N-acetylcysteine inhibits multiple signalling, regulatory, and virulence pathways/genes in Pseudomonas aeruginosa wound isolates

Pseudomonas aeruginosa is responsible for nosocomial and foodborne infections, and the WHO has graded P. aeruginosa as a high-priority pathogen and prioritizes developing new antimicrobial agents. In this study, through comprehensive RNA-Seq analysis and quorum-sensing reporter strains, for the first time, it has been identified that N-acetylcysteine (NAC) differentially expresses over 1000 genes in P. aeruginosa PAO1 and diabetic foot ulcer isolate DFU-53, and initiates inhibition of all three QS systems (Las, Rhl, and PQS) in P. aeruginosa . RNA-Seq pathway analysis revealed downregulation of critical genes, including the two-component regulatory system and various transcriptional regulators. Subsequently, resulted in downregulation of numerous extracellular toxin genes, including phenazine operons ( phzA1-G/phzA2-G2 ), peptides/enzymes such as elastase ( lasB ), rhamnolipids ( rhlABC ), hydrogen cyanide ( hcn ), biofilm formation genes ( wsp ), siderophore ( pvd and pch ), type III and VI secretion system, and lectin ( lec ) that are responsible for infecting host cells. NAC, at its intrinsically low pH, showed efficacy in disrupting existing biofilms. Experimental evidence further demonstrated that P. aeruginosa (GFP-tagged) grown in the presence of NAC remained ineffective to human fibroblast cells; in contrast, in the absence of NAC, P. aeruginosa PAO1 colonizes on the surface and removes pre-confluence fibroblast cells. These findings open new avenues for the development of a next-generation antibacterial strategy to tackle the challenge of multidrug-resistant infections.