N-acetylcysteine (NAC) attenuates quorum sensing regulated phenotypes in Pseudomonas aeruginosa PAO1

Emília Maria França Lima; Felipe Alves de Almeida; Marcelo Palma Sircili; Vanessa Bueris; Uelinton Manoel Pinto

doi:10.1016/j.heliyon.2023.e14152

N-acetylcysteine (NAC) attenuates quorum sensing regulated phenotypes in Pseudomonas aeruginosa PAO1

Heliyon. 2023 Feb 28;9(3):e14152. doi: 10.1016/j.heliyon.2023.e14152. eCollection 2023 Mar.

Authors

Emília Maria França Lima¹, Felipe Alves de Almeida², Marcelo Palma Sircili³, Vanessa Bueris⁴, Uelinton Manoel Pinto¹

Affiliations

¹ Food Research Center, Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of Sao Paulo (USP), São Paulo, SP, Brazil.
² Instituto de Laticínios Cândido Tostes (ILCT), Empresa de Pesquisa Agropecuária de Minas Gerais (EPAMIG), Juiz de Fora, MG, Brazil.
³ Genetics Laboratory, Butantan Institute, São Paulo, SP, Brazil.
⁴ Microbiology Department, Institute of Biomedical Science, University of São Paulo (USP), São Paulo, SP, Brazil.

Abstract

The expression of many virulence genes in bacteria is regulated by quorum sensing (QS), and the inhibition of this mechanism has been intensely investigated. N-acetylcysteine (NAC) has good antibacterial activity and is able to interfere with biofilm-related respiratory infections, but little is known whether this compound has an effect on bacterial QS communication. This work aimed to evaluate the potential of NAC as a QS inhibitor (QSI) in Pseudomonas aeruginosa PAO1 through in silico and in vitro analyses, as well as in combination with the antibiotic tobramycin. Initially, a molecular docking analysis was performed between the QS regulatory proteins, LasR and RhlR, of P. aeruginosa with NAC, 3-oxo-C12-HSL, C4-HSL, and furanone C30. The NAC sub-inhibitory concentration was determined by growth curves. Then, we performed in vitro tests using the QS reporter strains P. aeruginosa lasB-gfp and rhlA-gfp, as well as the expression of QS-related phenotypes. Finally, the synergistic effect of NAC with the antibiotic tobramycin was calculated by fractional inhibitory concentrations index (FIC_i) and investigated against bacterial growth, pigment production, and biofilm formation. In the molecular docking study, NAC bound to LasR and RhlR proteins in a similar manner to the AHL cognate, suggesting that it may be able to bind to QS receptor proteins in vivo. In the biosensor assay, the GFP signal was turned down in the presence of NAC at 1000, 500, 250, and 125 μM for lasB-gfp and rhlA-gfp (p < 0.05), suggesting a QS inhibitory effect. Pyocyanin and rhamnolipids decreased (p < 0.05) up to 34 and 37%, respectively, in the presence of NAC at 125 μM. Swarming and swimming motilities were inhibited (p < 0.05) by NAC at 250 to 10000 μM. Additionally, 2500 and 10000 μM of NAC reduced biofilm formation. NAC-tobramycin combination showed synergistic effect with FIC_i of 0.8, and the best combination was 2500-1.07 μM, inhibiting biofilm formation up to 60%, besides reducing pyocyanin and pyoverdine production. Confocal microscopy images revealed a stronger, dense, and compact biofilm of P. aeruginosa PAO1 control, while the biofilm treated with NAC-tobramycin became thinner and more dispersed. Overall, NAC at low concentrations showed promising anti-QS properties against P. aeruginosa PAO1, adding to its already known effect as an antibacterial and antibiofilm agent.

Keywords: GFP expression; Molecular docking; Pyocyanin; Rhamnolipids; Synergism; Tobramycin.