Nitric Oxide Donor Spermine-NONOate Elicits Endogenous Dispersal-Associated Transcriptional Responses to Promote Biofilm Dispersal in Pseudomonas aeruginosa

Background/Objectives: Bacterial biofilms are structured communities of sessile cells embedded in a self-produced extracellular matrix that protects against environmental stress, host immune responses and antimicrobial treatments. In response to specific cues, biofilm cells can revert to a planktonic free-swimming lifestyle through a process termed biofilm dispersal. When dispersed cells escape the biofilm matrix, they lose bio-film-associated antibiotic tolerance, a major barrier to treating medical biofilms. As such, dispersal-inducing compounds like nitric oxide (NO) are actively investigated as adjuvants to potentiate the biofilm eradicating activity of existing antibiotics. We recently characterised the transcriptomic responses elicited during spontaneous biofilm dispersal in closed culture-grown Pseudomonas aeruginosa biofilms. Here, we evaluated the tran-scriptional profile of P. aeruginosa biofilms treated with the NO donor Spermine-NONOate (SP-NONO) and the nitroxide C-TEMPO, an NO analogue to determine potential pathways involved in NO-mediated dispersal. Methods: Dispersal activity on P. aeruginosa PAO1 biofilms by SP-NONOate and C-TEMPO was quantified by crystal violet staining. Cellular responses to each compound were profiled by RNA-seq on treated and untreated cells. Results: While both compounds disrupted the transcription of ANR-regulated energy metabolism pathways, only SP-NONO activated canonical NO-regulated responses. Considering that only SP-NONO showed biofilm dispersal activity in this culture system, we investigated shared transcriptional shifts in SP-NONO-treated and spontaneously dispersed biofilms to identify pathways likely involved in central dispersal responses. These mostly included genes participating in the catabolism of leucine, valine, isoleucine and lysine, as well as 9 of 14 genes previously defined as transcriptional biomarkers of spontaneous biofilm dispersal. Conclusions: This study suggests that NO disrupts biofilm maturation by prematurely stimulating central pathways of spontaneous biofilm dispersal and highlights this set of biomarkers as robust indicators of dispersal responses.