The antimicrobial activity and cellular pathways targeted by p-anisaldehyde and epigallocatechin gallate in the opportunistic human pathogen Pseudomonas aeruginosa.

Plant-derived aldehydes are constituents of essential oils that possess broad-spectrum antimicrobial activity and kill microorganisms without promoting resistance. In our previous study, we incorporated p-anisaldehyde from star anise into a polymer network called PANDAs (Pro-Antimicrobial Networks via Degradable Acetals) and used it as a novel drug delivery platform. PANDAs released p-anisaldehyde upon a change in pH and humidity, and controlled growth of the multi-drug resistant pathogen Pseudomonas aeruginosa PAO1. In this study, we identified cellular pathways targeted by p-anisaldehyde, by generating 10,000 transposon mutants of PAO1 and screened them for hypersensitivity to p-anisaldehyde. To improve the antimicrobial efficacy of p-anisaldehyde, we combined it with epigallocatechin gallate (EGCG), a polyphenol from green tea, and demonstrated that it acts synergistically with p-anisaldehyde in killing P. aeruginosa. We then used RNA-seq to profile transcriptomic responses of P. aeruginosa to p-anisaldehyde, EGCG, and their combination. The exposure to p-anisaldehyde altered the expression of genes involved in the modification of cell envelope, membrane transport, drug efflux, energy metabolism, molybdenum cofactor biosynthesis, and stress response. We also demonstrated that the addition of EGCG reversed many p-anisaldehyde-coping effects and induced oxidative stress. Our results provide an insight into the antimicrobial activity of p-anisaldehyde and its interactions with EGCG and may aid in the rational identification of new synergistically-acting combinations of plant metabolites. Our study also confirms the utility of the thiol-ene polymer platform for the sustained and effective delivery of hydrophobic and volatile antimicrobial compounds. IMPORTANCE Essential oils (EOs) are plant-derived products that have been long exploited for their antimicrobial activities in medicine, agriculture, and food preservation. EOs represent a promising alternative to conventional antibiotics due to the broad-range antimicrobial activity, low toxicity to human commensal bacteria, and the capacity to kill microorganisms without promoting resistance. Despite the progress in the understanding of the biological activity of EOs, many aspects of their mode of action remain inconclusive. The overarching aim of this work was to address these gaps by studying molecular interactions between an antimicrobial plant aldehyde and the opportunistic human pathogen Pseudomonas aeruginosa. Results of this study identified microbial genes and associated pathways involved in response to antimicrobial phytoaldehydes and provided insights into molecular mechanisms governing the synergistic effects of individual constituents within essential oils.