Pyocyanin, a Metabolite of Pseudomonas Aeruginosa, Exhibits Antifungal Drug Activity Through Inhibition of a Pleiotropic Drug Resistance Subfamily FgABC3

Abstract

The fungus Fusarium graminearum is the causative agent of economically significant plant diseases such as Fusarium Healed Blight (FHB) of cereals, its mycotoxins as deoxynivalenol (DON), Nivalenol (NIV) and Zearalenone (ZEN) contaminate wheat and other grains. The objectives of the present study were to determine the mechanism by which the bacterium Pseudomonas aeruginosa inhibits the growth of F. graminearum. Our results indicate that P. aeruginosa metabolites as pyocyanin has effective antifungal properties. Pyocyanin was produced by P. aeruginosa when cultured on mineral salt medium and reached a maximum concentration after 72 h. Pyocyanin significantly decreased mycotoxins of F. graminearum, a 25 mg/ml of pyocyanin for 72 h decreased DON by 68.7% and NIV by 57.7%.

Real-Time PCR analysis demonstrated that the antifungal effect is mediated by downregulation of the Pleiotropic Drug Resistance (PDR) subfamily FgABC3. 25 mg/ml of pyocyanin decreased FgABC3-mRNA by 60%, inhibited the fungal growth and decreased the area of mycelial growth at 12, 24, 36 and 72 h post incubation by 40–50%. Deletion of FgABC3 led to enhanced accumulation of DON and NIV by 40 and 60%, respectively.

The data presented in this report may have significance in understanding mechanism by which certain bacterial metabolites exert a beneficial effect and for developing antifungal drugs.

