Insight into the antifungal mechanism of Neosartorya fischeri antifungal protein

Abstract

Small, cysteine-rich, highly stable antifungal proteins secreted by filamentous Ascomycetes have great potential for the development of novel antifungal strategies. However, their practical application is still limited due to their not fully clarified mode of action. The aim of this work was to provide a deep insight into the antifungal mechanism of Neosartorya fischeri antifungal protein (NFAP), a novel representative of this protein group. Within a short exposure time to NFAP, reduced cellular metabolism, apoptosis induction, changes in the actin distribution and chitin deposition at the hyphal tip were observed in NFAP-sensitive Aspergillus nidulans. NFAP did show neither a direct membrane disruptingeffect nor uptake by endocytosis. Investigation of A. nidulans signalling mutants revealed that NFAP activates the cAMP/protein kinase A pathway via G-protein signalling which leads to apoptosis and inhibition of polar growth. In contrast, NFAP does not have any influence on the cell wall integrity pathway, but an unknown cell wall integrity pathway-independent mitogen activated protein kinase A-activated target is assumed to be involved in the cell death induction. Taken together, it was concluded that NFAP shows similarities, but also differences in its mode of antifungal action compared to two most investigated NFAP-related proteins from Aspergillus giganteus and Penicillium chrysogenum.

Key words： Neosartorya fischeri antifungal protein; Aspergillus nidulans; cAMP/Pka signalling; Pkc/Mpk signalling; antifungal mechanism

References

1 Bencina M, Legisa M, Read ND (2005) Cross-talk between cAMP and calcium signalling in Aspergillus niger. Mol Microbiol56: 268-281

2 Binder U, Oberparleiter C, Meyer V, Marx F (2010) The antifungal protein PAF interferes with PKC/MPK and cAMP/PKA signalling of Aspergillus nidulans. Mol Microbiol75: 294-307

3 Binder U, Bencina M, Eigentler A, Meyer V, Marx F (2011) The Aspergillus giganteus antifungal protein AFP_NN5353 activates the cell wall integrity pathway and perturbs calcium homeostasis. BMC Microbiol11: 209

4 Bussink HJ, Osmani SA (1999) A mitogen-activated protein kinase (MPKA) is involved in polarized growth in the filamentous fungus, Aspergillus nidulans. FEMS Microbiol Lett173: 117-125

5 Colabardini AC, De Castro PA, De Gouvêa PF, Savoldi M, Malavazi I, Goldman MH, Goldman GH (2010) Involvement of the Aspergillus nidulans protein kinase C with farnesol tolerance is related to the unfolded protein response. Mol Microbiol78: 1259-1279

6 Duncan VMS, O’Neil DA(2013) Commercialization of antifungal peptides. Fungal Biol. Rev. 26: 156-165

7 Fischer R, Timberlake WE(1995) Aspergillus nidulans apsA (anucleate primary sterigmata) encodes a coiled-coil protein required for nuclear positioning and completion of asexual development. J Cell Biol128: 485-498

8 Fujioka T, Mizutani O, Furukawa K, Sato N, Yoshimi A, Yamagata Y, Nakajima T, Abe K (2007) MpkA-dependent and -independent cell wall integrity signaling in Aspergillus nidulans. Eukaryot Cell6: 1497-1510

9 Galgóczy L, Kovács L, Karácsony Z, Virágh M, Hamari Zs, Vágvölgyi Cs (2013) Investigation of the antimicrobial effect of Neosartorya fischeri antifungal protein (NFAP) after heterologous expression in Aspergillus nidulans. Microbiol-SGM159: 411-419

10 Gorovits R, Yarden O (2003) Environmental suppression of Neurospora crassa cot-1 hyperbranching: a link between COT1 kinase and stress sensing. Eukaryot Cell2: 699-707

11 Guest GM, Lin X, Momany M (2004) Aspergillus nidulans RhoA is involved in polar growth, branching, and cell wall synthesis. Fungal Genet Biol41: 13-22

12 Hagen S, Marx F, Ram AF, Meyer V (2007) The antifungal protein AFP from Aspergillus giganteus inhibits chitin synthesis in sensitive fungi. Appl Environ Microbiol73: 2128-2134

13 Hegedüs N, Marx F (2013) Antifungal proteins: More than antimicrobials? Fungal Biol. Rev.26: 132-145

14 Hegedus N, Leiter E, Kovács B, Tomori V, Kwon NJ, Emri T, Marx F, Batta G, Csernoch L (2011) The small molecular mass antifungal protein of Penicillium chrysogenum–a mechanism of action oriented review. J Basic Microbiol51: 561-571

