Discussion and Future Work Targeted Therapy for the Future: the Use of Novel Antimicrobial Peptides against P. aeurginosa Matthew M. Froid, Trevor Pentzien, Donald Rowen Ph.D. Department of Biology, University of Nebraska at Omaha, Omaha, NE 68182 Abstract Methods and Results Mutants were generated using transposon mutagenesis of the PAO1 strain. Inverse PCR was optimized, and after several trials, products were obtained. Sequencing of the resulting PCR products revealed the transposon was inserted into the mexT gene. (Fig. 3) Mutations in mexT have been observed to affect P. aerugionosa sensitivity of cells to other antimicrobial agents. https://www.ncbi.nlm.nih.gov/pubmed/27845 Pre-constructed plasmids were used to generate two triparental conjugate strains. Two triparental conjugates strains were generated. Constructs were selectively grown in tetracycline. An MIC was performed to determine if wild type sensitivity had been restored. (Fig. 5) Discussion and Future Work Pathogenic bacteria, such as the gram-negative bacterium Pseudomonas aeruginosa, are becoming resistant to our current arsenal of antibiotics at an alarming rate. P. aeruginosa is a leading cause of nosocomial acquired infections and is a primary co-morbidity in patients with compromised immune systems. One potential source of new antibiotic agents is antimicrobial peptides. Antimicrobial peptides (AMPs) are small proteins, and some have shown a high degree of efficacy and broad-spectrum activity against both Gram-positive and Gram-negative bacteria. An experimental AMP that has been developed by Dr. Wang at UNMC, DASamp2, has shown to be effective against virulent bacteria, including P. aeruginosa. To help assess the usefulness of DASamp2, members of the Rowen lab have isolated a mutant strain of P. aeruginosa with 8-fold resistance (RMB1) to the compound. This previous work established that the gene mexT might play a role in the increased resistance to DASamp2 observed in the RMB1 strain. We believe this gene is responsible for encoding a transcription activator that affects the expression of the MexEF-OprN efflux pump --a cellular structure that actively moves compounds out of a cell-- and that the target of DASamp2 must lay beyond the cell wall of P. aeruginosa. The goal of this project was to confirm that mexT was responsible for the increased resistance by restoring the wild type sensitivity to the experimental compound in the mutant strains. The results suggest: Expression levels of mexEF-oprN operon affects efflux pump activity of P. aeruginosa cells. The activity of efflux pumps will affect the sensitivity of bacteria to DASamp2. Future work: Confirm that a mutation in mexT is solely responsible for the increased resistance observed in RMB, optimize MIC, and perform qRT-PCR. * Fig. 4 – Triparental Conjugation (Image courtesy of Hirokazu Yano) Fig. 3 – Transposon insertion in the P. aeurginosa genome Fig. 6 –Graphical plot of results for the DASamp2 minimum inhibitory concentration. Introduction DASamp2 is a novel antimicrobial peptide developed by Dr. Wang at UNMC. To learn about its mechanism of action, mutants of P. aeruginosa cells with altered susceptibility were previously isolated. The mutant RMB1 showed a resistant phenotype to DASamp2 due to transposon mutagenesis. The mutation was identified using inverse PCR and sequencing. This project sought to confirm previously found results by restoring wild type sensitivity to the mutant strain RMB1. Acknowledgements Fig. 5 – MIC schematic with 4.0 µM DASamp2 We would like to thank the Poole lab at Queens University, Ontario and Dr. Wang at UNMC for their gracious contributions, as well as Zachary Scott, and Christopher Johnson for their willingness to provide technical assistance. We would also like to thank the UNO FUSE program for support and funding. References 1. Defezab, P. F., Fabbro-Perayab, N. B., Bouzigesc, A., et al. (2004). Risk factors for multidrug-resistant Pseudomonas aeruginosa nosocomial infection.Journal of Hospital Infection 2. McCarthy, K. L., Paterson, D. L. (2016). Long-term mortality following Pseudomonas aeruginosa bloodstream infection.Journal of Hospital Infections. 95(3): 292-299. 3. World Health Organization (WHO). (2017). Global Priority List of Antibiotic-Resistant Bacteria to Guide Research, Discovery, and Development of New Antibiotics. 4. Mishra, B.,&Wang, G. (2017). Individual and Combined Effects of Engineered Peptides and Antibiotics on Pseudomonas aeruginosa Biofilms.Pharmaceuticals.10(3): 58. 5. Fetar, H., Gilmour, C., Klinoski, R., Daigle, D. M., Dean, C. R., Poole, K., 2011.  MexEF-oprN Multidrug Efflux Operon of Pseudomonas aeruginosa: Regulation by the MexT Activator in Response to Nitrosative Stress and Chloramphenicol. Antimicrobial Agents and Chemotherapy, 55 (2) p. 508–5146. 6. Köhler, T., Epp, S. F., Curty, L. K.,&Pechère, J.-C. (1999). Characterization of MexT, the Regulator of the MexE-MexF-OprN Multidrug Efflux System of Pseudomonas aeruginosa.Journal of Bacteriology,181(20), 6300–6305. 7. Lister, P. D., Wolter, J. D., Hanson, N. D., Antibacterial-Resistant Pseudomonas aeruginosa: Clinical Impact and Complex Regulation of Chromosomally Encoded Resistance Mechanisms, Clinical Microbiology Reviews 2009 vol. 22 no. 4 582-6101 Fig. 1 In depth view of mexT’s role in the mexEF-oprN operon. (7) Fig. 2 Upregulation of an efflux pump can increased resistance to antibiotics. (7)