An emerging hallmark of bacterial QS activation is the alteration of metabolic states. Intervening in cellular metabolism, which regulates QS activation, emerges as a promising strategy to mitigate the production of QS-controlled virulence factors. In this study, we found fatty acid biosynthesis had the highest fold enrichment among all metabolic pathways regulated by PA1895-1897 operon, the QS anti-activator in P. aeruginosa. Exogenous administration of palmitoleic acid and acetic acid, which were the two major fatty acids regulated by PA1895-1897 operon, suppressed QS activation in PA1895-1897-knockout mutant. Inhibition of fatty acid biosynthesis promoted QS activation in PA1895-1897-overexpressed culture. These results indicate that promoted fatty acid synthesis is involved in the delayed QS activation by PA1895-1897 operon, which provides new insights into the bacterial QS regulation.

Palmitoleic acid and acetic acid were fatty acids that were significantly enriched by PA1895-1897 operon. Palmitoleic acid, a cis-monounsaturated n-7 fatty acid containing 16 carbon atoms, is classified as an omega-7 fatty acid, whereas acetic acid, comprising two carbon atoms, is categorized as a short-chain fatty acid within the group of volatile fatty acids (Viso and Marty 1993; He et al. 2020). Both fatty acids can be found in a wide variety of bacterial organisms, where the synthesis of these fatty acids requires various enzymatic reactions. In P. aeruginosa, the mechanism by which PA1895-1897 operon promotes the synthesis of these fatty acids remains to be clarified. In the Pfam database, the proteins of PA1895 and PA1897 have been predicted to be fatty acid desaturase and fatty acid hydroxylase, respectively (Finn et al. 2016), thus it’s probable that the gene products of PA1895-1897 had direct or indirect enzymatic effects on the synthesis of these fatty acids. Further study is needed to clarify the process. Since we only conducted differential metabolomics analysis in this study, the exact amount of palmitoleic acid and acetic acid in P. aeruginosa needs to be clarified in further studies.

Fatty acids have shown anti-QS activity in many bacterial species (Kumar et al. 2020). For example, both palmitoleic acid and myristoleic acids prevent A. baumannii motility and biofilm formation at sub-inhibitory concentrations (Nicol et al. 2018). In V. cholera, fatty acids reduced the QS-controlled virulence to various degrees, according to their chain length, molecule conformation, and the presence of unsaturated bonds (Withey et al. 2015; Hema et al. 2017). Some cis-unsaturated fatty acids (UFAs), known as Diffusible Signaling Factors (DSF), disrupt P. aeruginosa biofilm formation by meddling with AHL production (Zhou et al. 2017). Here, we showed that fatty acids, such as palmitoleic acid and acetic acid, delayed the expression of QS phenotypes in P. aeruginosa.

The mechanism of fatty acids delaying QS activation could be related to its suppressive impact on QS gene expression. In A. baumannii, UFAs hinder biofilm formation by suppressing abaR gene expression and consequently inhibiting abaI autoinducer synthase gene expression, resulting in the downregulation of long-AHLs production (Nicol et al. 2018). In Francisella novicida, Burkholderia diffusible signal factor (BDSF) inhibits biofilm formation by modulating the expression of biofilm-associated genes and concurrently enhancing relA expression and ppGpp level (Dean et al. 2015). Furthermore, BDSF delays QS activation in Burkholderia cenocepacia by decreasing the transcription of cepI (Wang et al. 2022). In Stenotrophomonas maltophilia, fatty acids influence the binding of the TetR-like transcriptional regulator Smlt2053 to its promoter, inhibiting gene expression even at low concentrations (Coves et al. 2023). Here, we show that in P. aeruginosa, the mRNA expression of lasR, rhlR, and rhlI were significantly suppressed by high doses of both palmitoleic acid and acetic acid, indicating that fatty acids had suppressive effects on the expression of P. aeruginosa QS genes. It’s noteworthy that in this study the mRNA expression of lasI was only significantly suppressed by acetic acid, and palmitoleic acid didn’t have a significantly suppressive effect on it, so it’s possible that a post-transcription gene regulation was involved in the inhibition of lasI expression by palmitoleic acid. Furthermore, according to our differential metabolism analysis, amino acid metabolism may also play a role in QS activation, and there might be an interaction between the metabolisms of fatty acids and amino acids, thus more studies are needed to clarify the related mechanism in the future.

