Introduction

Cystic Fibrosis (CF) is a genetic disease seen in the lungs, intestines, pancreas and sweat glands; it affects the respiratory and digestive systems through elevated viscosity of fluids. CF especially impairs the respiratory system and causes degraded lung activity (Caldas and Boisrame 2015). Pseudomonas aeruginosa infections in CF are presented as the main cause for high mortality and morbidity rates. Several antibiotics are utilized in treating CF for repressing chronic infections. However, medical treatments are not sufficient in some cases. Studies suggest that the failure to cure the disease might be caused by the biofilm form of P. aeruginosa’s resistance to antibiotics (Proesmans et al. 2013). This study investigates the possibility of an alternative treatment approach by utilizing lichen secondary metabolites to reduce the expressions of P. aeruginosa virulence factors.

P. aeruginosa is an opportunistic pathogen. It is resistant to multiple antibiotics and causes pneumonia, sepsis, acute infections and chronic lung infections (Jones et al. 2009; Lambert et al. 2011; Zhanel et al. 2004). P. aeruginosa can adapt and grow in diverse environmental conditions. They can receive signals from their surroundings and react to these signals by organizing their gene expressions. However, bacteria can also receive signals from other bacteria and regulate gene expressions as a population. This mechanism is called quorum sensing (QS) (Bassler 2015).

Gram-negative bacteria utilize the QS system to express virulence factors by constantly releasing signal molecules called N-acyl-homoserine lactones (AHLs) (Jakobsen et al. 2013). As the bacteria population grows, the concentration of AHLs increase and they reach a threshold. At this point the expression of the genes controlled by QS are induced. The QS system is known to regulate many physiological characteristics such as virulence, motility, conjugation and bioluminescence (Girard and Bloemberg 2008).

Pseudomonas aeruginosa QS consist of two important systems: LasIR and RhlIR. LasI synthesizes N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and RhlI synthesizes N-butanoyl homoserine lactone (C4-HSL). At high concentrations, these signal molecules called autoinducers (AIs) bind to their receptors and activate transcriptional regulators LasR and RhlR. The AI-Regulator complex then binds to the QS related genes’ promoter sites and start the expression of the genes (Zhang et al. 2002). P. aeruginosa has a third QS system that utilizes Pseudomonas quinolone signal (PQS). This system is believed to act as a mediatory between the other two systems (Diggle et al. 2003; McGrath et al. 2004).

Utilizing QS, P. aeruginosa form biofilm to protect them against harsh conditions such as pH, ultraviolet light, heat etc. (Boor 2006; Shannon and French 2004). The biofilm form gives them a substantial advantage over planktonic form, one of which is antibiotic resistance. (Brackman and Coenye 2015). Antibiotic resistance of microorganisms has become a significant problem in recent years. The novel approach to fight against bacteria without risking antibiotic resistance is to target their QS systems (Hentzer et al. 2003a). Inhibition of QS by employing quorum sensing inhibitors (QSIs) can leave the bacteria vulnerable and easier to fight against (Zeng et al. 2008).

At this stage, the need for determining natural sources that can inhibit quorum sensing with secondary metabolites arises (Pompilio et al. 2013). Plants or natural or semi-synthetic products obtained from them are preferred for the development of pharmaceutical raw materials. Like plants, lichens also have been used in the treatment of many diseases for their medicinal properties since ancient times. Therefore they can also be used for developing new pharmaceutical products.

Lichens are complex organisms composed of algae or cyanobacteria (photobiont) living together with fungi (mycobiont) in a symbiotic relationship (Nash 2008). Studies demonstrate that the healing properties of lichens used in the prevention of diseases results from their secondary metabolite components with acidic properties (Acikgoz et al. 2013; Nguyen et al. 2014). Lichens are exposed to a number of biotic and abiotic stresses and the production of secondary metabolites is one way of coping with this (Deduke et al. 2012; Fernandez-Moriano et al. 2016). Lichens have approximately 1150 secondary metabolites as a result of their metabolism (Shukla et al. 2010). These metabolites have antibacterial, antiviral, antimicrobial, antifungal, antioxidant, antitumor and enzyme inhibiting properties (Acikgoz et al. 2013; Basile et al. 2015; Bhattarai et al. 2008; Nguyen et al. 2014). Therefore, it is essential to determine QSI potentials and healing properties of lichens, and have specific knowledge of the active compounds. Such a compound is evernic acid: a secondary metabolite that can be isolated from Evernia species, and is known to have antifungal properties (Marijana and Branislav 2011).

In this study, a lichen secondary metabolite evernic acid was tested for its QSI activity against P. aeruginosa. Fluorescent monitor strains were utilized for QSI screens and quantitative reverse-transcriptase PCR (qRT-PCR) analyses were conducted for comparison.

