What is Pseudomonas aeruginosa?

Pseudomonas aeruginosa (PA) is a Gram-negative, opportunistic pathogen that typically causes illness in individuals with weakened immune systems or compromised defenses. The extreme adaptability to harsh environments, resistance to antibiotics, and wide range of defenses make P. aeruginosa one of the most difficult organisms to treat in modern medicine. [1]

Multi-drug-resistant (MDR) Pseudomonas aeruginosa strains are resistant to at least one antibiotic in three or more different classes of antibiotics and are associated with approximately 559,000 deaths globally each year. [2]

Our Question: How can we effectively disarm Pseudomonas aeruginosa and improve treatment outcomes for high-risk patient populations?

Given the wide range of solutions that Pseudomonas aeruginosa has to current treatment options, we aimed to design a solution to enhance current treatment options and reduce the ability of PA to harm high-risk patient groups in healthcare settings.

Our primary goal is to reduce the impact of P. aeruginosa on immunocompromised patients and alleviate the burden of AMR strains on hospital settings.

For this year’s project, we decided to gravitate towards PA’s mechanisms for attacking host cells, one of which is the release of extracellular toxins. Virulence factors, including extracellular toxins, allow PA to adhere to tissue surfaces, spread damaged tissues within the host, and improve the bacteria’s chances of survival by securing nutrients. [3]

Exotoxin A (ExoA) is the most toxic virulence factor of Pseudomonas aeruginosa [4] and causes disease by inhibition of protein synthesis, direct cytopathic effects, and interference with cellular immune functions of the host [5].

Our Solution: ExAway

ExAway: A novel protein inhibitor that blocks ExoA from entering human cells.

Through the use of deep learning-based tools such as RFdiffusion, ProteinMPNN, and AlphaFold, we aimed to develop a protein inhibitor that binds to the N-terminal receptor-binding domain of ExoA, thereby preventing ExoA from binding to host cell receptors.

Using the designs generated, our Wet Lab team conducted protein purification of ExoA domains and fragments, followed by binding studies to evaluate the affinity between ExoA and candidate inhibitors. This will reduce the toxicity of ExoA and increase patients’ ability to develop an immune response against PA, providing an alternative approach to more potent antibiotics.

Our Plans

• Design an inhibitor that binds to ExoA with high specificity and structural stability, minimizing potential off-target effects.
• Experimentally validate the modeling team’s mini-binders through assays such as western blots and cytotoxicity tests, involving DNA ordering, protein expression, and purification.
• Translate literature and experimental data into readable and contextualized modeling.
• Integrate our protein inhibitor as an adjunctive therapy into existing healthcare strategies to improve the treatment of Pseudomonas aeruginosa infections and healthcare-associated infections in high-risk patients, such as those with bronchiectasis and cystic fibrosis.

References

• [1] MaciáM. D., D. Blanquer, Bernat Togores, Jaume Sauleda, PérezJ. L., and A. Oliver, “Hypermutation Is a Key Factor in Development of Multiple-Antimicrobial Resistance in Pseudomonas aeruginosaStrains Causing Chronic Lung Infections,” Antimicrobial Agents and Chemotherapy, vol. 49, no. 8, pp. 3382–3386, Jul. 2005, doi: https://doi.org/10.1128/aac.49.8.3382-3386.2005.
• [2] M. G. Wilson and S. Pandey, “Pseudomonas aeruginosa,” Nih.gov, Aug. 08, 2023. https://www.ncbi.nlm.nih.gov/books/NBK557831/ (accessed Sep. 26, 2025).
• [3] G. Pierre, “GARDP,” GARDP, Nov. 14, 2024. https://gardp.org/stories/meet-pseudomonas-aeruginosa/ (accessed Sep. 26, 2025).
• [4] “Pseudomonas Infection: Causes, Symptoms & Treatment,” Cleveland Clinic, Aug. 14, 2023. https://my.clevelandclinic.org/health/diseases/25164-pseudomonas-infection (accessed Sep. 26, 2025).
• [5] Shahab, “Pseudomonas aeruginosa Infections: Practice Essentials, Background, Pathophysiology,” Medscape.com, Apr. 14, 2025. https://emedicine.medscape.com/article/226748-overview#a6 (accessed Sep. 26, 2025).