15 Kaiserer L, Oberparleiter C, Weiler-Görz R, Burgstaller W, Leiter E, Marx F (2003) Characterization of the Penicillium chrysogenum antifungal protein PAF. Arch Microbiol180: 204-210

16 Katayama T, Uchida H, Ohta A, Horiuchi H (2012) Involvement of protein kinase C in the suppression of apoptosis and in polarity establishment in Aspergillus nidulans under conditions of heat stress. PLoS One7: e50503

17 Kovács L, Virágh M, Takó M, Papp T, Vágvölgyi Cs, Galgóczy L (2011) Isolation and characterization of Neosartorya fischeri antifungal protein (NFAP). Peptides32: 1724-1731

18 Kuranda K, Leberre V, Sokol S, Palamarczyk G, François J (2006) Investigating the caffeine effects in the yeast Saccharomyces cerevisiae brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways. Mol Microbiol61: 1147-1166

19 Leiter E, Szappanos H, Oberparleiter C, Kaiserer L, Csernoch L, Pusztahelyi T, Emri T, Pócsi I, Salvenmoser W, Marx F (2005) Antifungal protein PAF severely affects the integrity of the plasma membrane of Aspergillus nidulans and induces an apoptosis-like phenotype. Antimicrob Agents Chemother49: 2445-2453

20 Marx F, Binder U, Leiter E, Pócsi I (2008) The Penicillium chrysogenum antifungal protein PAF, a promising tool for the development of new antifungal therapies and fungal cell biology studies. Cell Mol Life Sci65: 445-454

21 Meyer V (2008) A small protein that fights fungi: AFP as a new promising antifungal agent of biotechnological value. Appl Microbiol Biotechnol78: 17-28

22 Montesinos E (2007) Antimicrobial peptides and plant disease control. FEMS Microbiol Lett270: 1-11

23 Oberparleiter C, Kaiserer L, Haas H, Ladurner P, Andratsch M, Marx F (2003) Active internalization of the Penicillium chrysogenum antifungal protein PAF in sensitive aspergilli. Antimicrob Agents Chemother47: 3598-3601

24 Ouedraogo JP, Hagen S, Spielvogel A, Engelhardt S, Meyer V (2011) Survival strategies of yeast and filamentous fungi against the antifungal protein AFP. J Biol Chem286: 13859-13868

25 Ronen R, Sharon H, Levdansky E, Romano J, Shadkchan Y, Osherov N (2007) The Aspergillus nidulans pkcA gene is involved in polarized growth, morphogenesis and maintenance of cell wall integrity. Curr Genet51: 321-329

26 Semighini CP, Hornby JM, Dumitru R, Nickerson KW, Harris SD (2006) Farnesol-induced apoptosis in Aspergillus nidulans reveals a possible mechanism for antagonistic interactions between fungi. Mol Microbiol59: 753-764

27 Shimizu K, Keller NP (2001) Genetic involvement of a cAMPdependent protein kinase in a G protein signaling pathway regulating morphological and chemical transitions in Aspergillus nidulans. Genetics157: 591-600

28 Taheri-Talesh N, Horio T, Araujo-Bazán L, Dou X, Espeso EA, Peñalva MA, Osmani SA, Oakley BR (2008) The tip growth apparatus of Aspergillus nidulans. Mol Biol Cell19: 1439-1449

29 Theis T, Wedde M, Meyer V, Stahl U (2003) The antifungal protein from Aspergillus giganteus causes membrane permeabilization. Antimicrob Agents Chemother47: 588-593

30 Theis T, Marx F, Salvenmoser W, Stahl U, Meyer V (2005) New insights into the target site and mode of action of the antifungal protein of Aspergillus giganteus. Res Microbiol156: 47-56

31 Van Dijck P (2009) Nutrient sensing G protein-coupled receptors: interesting targets for antifungals? Med Mycol47: 671-680

32 Virágh M, Vörös D, Kele Z, Kovács L, Fizil Á, Lakatos G, Maróti G, Batta G, Vágvölgyi Cs, Galgóczy L (2014) Production of a defensin-like antifungal protein NFAP from Neosartorya fischeri in Pichia pastoris and its antifungal activity against filamentous fungal isolates from human infections. Protein Expr Purif94: 79-84

33 Yu JH, Wieser J, Adams TH (1996) The Aspergillus FlbA RGS domain protein antagonizes G protein signaling to block proliferation and allow development. EMBO J15: 5184-5190