Accessible Requires Authentication Published by De Gruyter November 2, 2016

The insect-derived antimicrobial peptide metchnikowin targets Fusarium graminearum β(1,3)glucanosyltransferase Gel1, which is required for the maintenance of cell wall integrity

Mohammad-Reza Bolouri Moghaddam, Andreas Vilcinskas and Mohammad Rahnamaeian
From the journal Biological Chemistry


Antimicrobial peptides (AMPs) are essential components of the insect innate immune system. Their diversity provides protection against a broad spectrum of microbes and they have several distinct modes of action. Insect-derived AMPs are currently being developed for both medical and agricultural applications, and their expression in transgenic crops confers resistance against numerous plant pathogens. The antifungal peptide metchnikowin (Mtk), which was originally discovered in the fruit fly Drosophila melanogaster, is of particular interest because it has potent activity against economically important phytopathogenic fungi of the phylum Ascomycota, such as Fusarium graminearum, but it does not harm beneficial fungi such as the mycorrhizal basidiomycete Piriformospora indica. To investigate the specificity of Mtk, we used the peptide to screen a F. graminearum yeast two-hybrid library. This revealed that Mtk interacts with the fungal enzyme β(1,3)-glucanosyltransferase Gel1 (FgBGT), which is one of the enzymes responsible for fungal cell wall synthesis. The interaction was independently confirmed in a second interaction screen using mammalian cells. FgBGT is required for the viability of filamentous fungi by maintaining cell wall integrity. Our study therefore paves the way for further applications of Mtk in formulation of bio fungicides or as a supplement in food preservation.


The authors acknowledge generous funding by the Hessen State Ministry of Higher Education, Research and the Arts (HMWK) via the ‘LOEWE Center for Insect Biotechnology and Bioresources’ and from the Federal Ministry of Education. We thank Dr. Richard M. Twyman for editing the manuscript.

  1. Conflict of interest statement: The authors declare there is no conflict of interest.


Al Souhail, Q., Hiromasa, Y., Rahnamaeian, M., Giraldo, MC., Takahashi, D., Valent, B., Vilcinskas, A., and Kanost, M. R. (2016). Characterization and regulation of expression of an antifungal peptide from hemolymph of an insect, Manduca sexta. Dev. Comp. Immunol. 61, 258–268. Search in Google Scholar

Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–402. Search in Google Scholar

Anisimova, M. and Gascuel, O. (2006). Approximate likelihood-ratio test for branches: a fast, accurate, and powerful alternative. Syst. Biol. 55, 539–552. Search in Google Scholar

Beauvais, A., Bruneau, J. M., Mol, P. C., Buitrago, M. J., Legrand, R., and Latge, J. P. (2001). Glucan synthase complex of Aspergillus fumigatus. J. Bacteriol. 183, 2273–2279. Search in Google Scholar

Bolouri Moghaddam, M. R., Vilcinskas, A., and Rahnamaeian, M. (2015). Cooperative interaction of antimicrobial peptides with the interrelated immune pathways in plants. Mol. Plant Pathol. 17, 464–71. Search in Google Scholar

Bolouri Moghaddam, M. R., Tonk, M., Schreiber, C., Salzig, D., Czermak, P., Vilcinskas, A., and Rahnamaeian, M. (2016). The potential of the Galleria mellonella innate immune system is maximized by the co-presentation of diverse antimicrobial peptides. Biol. Chem. 397, 939–945. Search in Google Scholar

Bowman, S. M. and Free, S. J. (2006). The structure and synthesis of the fungal cell wall. Bioessays. 28, 799–808. Search in Google Scholar

Brogden, K. A. (2005). Antimicrobial peptides: Pore formers or metabolic inhibitors in bacteria? Nat. Rev. Microbiol. 3, 238–250. Search in Google Scholar

Cabezas-Cruz, A., Tonk, M., Bouchut, A., Pierrot, C., Pierce, R. J., Kotsyfakis, M., Rahnamaeian, M., Vilcinskas, A., Khalife, J., and Valdes, J. J. (2016). Antiplasmodial activity is an ancient and conserved feature of tick defensins. Front. Microbiol. 7, 1682. Search in Google Scholar

Caracuel, Z., Martinez-Rocha, A. L., Di Pietro, A., Madrid, M. P., and Roncero, M. I. G. (2005). Fusarium oxysporum gas1 encodes a putative β-1,3-glucanosyltransferase required for virulence on tomato plants. Mol. Plant Microbe Interact. 18, 1140–1147. Search in Google Scholar

