cell death of small molecules produced by Pseudomonas aeruginosa against Acanthamoeba castellanii

> **NIH NIH F31** · YALE UNIVERSITY · 2024 · $48,974

## Abstract

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.

## Key facts

- **NIH application ID:** 10826918
- **Project number:** 1F31AI181569-01
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Rebecca Isabel Colón Ríos
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $48,974
- **Award type:** 1
- **Project period:** 2024-08-01 → 2027-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10826918

## Citation

> US National Institutes of Health, RePORTER application 10826918, Determining the mechanism of cell death of small molecules produced by Pseudomonas aeruginosa against Acanthamoeba castellanii (1F31AI181569-01). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10826918. Licensed CC0.

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