Investigation of Natural Products as Novel Antimicrobial Agents Against the Pseudomonas aeruginosa Type III Secretion System

As antibiotic resistance among bacterial pathogens continues to escalate, posing an immense threat to global health, the discovery and development of alternative antimicrobial therapies has become critical. The type III secretion system (T3SS) is a virulence factor used by dozens of Gram-negative bacterial pathogens to cause disease. The T3SS acts as a needle-like apparatus to modulate host defenses through the injection of effector proteins into target host cells. This extracellular appendage is an attractive target for new antimicrobial therapies due to its high conservation among pathogenic bacteria such as Salmonella, Chlamydia, and Pseudomonas, its widespread absence in commensal flora, and its critical role in causing disease. In a campaign to discover molecules that inhibit the T3SS, we have screened four unique and diverse natural product-derived libraries for their inhibitory bioactivity against a strain of P. aeruginosa harboring a T3SS promoter-controlled luciferase. One marine bacterial extract was shown to inhibit T3SS-driven bioluminescence and, importantly, did not act as a general luciferase inhibitor, affect bacterial viability, or perturb mammalian cell viability. The purified bioactive compound, identified as sebastenoic acid, inhibited T3SS effector protein secretion in a dose-dependent manner. Interestingly, sebastenoic acid was previously found to affect motility and biofilm formation in Vibrio cholera, both of which are linked to cyclic-di-GMP. In Pseudomonas, synthesis of the T3SS global regulator, ExsA, is inhibited by cyclic di-GMP through cAMP-Vfr signaling, suggesting that sebastenoic may play a role in modulating cyclic-di-GMP levels. In addition to sebastenoic acid, we also identified marine fungal-derived penicillic acid as a T3SS inhibitor. Penicillic acid inhibited T3SS effector secretion in a dose dependent manner without affecting bacterial viability, however, its known toxicity to mammalian cells complicates its further development as a potential therapeutic. Notably, penicillic acid was previously shown to be bioactive against both biofilm alginate biosynthesis and quorum sensing, two processes which are typically anti-regulated with the T3SS. Lastly, we identified an additional six bioactive extracts from Burkholderia-derived natural products and five bioactive extracts from marine-derived natural products for future small molecule inhibitor discovery