Materials and methods

Bacterial strains

Pseudomonas aeruginosa strains used in this study are listed in Table 1. Bacteria strains were grown in M9 minimal media supplemented with 2.5 mg of thiamine per liter, 0.5 % (wt/vol) glucose, and 0.5 % (wt/vol) Casamino Acids. lasB-gfp and rhlA-gfp strains were used for QSI screens and wild type P. aeruginosa strain PAO1 was used for qRT-PCR analyses. The monitor strains lasB-gfp and rhlA-gfp are developed by Hentzer et al. (2002) and Yang et al. (2009). They contain a mini-Tn5 insert containing well characterized lasB and rhlA promoters that are regulated by lasR and rhlR, and genes for an unstable green fluorescent protein (GFP). Strains are highly sensitive even with a single chromosomal copy and very low AHL concentrations. All strains were obtained from Tim Holm Jakobsen (University of Copenhagen, Copenhagen, Denmark).

Table 1 Strains used in this study. PAO1 strain was used as wild type rhlA-gfp and lasB-gfp strains were used as fluorescent monitors for QS inhibition
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P. aeruginosa QSI screens

QSI screenings were performed according to a modified method of that described by Bjarnsholt et al. (2010). Growth medium (100 µl M9 medium with 0.5 % [w/v] Casamino Acids, 2.5 mg of thiamine liter−1 and 0.5 % [w/v] glucose) were added to each well of 96-well black microplates (Nunc, Thermo Scientific) and two-fold serial dilutions of evernic acid were made. An overnight culture of the lasB-gfp or rhlA-gfp monitor strain were added to obtain a total volume of 200 µl and give an OD 450 nm of 0.1. As a result, 116 µM, 58 µM, 29 µM, 14.5 µM and 7.25 µM final concentrations of evernic acid were achieved. Growth and green fluorescent protein (GFP) expressions were monitored using Cytation 3 multimode microplate reader (Biotek) at 34 °C for 15 h, measuring every 15 min. Fluorescence of GFP expression were measured at 485 nm excitation and 535 nm emission wavelengths and growth was measured at OD 450 nm.

qRT-PCR

Overnight cultures of P. aeruginosa PAO1 strain were treated with same concentrations of evernic acid (Extrasynthese) as in the QSI screens. Cultures were incubated at 34 °C for 15 h. After incubation, samples were retrieved and RNA isolation was performed with Roche High Pure RNA Isolation Kit (Roche). RNA purities were measured and concentrations were calculated using Cytation 3 with Take3 plate (Biotek). cDNA synthesis was performed with Transcriptor First Strand cDNA Synthesis Kit (Roche). Gene expression analyses were performed using FastStart Essential DNA Probes Master Kit (Roche) and TaqMan probes. 45 cycles were run and the rpoD gene was used as control for normalization. The primer sequences are listed in Table 2.

Table 2 Primer sequences used in this study
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Biofilm inhibition assay

Overnight cultures of P. aeruginosa PAO1 strain were diluted 1:100 into growth medium (M9 medium with 0.5 % [w/v] Casamino Acids, 2.5 mg of thiamine liter−1 and 0.5 % [w/v] glucose) for biofilm assays. Serial dilutions were made and 116 µM, 58 µM, 29 µM, 14.5 µM and 7.25 µM final concentrations of evernic acid were achieved on microplate. After a day of incubation at 37 °C, plate was rinsed and stained with 0.1 % crystal violet solution for 5 min. Plate was rinsed again and then allowed to dry. Finally, stains in the plate was dissolved using ethanol and absorbance measurements were made at 590 nm.

Results

QSI activity of evernic acid

Evernic acid concentrations have presented QS inhibition for both las and rhl systems. The results of the GFP measurements are means of three experiments and shown in Fig. 1. The results are presented as GFP expression/bacterial cell growth to make sure the reduced fluorescence is due to QS inhibition. It was observed that evernic acid is capable of inhibiting approximately 54 % of gfp expression of lasB-gfp and 50 % of rhlA-gfp at 116 µM when compared to the untreated control group.

Fig. 1
figure 1

Dose response curves of lasB-gfp a and rhlA-gfp b monitor strains treated with evernic acid concentrations between 7.25 and 116 µM, compared to untreated samples

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qRT-PCR

The regulations of rhlA and lasB genes were analyzed and relatively quantified for treated and untreated PAO1 strain samples. The results are shown in Fig. 2. It is indicated that there is a downregulation of the genes depending on QSI concentration. Compared to lasB, expression of rhlA gene seem to be inhibited slightly more. According to results, the expression of lasB was downregulated by 74 % at 116 µM, 55 % at 58 µM, 46 % at 29 µM, 32 % at 14,5 µM and 18 % at 7,25 µM doses. The expression of rhlA was downregulated by 78 % at 116 µM, 63 % at 58 µM, 62 % at 29 µM, 44 % at 14,5 µM and 21 % at 7,25 µM doses.

