Antifungal Effects of Fusion Puroindoline B on the Surface and Intracellular Environment of Aspergillus flavus


Aspergillus flavus infection is a major issue for safe food storage. In this study, we constructed an efficient prokaryotic expression system for puroindoline B (PINB) protein to detect its antifungal activity. The Puroindoline b gene was cloned into pET-28a (+) vector and expressed in Escherichia coli. Treatment with fusion PINB revealed that it inhibits mycelial growth of A. flavus, a common grain mold. Moreover, fusion PINB-treated A. flavus mycelium withered and exhibited a sunken spore head. As fusion PINB concentration increased, electrical conductivity in mycelium also increased, indicative of cell membrane damage. Furthermore, intracellular malate dehydrogenase and succinate dehydrogenase activity decreased, revealing a disruption in the tricarboxylic acid cycle. Moreover, the dampened activity of the ion pump Na+K+-ATPase negatively affected the intracellular regulation of both ions. Catalase and superoxide dismutase activity decreased, thus reducing antioxidant capacity, a result confirmed with an increase in malondialdehyde content. Changes to the GSH/GSSG ratio indicated a shift to an intracellular oxidative state. At the same time, laser scanning confocal microscopy assay showed the accumulation of reactive oxygen species and nuclear damage. Therefore, the PINB fusion protein may have the potential to control A. flavus in grain storage and food preservation.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7


  1. 1.

    Lahouar A, Marin S, Crespo-Sempere A, Saïd S, Sanchis V (2016) Effects of temperature, water activity and incubation time on fungal growth and aflatoxin B1 production by toxinogenic Aspergillus flavus isolates on sorghum seeds. Rev Argent Microbiol 48(1):78–85.

    Article  PubMed  Google Scholar 

  2. 2.

    Astoreca A, Vaamonde G, Dalcero A, Marin S, Ramos A (2014) Abiotic factors and their interactions influence on the co-production of aflatoxin B1 and cyclopiazonic acid by Aspergillus flavus isolated from corn. Food Microbiol 38:276–283.

    CAS  Article  PubMed  Google Scholar 

  3. 3.

    Masiello M, Somma S, Ghionna V, Logrieco AF, Moretti A (2019) In vitro and in field response of different fungicides against Aspergillus flavus and Fusarium species causing ear rot disease of maize. Toxins 11(1):18.

    CAS  Article  Google Scholar 

  4. 4.

    Casquete R, Benito MJ, Córdoba MG, Ruiz-Moyano S, Martín A (2017) The growth and aflatoxin production of Aspergillus flavus strains on a cheese model system are influenced by physicochemical factors. J Dairy Sci 100(9):6987–6996.

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    Prencipe S, Siciliano I, Contessa C, Botta R, Garibaldi A, Gullino ML, Spadaro D (2018) Characterization of Aspergillus section Flavi isolated from fresh chestnuts and along the chestnut flour process. Food Microbiol 69:159–169.

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Al-Dhabi NA, Valan Arasu M (2018) Environmentally-friendly green approach for the production of zinc oxide nanoparticles and their anti-fungal, ovicidal, and larvicidal properties. Nanomaterials 8(7):500.

    CAS  Article  PubMed Central  Google Scholar 

  7. 7.

    Kimura H, Asano R, Tsukamoto N, Tsugawa W, Sode K (2018) Convenient and universal fabrication method for antibody–enzyme complexes as sensing elements using the SpyCatcher/SpyTag system. Anal Chem 90(24):14500–14506.

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Abdel-Kareem MM, Rasmey AM, Zohri AA (2019) The action mechanism and biocontrol potentiality of novel isolates of Saccharomyces cerevisiae against the aflatoxigenic Aspergillus flavus. Lett Appl Microbiol 68(2):104–111.

