From farm shop to the Pharmacy. Using the novel organism Dictyostelium discoideum to elucidate the antimicrobial action of garlic-derived ajoene.

Patients with cystic fibrosis often experience chronic bacterial infections, contributing to increased
morbidity and mortality. In these cases, colonies of bacteria produce a protective layer known as a
biofilm which helps to evade the host immune system and cause antibiotic resistance. One potential
treatment for bacterial infection in these patients is ajoene, a garlic-derived compound, which has
been demonstrated to be effective in clinical trials and has been shown to disrupt bacterial biofilm
formation through inhibition of the cell-to-cell communication process known as quorum sensing. This
project aims to identify potential mechanisms and targets of ajoene using the model organism
Dictyostelium discoideum. We showed that Dictyostelium growth is sensitive to ajoene (concentration
for 50% inhibition (IC50) =8 μM) at a physiologically relevant concentration. We then identified ajoene
resistant mutants and characterised the proteins lost in these mutants in order to understand the
cellular effect of ajoene using both chronic treatment and high throughput sequencing driven
screening methods. One selected mutant was the induced after Legionella infection G (iliG) gene;
however, independent ablation of this gene displayed no change in phenotypic response to ajoene
treatment. Other candidate proteins were assessed using in silico ligand docking modelling to
prioritise candidates for future experimentation. The quorum sensing protein GacS was identified as
a probable target through literature review and the Dictyostelium histidine kinase protein J (DhKJ) was
identified as the closest related Dictyostelium protein. Ablation of DhKJ reduced ajoene sensitivity
with the IC50 increased from 5.9 μM to 10.4 μM (p < 0.001). Introducing a truncated DhKJ gene to the
DhKJ null cell line restored ajoene sensitivity, reducing the IC50 from 10.4 μM to 5.7 μM (p < 0.001). To
further validate the potential targets DhKJ and GacS, ligand-based virtual screening was used to
identify compounds that could potentially interact with DhKJ and GacS. After independent docking
analysis, 23 candidates were identified and 21 compounds were screened against wildtype cells, DhKJ
null and DhKJ rescue cell lines. Twelve compounds were identified to affect cellular proliferation in a
way that suggests interaction with DhKJ, and therefore are hypothesised to inhibit biofilm formation
in Pseudomonas aeruginosa through interaction with the quorum sensing inhibitor GacS. Thus, our
research strongly suggests that ajoene inhibits biofilm through direct interaction with P. aeruginosa
quorum sensing receptor GacS, inhibiting quorum sensing.