Overproduction of Pyocyanin in Pseudomonas aeruginosa by Supplementation of Pathway Precursor Shikimic acid and Evaluation of its Activity

 

Vipin C¹, Ashwini P², Kavya AV³, Rekha PD⁴*

¹Junior Research Fellow, Yenepoya Research Centre, Yenepoya University

²Assistant Professor, Yenepoya Research Centre, Yenepoya University

³ M.Sc. Department of Bioscience, Mangalore University

⁴ Professor and Deputy Director, Yenepoya Research Centre, Yenepoya University

 

ABSTRACT:

Pyocyanin is a fluorescent phenazine compound with enormous biotechnological applications. In this study, the production of pyocyanin by P. aeruginosa PAO1 was investigated under different growth conditions. Optimization of growth media was carried out using nutrient broth, tryptic soy broth, Pseudomonas broth and Luria bertani broth. The effect of incubation time, and temperature on production of pyocyanin was investigated. Subsequently, shikimic acid pathway mediated pyocyanin overproduction was targeted using Illicium verum hot water extract as a source of shikimic acid for phenazine biosynthesis. Pyocyanin was purified and tested for its activity against glioma cells. Optimum yield of pyocyanin was obtained in nutrient broth at 37 ºC and pH 7.2. We also found that supplementation of nutrient media with Illicium verum extract increased the pyocyanin production. Pyocyanin exhibited cytotoxicity of 66.34% at 200µg/ml concentration against glioma cells.

 

 

INTRODUCTION:

Pseudomonas aeruginosa is a Gram negative bacteria belonging to the family Pseudomonadaceae. One of the characteristic features of P. aeruginosa is the production of extracellular pigments such as pyocyanin, a fluorescent phenazine compound. Pyocyanin is a water-soluble blue-green, redox-active pigment having a molecular weight of 210.23 kDa (Sudhakar et al., 2013). Pyocyanin has a broad spectrum antibiotic activity with diverse pharmacological, aquaculture, agriculture and industrial applications (Jayaseelan et al., 2013).

 

It can selectively inhibit Gram-positive and Gram negative bacteria other than Pseudomonas species (El-Shouny et al., 2011; EL-Fouly et al., 2015). Pyocyanin also has application as food colorant (Saha et al., 2008; Venil et al., 2013). Its ability to cause cytotoxicity on eukaryotic cells has been utilized to target cancer cells with promising results (Zhao et al., 2014). It can cause premature cellular senescence by interfering with oxidative response and destruct the cancer cells (Muller et al., 2009). The biotechnological applications of pyocyanin hold promise for exploiting P. aeruginosa as a source of pyocyanin for large scale production.  Shikimic acid acts as a precursor for the simultaneous biosynthesis of phenazines. DAHP (3-deoxy-7-phosphoheptulonate) synthase is the first enzyme of the shikimate pathway and catalyses the condensation of phosphoenol pyruvate and erythrose-4-phosphate leading to the synthesis of pyocyanin (MacDonald et al., 2001). Illicium verum L., commonly known as star aniseed is a source of shikimic acid. Since shikimic acid acts as a precursor for the biosynthesis of phenazines, in this study, Illicium verum L seeds were used as a source of shikimic acid for pyocyanin production by P. aeruginosa.  Pyocyanin production by P. aeruginosa is dependent on growth conditions selected for culturing such as media composition, temperature and incubation time. Product recovery and its activity are important factors affecting the quality and biotechnological viability of the process and product. Hence, in this study, production of pyocyanin by P. aeruginosa was investigated under different growth conditions and cytotoxicity of purified pyocyanin was tested against glioma cells.

 

MATERIALS AND METHODS:

Media and Chemicals:

Nutrient broth (NB), Tryptic soy broth (TSB), Pseudomonas broth (PB) and Luria Bertani (LB) broth were purchased from Hi-media (Mumbai, India). Chloroform, hydrochloric acid, Tris base and sodium hydroxide were purchased from MERCK (Mumbai, India). Star aniseeds (Illicium verum) were purchased from a local store. 

 

Bacteria and Culture Conditions:

Pseudomonas aeruginosa (PAO1) was used for the experiments. Culture was maintained in nutrient agar (NA) media at room temperature. For inoculation, overnight culture was prepared in 5 ml NB by incubating at 37^°C on a shaker incubator (100 rpm). Cell density was measured using spectrophotometer as OD₆₀₀.