Key words

Fusarium graminearum - Pseudomonas aeruginosa - Pyocyanin - mycotoxins - FgABC3

• References

• 1 Amedei A, D’Elios MM. New therapeutic approaches by using microorganisms-derived compounds. Curr Med Chem 2012; 19: 3822-3840
• 2 Velusamy P, Gnanamanickam SS. The Effect of Bacterial Secondary Metabolites on Bacterial and Fungal Pathogens of Rice. In Karlovsky P. (eds) Secondary Metabolites in Soil Ecology. Soil Biology. vol 14 Springer; Berlin, Heidelberg: 2008
• 3 Becher R, Miedaner T, Wirsel SGR. Biology, diversity and management of FHB-causing Fusarium species in small-grain cereals. In Kempken F. editor The Mycota XI, Agricultural Applications. 2 ed. Berlin Heidelberg: Springer- Verlag; 2013
• 4 Stepien L, Chelkowski J. Fusarium head blight of wheat: Pathogenic species and their mycotoxins. World Mycotoxin 2010; J 3: 107-119
• 5 Paul PA, McMullen MP, Hershman DE. et al. Meta-analysis of the effects of triazole-based fungicides on wheat yield and test weight as influenced by Fusarium head blight intensity. Phytopathology 2010; 100: 160-171
• 6 Klix MB, Verreet JA, Beyer M. Comparison of the declining triazole sensitivity of Gibberella zeae and increased sensitivity achieved by advances in triazole fungicide development. Crop Prot 2007; 26: 683-690
• 7 Yin Y, Liu X, Li B. et al. Characterization of sterol demethylation inhibitor-resistant isolates of Fusarium asiaticum and F. graminearum collected from wheat in China. Phytopathology 2009; 99: 487-497
• 8 Schoonbeek HJ, Raaijmakers JM, De Waard MA. Fungal ABC transporters and microbial interactions in natural environments. Mol Plant Microbe Interact 2002; 15: 1165-1172
• 9 Baral B. Evolutionary Trajectories of Entomopathogenic Fungi ABC Transporters. Adv Genet 2017; 98: 117-154
• 10 Mendez C, Salas JA. The role of ABC transporters in antibiotic-producing organisms: Drug secretion and resistance mechanisms. Res Microbiol 2001; 152: 341-350
• 11 Del Sorbo G, Schoonbeek H, De Waard MA. Fungal transporters involved in efflux of natural toxic compounds and fungicides. Fungal Genet Biol 2000; 30: 1-15
• 12 Dassa E, Bouige P. The ABC of ABCs: A phylogenetic and functional classification of ABC systems in living organisms. Res Microbiol 2001; 152: 211-229
• 13 Kretschmer M, Leroch M, Mosbach A. et al. Fungicide-driven evolution and molecular basis of multidrug resistance in field populations of the grey mould fungus Botrytis cinerea. PLoS Pathog 2009; 5: e1000696
• 14 Kovalchuk A, Driessen A. Phylogenetic analysis of fungal ABC transporters. BMC Genom 2010; 11: 177
• 15 Qi PF, Zhang YZ, Liu CH et al. Fusarium graminearum ATP-Binding Cassette Transporter Gene FgABCC9 Is Required for Its Transportation of Salicylic Acid, Fungicide Resistance, Mycelial Growth and Pathogenicity towards Wheat. Int J Mol Sci 2018; 19:
• 16 Abou Ammar G, Tryono R, Döll K. et al. Identification of ABC transporter genes of Fusarium graminearum with roles in azole tolerance and/or virulence. PLoS One 2013; 11: 8-e79042
• 17 Stead P, Rudd BA, Bradshaw H. et al. Induction of phenazine biosynthesis in cultures of Pseudomonas aeruginosa by L-N-(3-oxohexanoyl) homoserine lactone. FEMS Microbiology Letters 1996; 140: 15-22
• 18 Kerr J. Phenazine pigments, antibiotics and virulence factors. The Infectious Disease Review—microbes of man, animals and the environment 2000; 2: 184-194
• 19 Michel-Briand Y, Baysee C. The pyocins of Pseudomonas aeruginosa. Biochimie 2002; 84: 499-510
• 20 Morales DK, Jacobs NJ, Rajamani S. et al. Antifungal mechanisms by which novel Pseudomonas aeruginosa phenazine toxin kills Candida albicans in biofilms. Mol Microbiol 2010; 78: 1379-1392
• 21 Rahme LG, Stevens EJ, Wolfort SF. et al. Common virulence factors for bacterial pathogenicity in plants and animals. Science 1995; 268: 1899-1902
• 22 Frank LH, DeMoss RD. On the biosynthesis of pyocyanine. J Bacteriol 1959; 77: 776-782
• 23 Nutz S, Döll K, Karlovsky P. Determination of the LOQ in real-time PCR by receiver operating characteristic curve analysis: Application to qPCR assays for Fusarium verticillioides and F. proliferatum. Anal Bioanal Chem 2011; 401: 717-726
• 24 Adejumo TO, Hettwer U, Karlovsky P. Occurrence of Fusarium species and trichothecenes in Nigerian maize. Int J Food Microbiol 2007; 116: 350-357
• 25 Fang S, Yan X, Liao H. 3D reconstruction and dynamic modeling of root architecture in situ and its application to crop phosphorus research. Plant J 2009; 60: 1096-1108
• 26 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2^−ΔΔCT method. Methods 2001; 25: 402-408
• 27 Saha S, Thavasi R, Jayalakshmi S. Phenazine pigments from Pseudomonas aeruginosa and their application as antibacterial agent and food colourants. Research. Journal of Microbiology 2008; 3: 122-128
• 28 Essar DW, Eberly L, Hadero A. et al. Identification and characterization of genes for a second anthranilate synthase in Pseudomonas aeruginosa: interchangeability of the two anthranilate synthases and evolutionary implications. Journal of Bacteriology 1990; 172: 884-900
• 29 Lodhi MA, Ye GN, Veeden NF. et al. A simple and efficient method for DNA extraction from grapevine cultivars and Vitis species. Plant Mol Biol Rep 1994; 12: 6-13
• 30 Singer RS, Finch R, Wegener HC. et al. Antibiotic resistance - the interplay between antibiotic use in animals and human beings. Lancet Infect Dis 2003; 3: 47-51
• 31 Tawiah AA, Gbedema SY, Adu F. Antibiotic producing microorganisms from River Wiwi, Lake Bosomtwe and the Gulf of Guinea at Doakor Sea Beach, Ghana. BMC Microbiol 2012; 12: 234
• 32 Jansen C, von Wettstein D, Schäfer W. et al. Infection patterns in barley and wheat spikes inoculated with wild-type and trichodiene synthase gene disrupted Fusarium graminearum. Proc Natl Acad Sci 2005; 102: 16892-16897
• 33 Maier FJ, Miedaner T, Hadeler B. et al. Involvement of trichothecenes in fusarioses of wheat, barley and maize evaluated by gene disruption of the trichodiene synthase (Tri5) gene in three field isolates of different chemotype and virulence. Mol Plant Pathol 2006; 7: 449-461
• 34 Edwards SG, Pirgozliev SR, Hare MC. et al. Quantification of trichothecene-producing Fusarium species in harvested grain by competitive PCR to determine efficacies of fungicides against Fusarium head blight of winter wheat. Appl Environ Microbiol 2001; 67: 1575-1580
• 35 Kandela SA, al-Shibib AS, al-Khayat BH. A study of purified pyorubin produced by local Pseudomonas aeruginosa. Acta Microbiol Pol 1997; 1: 37-43
• 36 Kerr JR, Taylor GW, Rutman A. et al. Pseudomonas aeruginosa pyocyanin and 1-hydroxyphenazine inhibit fungal growth. J Clin Pathol 1999; 52: 385-387
• 37 Tan MW, Rahme LG, Sternberg JA. et al. Pseudomonas aeruginosa killing of Caenorhabditis elegans used to identify P-aeruginosa virulence factors. Proc Natl Acad Sci 1999; 96: 2408-2413
• 38 Xu L, Wang F, Shen Y. et al. Pseudomonas aeruginosa inhibits the growth of pathogenic fungi: In vitro and in vivo studies. Exp Ther Med 2014; 7: 1516-1520
• 39 Kaur J, Pethani BP, Kumar S. et al. Pseudomonas aeruginosa inhibits the growth of Scedosporium aurantiacum, an opportunistic fungal pathogen isolated from the lungs of cystic fibrosis patients. Front Microbiol 2015; 6: 866
• 40 Keçeli Özcan S, Dündar D, Sönmez Tamer G. Anti-candidal activity of clinical Pseudomonas aeruginosa strains and in vitro inhibition of Candida biofilm formation. Mikrobiyol Bul. 2012; 46: 39-46
• 41 Krishnan HB, Kang BR, Hari Krishnan A. et al. Rhizobium etli USDA9032 engineered to produce a phenazine antibiotic inhibits the growth of fungal pathogens but is impaired in symbiotic performance. Appl Environ Microbiol 2007; 73: 327-330