Castresana, J. (2000). Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol. Biol. Evol. 17, 540–552. Search in Google Scholar

Chevenet, F., Brun, C., Banuls, A. L., Jacq, B., and Christen, R. (2006). TreeDyn: towards dynamic graphics and annotations for analyses of trees. BMC Bioinformatics 7, 439. Search in Google Scholar

Dereeper, A., Guignon, V., Blanc, G., Audic, S., Buffet, S., Chevenet, F., Dufayard, J. F., Guindon, S., Lefort, V., Lescot, M., et al. (2008). robust phylogenetic analysis for the non-specialist. Nucleic Acids Res. 36, W465–W469. Search in Google Scholar

Edgar, R. C. (2004). MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797. Search in Google Scholar

Guindon, S., Dufayard, J. F., Lefort, V., Anisimova, M., Hordijk, W., and Gascuel, O. (2010). New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst. Biol. 59, 307–321. Search in Google Scholar

Henrissat, B. and Davies, G. (1997). Structural and sequence-based classification of glycoside hydrolases. Curr. Opin. Struct. Biol. 7, 637–644. Search in Google Scholar

Henrissat, B. and Davies, G. J. (2000). Glycoside hydrolases and glycosyltransferases. Families, modules, and implications for genomics. Plant Physiol. 124, 1515–1519. Search in Google Scholar

Kamei, M., Yamashita, K., Takahashi, M., Fukumori, F., Ichiishi, A., and Fujimura, M. (2013). Deletion and expression analysis of β-(1,3)-glucanosyltransferase genes in Neurospora crassa. Fungal Genet. Biol. 52, 65–72. Search in Google Scholar

Langen, G., Imani, J., Altincicek, B., Kieseritzky, G., Kogel, K. H., and Vilcinskas, A. (2006). Transgenic expression of gallerimycin, a novel antifungal insect defensin from the greater wax moth Galleria mellonella, confers resistance to pathogenic fungi in tobacco. Biol. Chem. 387, 549–557. Search in Google Scholar

Langfelder, K., Jahn, B., Gehringer, H., Schmidt, A., Wanner, G., and Brakhage, A. A. (1998). Identification of a polyketide synthase gene (pksP) of Aspergillus fumigatus involved in conidial pigment biosynthesis and virulence. Med. Microbiol. Immunol. 187, 79–89. Search in Google Scholar

Levashina, E. A., Ohresser, S., Bulet, P., Reichhart, J. M., Hetru, C., and Hoffmann, J. A. (1995). Metchnikowin, a novel immune-inducible proline-rich peptide from Drosophila with antibacterial and antifungal properties. Eur. J. Biochem. 233, 694–700. Search in Google Scholar

Liu, J. and Balasubramanian, M. K. (2001). 1,3-β-Glucan synthase: a useful target for antifungal drugs. Curr. Drug. Targets Infect. Disord. 1, 159–169. Search in Google Scholar

Marchler-Bauer, A. and Bryant, S. H. (2004). CD-Search: protein domain annotations on the fly. Nucleic Acids Res. 32, W327–W331. Search in Google Scholar

Marchler-Bauer, A., Zheng, C. J., Chitsaz, F., Derbyshire, M. K., Geer, L. Y., Geer, R. C., Gonzales, N. R., Gwadz, M., Hurwitz, D. I., Lanczycki, C. J., et al. (2013). CDD: conserved domains and protein three-dimensional structure. Nucleic Acids Res. 41, D348–D352. Search in Google Scholar

Mazan, M., Ragni, E., Popolo, L., and Farkas, V. (2011). Catalytic properties of the Gas family β-(1,3)-glucanosyltransferases active in fungal cell-wall biogenesis as determined by a novel fluorescent assay. Biochem. J. 438, 275–282. Search in Google Scholar

Moretti, A., Panzarini, G., Somma, S., Campagna, C., Ravaglia, S., Logrieco, A. F., and Solfrizzo, M. (2014). Systemic growth of F. graminearum in wheat plants and related accumulation of deoxynivalenol. Toxins (Basel) 6, 1308–1324. doi:10.3390/toxins6041308. Search in Google Scholar