 

Optimization of growth conditions for pyocyanin production:

Four different media namely, NB, TSB, PB and LB (pH 7.2) were inoculated with overnight cultures of P. aeruginosa PAO1. The inoculated tubes were incubated at 37^°C in a bacteriological incubator. Pyocyanin was extracted from the spent cultures at 48, 72 and 96 h.

 

Pyocyanin production at different temperatures:

To study the effect of temperature on pyocyanin production, NB and LB media inoculated with P. aeruginosa (~10⁶ cfu/ml) were incubated at three different temperatures viz., 30^°C, 37^°C and 42 ^°C. Pyocyanin was extracted from the spent cultures at 48, 72 and 96 h intervals after incubation.

 

Effect of Illicium verum extract on pyocyanin production:

Dried florets of I. verum were powdered in a laboratory mill and extracted in hot water for 3 h. The extract obtained was concentrated and supplemented to NB media at two different concentrations (250 µg and 500 µg). The tubes were then inoculated with P. aeruginosa and incubated at 37^°C. Pyocyanin was extracted from the spent cultures at intervals of 48, 72 and 96 h after incubation.

 

Extraction and Purification of Pyocyanin:

Extraction of pyocyanin was carried out according to previously described methods (Das and Manefield 2012). Briefly, culture contents were centrifuged at 8000 rpm for 15 min at 4 ^°C to separate the cells. Supernatant collected was mixed with chloroform (2:1), vortexed and allowed to stand for one hour. Supernatant was discarded and chloroform layer was collected. Optical density (OD) of each sample in chloroform was measured at 318 nm using a spectrophotometer (UV-Vis 1100, Shimadzu, Japan). Simultaneously, the chloroform layer containing pyocyanin was acidified with 1 ml of 0.2 N HCl and the absorbance was recorded at 490 nm. For further purification of pyocyanin, acidified layer was collected and pH was neutralized to 7.0. Chloroform was added to separate pyocyanin and acidified layer was discarded. This procedure was repeated 3-4 times, followed by the addition of NaOH. Pyocyanin obtained was concentrated for further experiments.

 

Cytotoxicity studies of pyocyanin on Glioblastoma multiforme cell line:

Cytotoxicity of pyocyanin was tested against glioma cell line using MTT assay (Mosmann, 1983). Glioblastoma cells (U87MG) were procured from National Centre for Cell Sciences (NCCS), Pune. They were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS and 1% antibiotic antimycotic solution. The cells were used for the experiments after three consecutive passages. Cells were seeded on to 96 well micro titre plate at a seeding density of 5000 cells/well, and incubated in a CO₂ incubator at 37 ^°C and 5% CO₂ levels. Pyocyanin at concentrations of 50, 100 and 200 µg /ml was added to the cells and further incubated for 48 h. 100 µl of methyl thiazolyltetrazolium (MTT) solution was added to the wells and incubated for 4 h. The formazan crystals formed were dissolved by the addition of 100 µl of DMSO and OD₅₇₀ was recorded using a microplate reader (Floustar Omega, BMG Labtech). The percentage cytotoxicity was calculated with reference to untreated control.

 

RESULTS:

Effect of media on pyocyanin yield:

The highest pyocyanin production was observed in NB media (7.2 µg/ml) followed by LB (6.87 µg/ml), PB (4.6 µg/ml) and TSB (3.5 µg/ml) medium (Fig 1). However, no significant difference was observed in pyocyanin yield in NB and LB media. Compared to other media, pyocyanin yield in TSB media was significantly lower.

 

Fig 1: Pyocyanin production in different media at different incubation times.  NB-Nutrient broth, TSB- Trypticase soy broth, PB- Pseudomonas broth, LB- Luria bertani broth (Data points are Mean + SD; n=3).

 

Effect of Temperature on Pyocyanin Production:

Pyocyanin yield at 32^°C was the lowest in all the culture media, while significantly higher quantities of pyocyanin were recovered from the cultures grown at 37 ^°C .  In LB medium, at 32^°C the highest pyocyanin yield was 1.87 µg/ml at 96 h. However, at 37 ^°C, it produced significantly higher amounts of pyocyanin in both NB and LB medium. The cultures incubated at 42^°C showed significantly lower pyocyanin yield compared to 37^°C (Fig 2A, B and C).

