0 10 20 30 40 50 0 10 20 30 40 50 60 70 80 90 Control 12 M TrcA 12 M TrcB 12 M TrcC Triton X100 7 M GS Time (min) Sytox green Fluorescence Antifungal membranolytic activity of the tyrocidines against filamentous plant fungi Food shortages are augmented by pathogenic microorganisms, including fungi, which can lead to an annual crop loss of up to 16% and a further 50% post-harvest loss, especially in developing countries with limited infrastructure. [1] The health risks, environmental impact and microbial resistance against conventional chemical fungicides necessitate the development of novel fungicides. [2] The broad range of antimicrobial activity, including the noteworthy inhibition of Listeria monocytogenes [3] and Plasmodium falciparum [4] , of a group of cyclic decapeptides produced by Bacillus aneurinolyticus, the tyrocidines (Trcs) and analogous gramicidin S (GS) (Fig. 1, Table 1), encouraged the investigation into their potential as fungicides. [5] This study focused on their structure-activity relationships and mode of action related to their membrane activity. Figure 4 Model membrane activity. (A) Rapid dequenching kinetics of calcein fluorescence via permeabilisation induced by TrcA in large unilamillar vesicles consisting of 25 µM 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) membranes containing ergosterol (Erg) (70:30). (B) Comparison of the membranolytic activity of selected peptides on different model membranes containing 70% 1, 2-dioleoyl-phosphatidylcholine (DOPC). Statistical differences between membranes for each peptide are indicated with ***P<0.001; **P<0.01 and *P<0.05. INTRODUCTION RESULTS The Trcs and GS exhibited potent antifungal activity against Botrytis cinerea and Fusarium solani with IC max values (MICs calculated from dose-response curves) as low as 3.5 µM (Fig. 2). We also found that the Trc antifungal activity were generally salt tolerant (results not shown). However, the TrcA analogue, PhcA, lost activity in the high salt yeast supplemented TSB medium, while the TrcC analogue, TpcC, lost activity in low salt PDB medium, whereas the tyrocidines and GS showed similar activities in both media (Fig. 2). This indicates the importance of the Tyr 7 residue in the variable pentapeptide and the conserved VOLfP sequence for antifungal activity, possibly relating to aggregation in different media. No overt QSAR was further observed against the two target fungi in the two media. Light microscopy revealed that the tyrocidines are morphogenic, inducing retarded germination and hyperbranching of fungal spores indicating a possible interference with the cell cycle and/or hyphal growth (Fig 3A). At higher concentrations the Trc caused swelling, cytoplasmic disorganisation and cell wall/membrane disintegration of fungal hyphae (results not shown). Studies with membrane impermeable dyes suggest that the membrane integrity of the spores, as well as the hyphae of both B. cinerea and F. solani, is compromised by Trcs and GS treatments (Fig. 3C, D). The Trcs and GS induced rapid uptake of the dye SYTOX green into hyphae of B. cinerea (Fig. 3E) and F. solani (results not shown) indicating the primary targets could be the either the cell wall and/or membrane. Our model fungal membrane studies showed that at 3.2 µM the peptides lead to rapid (±5 seconds) and total lysis of 25 µM POPC:ergosterol liposomes (Fig. 4A) TrcA and TrcB were observed to be selective for ergosterol- above cholesterol-containing membranes (Fig. 4B). Using three different spin labeled lipids in model membranes the Trp-containing tyrocidines (TrcB and TrcC) were further observed to insert 11-12Å into the ergosterol containing membrane (Fig. 5). Glucosylceramides (GlcCer) in membranes antagonised the activity of these Trcs and GS (Fig. 4B). The antagonism, particularly for TrcA and GS, relates to the slower SYTOX green kinetics observed for B. cinerea hyphae (refer to Fig 3E). GlcCer and/or glucans could be Trc target(s) that is not dependent on membrane permeabilisation, as we also found that -glucanase treatment of fungi also antagonised the Trc activity (results not shown.) The significant antifungal activity of the tyrocidines, their media independent/salt tolerant activity, their ability not only to disrupt membrane integrity, but also to interfere with cell morphology, renders