Determination of Pseudomonas aeruginosa quorum sensing inhibitor lichen secondary metabolites and inhibition mechanisms

Opportunistic pathogen Pseudomonas aeruginosa regulates its pathogenicity through communication via small and diffusible molecules, that is called "quorum sensing" (QS). Inhibition of QS systems without killing the bacteria and thus leaving them ineffective and vulnerable is called "quorum quenching" (QQ), which is considered an important method for anti-virulence approach. Natural resources are used in search for new QS inhibitor metabolites. Lichens have high potential in this context with their unique and broad range of secondary metabolites. The aim of this study is to determine the QQ effects of extracts from lichen species in our country on P. aeruginosa, to analyze secondary metabolite contents of the effective species, to examine the effects of dominant secondary metabolites on QS-related virulence factors and to investigate the changes in P. aeruginosa transcriptome. To this end, 34 samples belonging to 17 lichen species from different regions were collected and the effective samples that may contain QS and biofilm inhibitors were determined by reference and GFP-based biomonitor strains. The metabolite ingredients of the effective extracts were analyzed via chromatographic methods. Inhibitions of selected pure metabolites of atranorine and psoromic acid against P. aeruginosa QS, biofilm and virulence factors were quantitatively determined using monitor strains, biochemical analyses and qRT-PCR methods. Both metabolites performed high virulence inhibition for the applied concentrations. In addition, the effects on viability in human fibroblast cell line were investigated and it was found that atranorine reduced viability by 50%. All changes in P. aeruginosa transcriptome treated with psoromic acid were analyzed by next-generation RNA sequencing. According to the results, a total of 533 genes, including QS related genes, were down-regulated and 664 genes were upregulated. The results obtained suggest that lichens can be an important source for QS inhibition and that the psoromic acid metabolite can be used against P. aeruginosa for this purpose.