 

Fig. 2: Concentration of pyocyanin in LB and NB media at A) 32°C, B) 37°C and C) 42°C (Data points are Mean + SD; n=3)

 

Effect of Illicium verum extract on pyocyanin production

Pyocyanin production was enhanced in NB supplemented with different concentrations of star aniseed extract (Fig 3). The yield was 9.38 µg/ml and 11.6 µg/ml in the culture supplemented with 250 µg/ml and 500 µg/ml of Illicium verum extract respectively at 96 h of incubation at 37^°C.

 

Fig. 3: Pyocyanin production in NB media supplemented with Illicium verum at different incubation times. (Data points are mean + SD; n=3).

 

Cytotoxicity of pyocyanin against glioblastoma cells

Pyocyanin exhibited toxicity against U87MG cells in a concentration dependent manner (Fig. 4).  Pyocyanin exhibited 33.41%, 36.89% and 66.34% cytotoxicity at 50 µg/ml, 100 µg/ml and 200 µg/ml concentrations respectively. No significant difference was observed in cytotoxicity between 50 µg/ml and 100 µg/ml concentrations of pyocyanin.

 

Fig. 4: Percentage cytotoxicity of pyocyanin against glioblastoma cells at different concentrations. (Data points are Mean + SD; n=3)

 

DISCUSSION:

Results of this study revealed that pyocyanin production by P. aeruginosa can be achieved in NB medium at pH 7.2 and incubation temperature of 37ºC. Pyocyanin yield increased with longer incubation time because the phenazine formation commences after the exponential phase of microbial growth along with associated aromatic amino acid biosynthesis (Chin et al., 2001). At 96 h, P. aeruginosa produced higher concentration of pyocyanin in all the four culture media studied. Temperature also plays an important role in the pyocyanin biosynthesis by P. aeruginosa. At 37°C it produced high concentration of pyocyanin compared to 42°C and 32°C. Optimum temperature for growth is 37 ºC, and it is able to grow at temperatures as high as 42ºC. The ability to grow at 42ºC distinguishes P. aeruginosa from other Pseudomonas species (Krieg and Holt, 2001; Garrity, 2004). Shikimic acid is the key intermediate in the common pathway of aromatic amino acid biosynthesis (Borah, 2015; Maeda and Dudareva, 2012). Media supplemented with I. verum as a source of shikimic acid increased the pyocyanin yield. This is because of the role of shikimic acid in the phenazinebiosynstheis. In P. aeruginosa,the phenazine biosynthetic pathway branches off from the shikimic acid pathway (Chin et al., 2001). Shikimic acid acts as a precursor for the simultaneous biosynthesis of phenazines. I. verum contains 17.14% shikimic acid on dry weight basis and is the main source for commercial production of shikimic acid. Supplementing precursors for the production biotechnologically important secondary metabolites are often effective. I. Verum extract did not inhibit the growth of the bacteria at these concentrations. Pyocyanin showed highest cytotoxicity at a concentration of 200 μg/ml against glioma cells. Cytotoxicity of pyocyanin is mainly due to its redox activity, which increases intracellular reactive oxygen species (ROS) as the pigment gets reduced in the cell by NADH and NADPH. The reduced form of pyocyanin then transfers electrons to oxygen, generating superoxide (O₂^º) and hydrogen peroxide (H₂O₂) radicals. The formation of ROS occurs within and around the mitochondria (O'Malley et al., 2003). It also targets the apoptotic pathways thereby causing cellular death. However, pyocyanin at lower concentration did not exert cytotoxicity probably due the cellular enzyme activity neutralizing the oxidative effect. Glutathione in the cell forms a cell-impermeant conjugate with pyocyanin and availability of the thiol is critical to minimizing the toxicity (Muller and Merett, 2015). Anticancer activity of pyocyanin on HepG2 human hepatoma cells was reported by Zhao et al. (2014) showing accelerated cellular senescence, apoptosis and induced oxidative stress associated DNA damage. The potential of pyocyanin as an anticancer moiety needs further pre-clinical and clinical investigations.

 

CONCLUSION:

This study demonstrated the effect of culture conditions, temperature and supplementation of star aniseed extract on pyocyanin yield. This is the first report on the use of shikimic acid for pyocyanin production. As pyocyanin is implicated to have many pharmacological, biotechnological applications, it can be produced in large quantities under certain growth conditions as investigated in this study. The study also prospects the development of pyocyanin into a potential cancer therapeutic.

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Accepted on 09.01.2017           © RJPT All right reserved