Signaling molecules (3OC12-HSL, C4-HSL) and pyocyanin are some major phenotypes for P. aeruginosa QS activation, and in PA1895-1897-knockout mutant, these phenotypes were significantly enhanced. In the time-course experiments, we observed that high doses of palmitoleic acid and acetic acid delayed the expression of these phenotypes in PA1895-1897-knockout culture, supporting that enriched fatty acids, such as palmitoleic acid and acetic acid, took an important part in the delayed QS activation by PA1895-1897 operon. Furthermore, when fatty acid biosynthesis was inhibited by triclosan in PA1895-1897-overexpressed mutant, the levels of signaling molecules and pyocyanin were significantly promoted. We chose the PA1895-1897-overexpressed mutant for triclosan treatment experiments, since its phenotype of delayed QS activation was more dramatic compared with that of wildtype strain, as well as it synthesized more abundant fatty acids, which would have a higher possibility to result in significant difference in QS phenotypes between triclosan treatment group and control group. Although triclosan may have multiple targets against PAO1, it has a more specific target in the fatty acid synthesis (Shrestha et al. 2020). So far, it is not clear whether triclosan would have indirect effects on autoinducers production through other mechanisms, such as interfering with the stability and topology of lipid and protein structures. However, these mechanisms are more likely to have an inhibitory effect on QS function rather than a promoting effect, and therefore are not likely to play a critical role in the enhanced QS phenotypes we observed. Furthermore, in this study we didn’t observe any significant impact on the bacterial growth by triclosan (0.5 μg/mL), suggesting at this concentration triclosan didn’t significantly affect the physiological features of PAO1. Although our data showed some variations in the effects of fatty acids on QS signal levels between PA1895-1897-knockout and -overexpressed cultures under different intervention conditions (fatty acids or triclosan), on the whole, these data supported the important role of fatty acids in the delayed QS activation by PA1895-1897 operon.

Fatty acids may suppress the production of P. aeruginosa virulence factors, which can be observed in an in vitro cell experiment that filtrates of PAO1 cultures with palmitoleic acid or acetic acid treatment improved the survival of human bronchial epithelial BEAS-2B cells (Figure S1). Pyocyanin is the virulence factor used in previous QscR studies (Chugani et al. 2001; Ding et al. 2018), and it has been confirmed that the genes that encode pyocyanin, such as phzA1 and phzB1, were significantly repressed by QscR, so we selected it in phenotype analysis and subsequent fatty acid intervention study. For future virulence studies, more experiments are needed regarding the effects of fatty acids on virulence factors other than pyocyanin, such as proteases, rhamnolipid, hydrogen cyanide, and biofilm formation. Furthermore, whether fatty acids would affect the production of Pseudomonas quinolone signals and related virulence factors also need to be clarified in future studies.

In the last years, numerous natural and synthetic compounds have been recognized for their effective quorum quenching capabilities (Vadakkan et al. 2019, 2020; Asif and Imran 2019). Among these, the antimicrobial potential of fatty acids is getting attention (Huang et al. 2010). In most pathogens, the antimicrobial action is usually restricted to the early period of incubation due to the formation of an absorption layer of fatty acid that causes increased cell membrane permeability (Nieman et al. 1954). In addition to the above mechanism, we found that in the early stage of bacterial growth, an increase of fatty acid synthesis in bacteria can delay the QS activation, which is the important metabolic mechanism underlying the suppressive effect of PA1895-1897 operon on QS function. These findings suggest fatty acids could serve as intermediate metabolic products of QS inhibitors, exerting inhibitory effects on bacterial virulence in the treatment of lung infection. In contrast to conventional antibiotics that directly kill or inhibit bacterial growth, therapies that promote fatty acid synthesis in bacteria cause potentially less selective pressure on the growth of drug-resistant mutants, thus alleviating therapeutic pressure from the development of antibiotic resistance (Allen et al. 2014; Pan and Lee 2023).

In conclusion, our work showed that fatty acid synthesis is promoted by PA1895-1897 operon and contributes to the delayed QS activation in P. aeruginosa. Our data supported the potential of fatty acids as anti-QS metabolites in tackling the challenge of P. aeruginosa lung infection.