SJSU Undergraduate Research Grants

Identification of the Target of an

Antipyocyanin Compound in P. aeruginosaThu Nguyen, Laura Miller Conrad

Department of Chemistry, San Jose State University

Citations

Abstract

Synthesis SchemeEnrichable PAL Approach

Project Activities

Research Questions Acknowledgement

Antibiotic resistance is an ever worsening problem. According to the Center for Disease Control, there were at least 2 million illnesses caused by antibiotic resistant bacteria, resulting in at least 23,000 deaths in the U.S. per year.(1) Pseudomonas aeruginosa is an opportunistic pathogen, meaning it infrequently causes infection in healthy individuals but is acquired by immune-compromised hospitalized patients.(2) While P. aeruginosa has evolved to become more resistant to antibiotics, an antivirulence approach is a new strategy developed to be an effective and long lasting treatment option. Pyocyanin is a key virulence factor important to P. aeruginosa. Dr. Miller Conrad has successfully synthesized an inhibitor for pyocyanin production, however, its molecular target has not been identified.(3) The purpose of my project is to develop an antipyocyanin analog with a photoaffinity ligand and a terminal alkyne. The photoaffinity label on the ligand has the ability to make covalent bond with the target protein and the terminal alkyne will allow us to attach biotin to the target protein/inhibitor complex. The target protein can then be isolated on a streptavidin column. The protein can be characterized and identified by mass spectrometry and proteomic analysis.

1. Antibiotic Resistance Threats in the United States, 2013(Centers for Disease Control and Prevention).

2. Fujitani, S.; Moffett, K. S.; Yu, V. L. Pseudomonas aeruginosa. 2015, Antimicrobe.com.

3. Miller, L. C.; O’Loughlin, C. T.; Zhang, Z.; Siryaporn, A.; Silpe, J. E.; Bassler, B. L.; Semmelhack, M. F. J. Med. Chem. 2015, 58 (3), 1298–1306.

4. O’Loughlin, C. T.; Miller, L. C.; Siryaporn, A.; Drescher, K.; Semmelhack, M. F.; Bassler, B. L. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 17981–17986.

After studying the antipyocyanin compound’s structure and its relationship to its antipyocyanin activity, (4) the model compound was designed to incorporate the terminal alkyne handle. Based on previous studies, the incorporation of the diazirine (PAL) does not affect activity greatly. Therefore, this compound will be tested in vivo to determine if the alkyne has any effect on the compound’s antipyocyanin activity. If the antipyocyanin activity remains the same as previously tested antipyocyanin compound, we can proceed to synthesize an enrichable PAL compound, then perform proteomics analysis to identify its target protein, and finally perform mass spectroscopy to verify the target protein.

Special thanks to:- Dr. Laura Miller Conrad- The Miller Conrad Research Group- San Jose State University- SJSU Undergraduate Research Grant Program

1. Will attaching a terminal alkyne to our potent antipyocyanin compound affect its antipyocyanin activity?

2. Is blocking pyocyanin production enough to disable pathogenicity in P. aeruginosa, allowing the host’s immune system to clear the bacteria?

3. How is pyocyanin produced and regulated in P. aeruginosa?