tyrocidines potential bio-fungicides with minimal likelihood of inducing resistance. DISCUSSION and CONCLUSIONS Tyrocidine activity towards and interaction with model membranes mimicking characteristics of fungal membranes Figure 5 Depth of membrane insertion as probed by different fluorescently labelled phospholipids and their interaction with Trp residue(s) in TrcB and TrcC. (A) A representative set of spectra with 200 µM POPC:Erg; 2 µM TrcC and the spin-labelled 1-palmitoyl-2-stearyl(5-doxyl)-sn-glycero-3-phosphatidyl-choline (5-DOX) (fractions of 5-DOX was 10, 15, 20, 30 and 40 mol %). The dotted line represents vesicles without spin-labelled lipids. (B) A scheme showing the depth of membrane insertion of the Trp-containing tyrocidines determined using the three labelled lipids. Influence of media and peptide structure on the antifungal activity of the tyrocidines and analogues REFERENCES [1] Chakraborty S & Newton AC (2011). Plant Pathol 60: 2-14. [2] Montesinos E & Bardaji E (2008). Chem Biodivers 5: 1225-1237. [3] Spathelf BM & Rautenbach M (2009). Bioorg Med Chem 17: 5541-5548. [4] Rautenbach M et al. (2007) Biochim Biophys 1768: 1488-1497. [5] Rautenbach M et al., Antimicrobial peptide compositions for plants, PCT Patent, WO 2013/150394 A1 This project was funded by a the BIOPEP Peptide Fund and NRF SA-Germany exchange grant Activity of the tyrocidines on the cell wall/membrane of fungi TpcC 1 TrcC TrcC 1 TrcB TrcB 1 TrcA TrcA PhcA 0 10 20 30 40 B. cinerea F. solani *** *** Pepide Ihibitory concentration IC max ( M) in PDB Tyr 7 Phe 7 Trp 7 Tyr 7 Phe 7 Trp 7 TpcC 1 TrcC TrcC 1 TrcB TrcB 1 TrcA TrcA PhcA 0 10 20 30 40 50 B. cinerea F. solani ** *** Peptide Inhibitory concentration IC max ( M) in YTSB N NH HN HN HN NH HN NH NH HN H 2 N O H 2 N H 2 N O O O O O O O O O O OH O D-Phe 4 / D-Trp 4 Orn 9 / Lys 9 Pro 2 Val 8 Leu 10 Gln 6 Asn 5 Phe 3 / Trp 3 Tyr 7 / Trp 7 / Phe 7 D-Phe 1 Identity Abbr. Sequence Major natural tyrocidines Tyrocidine C 1 TrcC 1 Cyclo-(fPWw NQYVK L) Tyrocidine C TrcC Cyclo-(fPWw NQYVO L) Tyrocidine B 1 TrcB 1 Cyclo-(fPWf NQYVK L) Tyrocidine B TrcB Cyclo-(fPWf NQYVO L) Tyrocidine A 1 TrcA 1 Cyclo-(fPFf NQYVK L) Tyrocidine A TrcA Cyclo-(fPFf NQYVO L) Natural tyrocidine analogues Tryptocidine C TpcC Cyclo-(fPWw NQWVO L) Phenycidine A PhcA Cyclo-(fPFf NQFVO L) Gramicidin S GS Cyclo-(fPVOLfP VOL) Figure 3 Micrographs of 24 hour old fungal cultures with (A) untreated control of F. solani and (B) treated with 9 g/mL Trc mixture, (C) uptake of SYTOX green in tyrocidine treated F. solani hyphae and (D) uptake of propridium iodide into B. cinerea hyphae. (E) Kinetics of SYTOX green uptake into B. cinerea hyphae. Trp 11.9 Å TrcB’s Trp 3 membrane insertion POPC:Erg: 10.9 Å POPC:GlcCer: 11.3 Å TrcC’s Trp 3 / Trp 4 membrane insertion POPC:Erg: 11.9 Å POPC:GlcCer: 11.9 Å Table 1 Summary of the peptides in the library used in this study. Standard abbreviations are used for amino acids, except O = ornithine, and lowercase for D-residues Figure 1 Primary structure of tyrocidine A (TrcA). The alternative amino acids indicated at positions 3, 4, 7 and 9 can be found in the analogues of TrcA. A D B C Figure 2 The activities of the tyrocidines in PDB (A) and yeast supplemented TSB (YTSB) (B) against F. solani and B. cinerea. TpcC activity in PDB was significantly less than that of the major Trcs (***P<0.001). In YTSB PhcA’s activity was significantly less than that of the major tyrocidines (**P<0.01 for B. cinerea; ***P<0.001 for F. solani ). A B TrcA TrcB TrcC GS 0 1 2 3 4 5 6 7 8 9 10 11 12 13 70 DOPC: 30 Cholesterol 70 DOPC: 30 Ergosterol 70 DOPC 30 GlcCeramide *** * * ** ** Peptide M EC 50 (calcein release) 0 50 100 150 200 250 300 0 50 100 150 200 250 300 350 400 3.2 M Control 1.6 M 0.8 M 0.48 M Triton X100 addition 0.32 M 0.16 M Seconds after TrcA addition Fluorescence (au) 300 350 400 450 500 -10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 (nm) F (a.u.) A B A B Marina Rautenbach 1 *, Anscha M. Troskie 1 , J. Arnold Vosloo 1 , Abré de Beer 1 and Margitta Dathe 2 1 BIOPEP Peptide Group, Department of Biochemistry, University of Stellenbosch, Stellenbosch, South Africa 2 Leibniz Institute of Molecular Pharmacology, Berlin, Germany Corresponding Author. Tel: +27-21-8085872, Email: mra@sun.ac.za E View publication stats View publication stats