Assessment of Anticancer and Antimicrobial Potential of Bioactive Metabolites and Optimization of Culture Conditions of Pseudomonas aurantiaca PB-St2 for High Yields

Abstract

The following study aimed to characterize the biological potential of the purified compounds of Pseudomonas aurantiaca PB-St2. Optimization of temperature and incubation time of 32oC and 72 h yielded the highest crude extract weight and optical density of bacterial culture. HPLC analysis of the crude metabolite extract (purified using gravitational column chromatography) showed three fractions named as PC1, PC2, and PC3. HPLC-purified fractions were subjected to LC-MS/MS analysis and the data was compared using reference library. Fraction PC1 was identified as mupirocin, PC2 as phenazine-1-carboxylic acid (PCA), and PC3 as the mixture of three compounds including pyoluteorin, PCA and 2-hydroxyphenazine (2-OH-phz). Fungicidal potential of the purified compounds was assessed against phytopathogens including Fusarium equiseti, Fusarium incarnatum, Alternaria alternata, and Colletotrichum falcatum. Fraction PC3 showed the highest fungicidal activity of ~89%, whereas, the least antifungal activity (~27%) was noted for mupirocin. Antibacterial activity of the purified compounds against Gram-positive pathogen Bacillus cereus, and Gram-negative pathogens Pseudomonas aeruginosa, Salmonella enterica, and Klebsiella oxytoca was also assessed. Fraction PC3 demonstrated the highest antibacterial activity against B. cereus and P. aeruginosa showing 1.8 cm, and 0.9 cm zones of inhibition, respectively. Against K. oxytoca and S. enterica, the antibacterial activity of PB-St2 crude extract was slightly higher than the fraction PC3. The fraction PC3 also demonstrated the highest IC₅₀ against HepG-2 and SF767 cancer cell lines at 25 μg and 20 μg concentrations, respectively. The multifaceted attributes of P. aurantiaca PB-St2 make it an ideal candidate for agricultural and pharmacological applications.

Keywords: Anticancer; P. aurantiaca; antimicrobial; bioactive metabolites; chromatography.

Fig. 1. Graphical representation of optimization of high yields of crude extract weight, optical density and PCA production of PB-St2.

(A) measurement of cell density and crude extract weight at different temperatures, (B) measurement of cell density and crude extract weight at different incubation times, (C) average quantification of PCA at different temperature ranges, (D) average quantification of PCA at different incubation times. The error bars represent standard deviation and results are the means of three replicates.

Fig. 2. Three dimensional (3D) surface plots based on experimental data showing the effect of temperature and incubation period on PCA quantification (A) extract weight (B) and optical density (C).

Results are the means of two replicates.

Fig. 3. Extraction and isolation of compounds from P. aurantiaca PB-St2.

Thin layer chromatography (TLC) of PB-St2 crude extract with mobile phase ethyl acetate: n-hexane (7:3) showing three compounds PC1, PC2 and PC3 (A) concentrated fractions revealing PC1 as a greasy textured compound (B) yellow-color crystals of PC2 (C) and dark orange colored crystals of PC3 (D).

Fig. 4. LC-MS chromatograms of purified compounds.

PC1 as mupirocin m/z [M+H]⁺ 501.9 (A), PC2 and P2 as phenazine-carboxylic acid m/z [M+H]⁺ 225.0 (B), P1 as pyoluteorin m/z [M+H]⁺ 271.1 (C), and P3 as 2-hydroxyphenazine (2- OH-phz) m/z [M+H]⁺ 197.0 (D).

Fig. 5. Antifungal plate assay of P. aurantiaca compounds showing antagonistic activity of PCA against Alternaria alternata1 (A) PC3 against Fusarium equiseti (B) 2-OH-phz against Colletotrichum falcatum (C) and PCA against Fusarium incarnatum (D). Graph representing average percent inhibition of compounds against selected fungal phytopathogens (E).

The error bars represent standard deviation where results are the means of two replicates.

Fig. 6. Stereomicroscopy (40X) observations revealing antagonistic activities of compounds against Alternaria alternata 1 (A) and Fusarium equiseti (B).

Elongated hyphae of Alternaria alternata 1, rosette formation and hyphal deformation by growing column fractions (A1). PB-St2 crude extract (A2), PCA (A3), PC3 (A4), mupirocin (A5), 2-OH-phz (A6), pyoluteorin (A7) with A. alternata 1. Elongated hyphal morphology of Fusarium equiseti (B1), branching of Fusarium hyphae by growing with mupirocin (B2). Deformed spiral shaped morphology of Fusarium with PCA (B3), PC3 (B4), pyoluteorin (B5), 2-OH-phz (B6), and PB-St2 crude extract (B7).

Fig. 7. Fluorescent microscopy (254 nm, 40X) observation revealing the effect of PC3 compound on A. alternata 1 and F. equiseti.

A. alternata 1 with septate in hyphae and high sporulation (A) PC3 treated A. alternata 1 showed granulations in hyphae, rosette formation and deformations (B) normal septate hyphae of Fusarium (C) effect of PC3 compound on hyphae of Fusarium (D). St: septate, Rzt: rosette formation, Df: deformations, Gr: granulations

Fig. 8. Antagonistic activity of purified compounds against B. cereus, P. aeruginosa, K. oxytoca and S. enterica (A). Antagonistic activity of HPLC fractions against B. cereus; control (1), pyoluteorin (2), 2-OH-phz (3) (B).

Fig. 9. Measurement of IC₅₀ of PB-St2 compounds against HepG-2 (A) and, SF767 (B).

The error bars represent standard deviation and results are the means of two replicates.