Fig. 2
figure 2

Gene expression levels of lasB a and rhlA b compared to the untreated control group. Data show representative experiment from three individual qRT-PCR determinations. Gene expressions were normalized to expression of rpoD

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Biofilm assays

Results indicate that in samples treated with evernic acid, biofilm formation was significantly less than the untreated control group. Measured absorbance (590 nm) levels are shown in Fig. 3. Results are means of 5 experiments.

Fig. 3
figure 3

Biofilm formation of PAO1 strain treated with evernic acid concentrations, compared to untreated control sample. Results are means of 5 individual experiments

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Discussion

The antibiotic resistance of bacteria and resulting ineffective treatments are a growing concern worldwide. Numerous studies focus on this problem and seek alternative treatment solutions. QS studies carry weight in current studies to find an alternative treatment to antibiotics. Trying to exterminate the bacteria induces stress and causes them to protect themselves via biofilm. The biofilm forms enable bacteria to grow in more secure conditions and gain antibiotic resistance (Brackman and Coenye 2015; Fux et al. 2005). Instead of attacking bacteria, the safer approach appears to be to block their communication and thus reduce virulence factors by inhibiting QS (Hentzer et al. 2003a). In this regard, it is important to discover potential QSIs from natural resources.

Plants and fungi are notable resources with their natural products for drug research and QS inhibition studies. Several strong QSIs have been identified to date (Al-Ani et al. 2015; Azimi et al. 2012; Savo et al. 2015; Truchado et al. 2012). Lichens, a symbiotic relationship between fungi and algae or cyanobacteria, are also known to have ethnobotanical value and produce unique secondary metabolites. Therefore, it is necessary to explore the QSI properties of lichen secondary metabolites such as evernic acid.

Pseudomonas aeruginosa has become a model organism for QS studies (Hentzer et al. 2002). As in many bacteria, P. aeruginosa can also determine its population density by excreting and translating communication signals with the QS mechanism (De Kievit 2009). Moreover, this cell density centered system regulates various genes most of which are virulence factors of P. aeruginosa. These genes include lasB (elastase), the rhlAB operon (rhamnolipid production), the hcnABC operon (hydrogen cyanide production) and chiC (chitinase) (Hentzer et al. 2003b).

In addition to two important QS systems of P. aeruginosa, there are two other systems that are activated by their unique signals: Pseudomonas Quinolone Signal (PQS) and Integrated Quorum Sensing Signal (IQS) (Pesci et al. 1999; Lee et al. 2013). PQS system has a role in regulation of virulence factors by mediating between las and rhl systems, but its importance in virulence remains debatable (Lee and Zhang 2015). Since both rhl and PQS systems are governed by the las system, its inhibition takes priority regarding virulence. Recently discovered IQS on the other hand, is reported to take over las system under phosphate depletion stress conditions (Lee et al. 2013).

In this study, two monitor strains lasB-gfp and rhlA-gfp were treated with evernic acid concentrations between 7.25 and 116 µM, and monitored for QSI screenings. PAO1 strain was used in biofilm assays. In addition, the expression levels of lasB and rhlA genes were analyzed using qRT-PCR method for confirmation. These genes are pivitol due to the virulence factors elastase and rhamnolipids encoded by them.

Measurements with fluorescent monitor strains indicate that both QS systems (LasIR and RhlIR) of P. aeruginosa were inhibited to approximately 50 % at 116 µM evernic acid concentration. In 2012, Jakobsen et al. have tested “iberin” from horseradish on P. aeruginosa and present that iberin can inhibit the expression of lasB-gfp fusion completely at 100 µM without affecting growth (Jakobsen et al. 2012). In this regard, we couldn’t achieve total inhibition with evernic acid, but it seems possible if it is applied in higher concentrations. According to the results, qRT-PCR method also confirms the down-regulation of lasB and rhlA genes and thus the inhibition of QS systems by evernic acid. This suggests that PQS system is also presumably inhibited since its regulation is also governed by the las system. Therefore, the outcomes of this study presents unique opportunities concerning the utilization of lichen secondary metabolites for QS inhibition studies.

Inhibition of QS is an effective strategy against infections caused by pathogenic bacteria. Determining potential QSIs and effective screenings are essential for this strategy. This study shows that a lichen secondary metabolite evernic acid is capable of inhibiting QS systems of the opportunistic pathogen P. aeruginosa. Findings of this study indicate that lichens are probably a wide resource for secondary metabolites capable of inhibiting QS. Their unique secondary metabolites present an opportunity for discovering new therapeutic agents and raw materials for future anti-QS drugs. Therefore, with additional research it is safe to assume that an alternative treatment can be developed for of CF patients with P. aeruginosa infections.