Mouyna, I., Fontaine, T., Vai, M., Monod, M., Fonzi, W. A., Diaquin, M., Popolo, L., Hartland, R. P., and Latge, J. P. (2000). Glycosylphosphatidylinositol-anchored glucanosyltransferases play an active role in the biosynthesis of the fungal cell wall. J. Biol. Chem. 275, 14882–14889. Search in Google Scholar

Mylonakis, E., Podsiadlowski, L., Muhammed, M., and Vilcinskas, A. (2016). Diversity, evolution and medical applications of insect antimicrobial peptides. Phil. Trans. R. Soc. B 371, 20150290. Search in Google Scholar

Pöppel, A. K., Koch, A., Kogel, K. H., Vogel, H., Kollewe, C., Wiesner, J., and Vilcinskas, A. (2014). Lucimycin, an antifungal peptide from the therapeutic maggot of the common green bottle fly Lucilia sericata. Biol. Chem. 395, 649–656. Search in Google Scholar

Rahnamaeian, M. and Vilcinskas, A. (2012). Defense gene expression is potentiated in transgenic barley expressing antifungal peptide metchnikowin throughout powdery mildew challenge. J. Plant Res. 125, 115–124. Search in Google Scholar

Rahnamaeian, M. and Vilcinskas, A. (2015). Short antimicrobial peptides as cosmetic ingredients to deter dermatological pathogens. Appl. Microbiol. Biotechnol. 99, 8847–8855. Search in Google Scholar

Rahnamaeian, M., Cytrynska, M., Zdybicka-Barabas, A., and Vilcinskas, A. (2016). The functional interaction between abaecin and pore-forming peptides indicates a general mechanism of antibacterial potentiation. Peptides 78, 17–23. Search in Google Scholar

Rahnamaeian, M., Langen, G., Imani, J., Khalifa, W., Altincicek, B., von Wettstein, D., Kogel, K. H., and Vilcinskas, A. (2009). Insect peptide metchnikowin confers on barley a selective capacity for resistance to fungal ascomycetes pathogens. J. Exp. Bot. 60, 4105–4114. Search in Google Scholar

Samalova, M., Mélida, H., Vilaplana, F., Bulone, V., Soanes, D. M., Talbot, N. J., and Gurr, S. J. (2016). The β-1,3-glucanosyltransferases (Gels) affect the structure of the rice blast fungal cell wall during appressorium-mediated plant infection. Cell. Microbiol. doi: 10.1111/cmi.12659. Search in Google Scholar

Sheehan, D. J., Hitchcock, C. A., and Sibley, C. M. (1999). Current and emerging azole antifungal agents. Clin. Microbiol. Rev. 12, 40–79. Search in Google Scholar

Tonk, M., Cabezas-Cruz, A., Valdes, J. J., Rego, R. O. M., Chrudimska, T., Strnad, M., Sima, R., Bell-Sakyi, L., Franta, Z., Vilcinskas, A., et al. (2014). Defensins from the tick Ixodes scapularis are effective against phytopathogenic fungi and the human bacterial pathogen Listeria grayi. Parasit. Vectors 7, 554. Search in Google Scholar

Tonk, M., Vilcinskas, A., and Rahnamaeian, M. (2016). Insect antimicrobial peptides: potential tools for the prevention of skin cancer. Appl. Microbiol. Biotechnol. 100, 7397–7405. Search in Google Scholar

Vilcinskas, A. and Gross, J. (2005). Drugs from bugs: the use of insects as a valuable source of transgenes with potential in modern plant protection strategies. J. Pest Sci. 78, 187–191. Search in Google Scholar

Yurlova, L., Derks, M., Buchfellner, A., Hickson, I., Janssen, M., Morrison, D., Stansfield, I., Brown, C. J., Ghadessy, F. J., Lane, D. P., et al. (2014). The fluorescent two-hybrid assay to screen for protein-protein interaction inhibitors in live cells: targeting the interaction of p53 with Mdm2 and Mdm4. J. Biomol. Screen. 19, 516–525. Search in Google Scholar

Zolghadr, K., Mortusewicz, O., Rothbauer, U., Kleinhans, R., Goehler, H., Wanker, E. E., Cardoso, M. C., and Leonhardt, H. (2008). A fluorescent two-hybrid assay for direct visualization of protein interactions in living cells. Mol. Cell. Proteomics 7, 2279–2287. Search in Google Scholar

Received: 2016-9-21
Accepted: 2016-10-28
Published Online: 2016-11-2
Published in Print: 2017-4-1

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