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Chaudhari AK, Singh VK, Dwivedy AK, Das S, Upadhyay N, Singh A, Dkhar MS, Kayang H, Prakash B, Dubey NK (2020) Chemically characterised Pimenta dioica (L.) Merr. essential oil as a novel plant based antimicrobial against fungal and aflatoxin B1 contamination of stored maize and its possible mode of action. Nat Prod Res 34(5):745–749.

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Devipriya D, Roopan SM (2019) UV-light intervened synthesis of imidazo fused quinazoline and its solvatochromism, antioxidant, antifungal and luminescence properties. J Photochem Photobiol B 190:42–49.

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Kim JE, Oh YJ, Song AY, Min SC (2019) Preservation of red pepper flakes using microwave-combined cold plasma treatment. J Sci Food Agric 99(4):1577–1585.

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Hu Y, Zhang J, Kong W, Zhao G, Yang M (2017) Mechanisms of antifungal and anti-aflatoxigenic properties of essential oil derived from turmeric (Curcuma longa L.) on Aspergillus flavus. Food Chem 220:1–8.

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Yadav SKR, Sahu T, Dixit A (2016) Structural and functional characterization of recombinant napin-like protein of momordica charantia expressed in methylotrophic yeast Pichia pastoris. Appl Microbiol Biotechnol 100(15):6703–6713.

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Yu Y, Zhang G, Li Z, Cheng Y, Gao C, Zeng L, Chen J, Yan L, Sun X, Guo L, Yan Z (2017) Molecular cloning, recombinant expression and antifungal activity of BnCPI, a Cystatin in Ramie (Boehmeria nivea L.). Genes 8(10):265.

    CAS  Article  PubMed Central  Google Scholar 

  15. 15.

    Bogdanov IV, Shenkarev ZO, Finkina EI, Melnikova DN, Rumynskiy EI, Arseniev AS, Ovchinnikova TV (2016) A novel lipid transfer protein from the pea Pisum sativum: isolation, recombinant expression, solution structure, antifungal activity, lipid binding, and allergenic properties. BMC Plant Biol 16(1):107.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Liang Y, Kong Q, Yao Y, Xu S, Xie X (2019) Fusion expression and anti-Aspergillus flavus activity of a novel inhibitory protein DN-AflR. Int J Food Microbiol 290:184–192.

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    Ishida H, Nguyen LT, Gopal R, Aizawa T, Vogel HJ (2016) Overexpression of antimicrobial, anticancer, and transmembrane peptides in Escherichia coli through a calmodulin-peptide fusion system. J Am Chem Soc 138(35):11318–11326.

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Blochet JE, Chevalier C, Forest E, Pebay-Peyroula E, Gautier MF, Joudrier P, Pézolet M, Marion D (1993) Complete amino acid sequence of puroindoline, a new basic and cystine-rich protein with a unique tryptophan-rich domain, isolated from wheat endosperm by Triton X-114 phase partitioning. FEBS Lett 329(3):336–340.

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Haney EF, Petersen AP, Lau CK, Jing W, Storey DG, Vogel HJ (2013) Mechanism of action of puroindoline derived tryptophan-rich antimicrobial peptides. Biochim Biophys Acta 1828(8):1802–1813.

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Sanders MR, Clifton LA, Frazier RA, Green RJ (2017) Tryptophan to arginine substitution in puroindoline-b alters binding to model eukaryotic membrane. Langmuir 33(19):4847–4853.

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Alfred RL, Palombo EA, Panozzo JF, Bhave M (2013) The antimicrobial domains of wheat puroindolines are cell-penetrating peptides with possible intracellular mechanisms of action. PLoS One 8(10):e75488.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Miao Y, Chen L, Wang C, Wang Y, Zheng Q, Gao C, Yang G, He G (2012) Expression, purification and antimicrobial activity of puroindoline a protein and its mutants. Amino Acids 43(4):1689–1696.

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Dubreil L, Gaborit T, Bouchet B, Gallant DJ, Broekaert WF, Quillien L, Marion D (1998) Spatial and temporal distribution of the major isoforms of puroindolines (puroindoline-a and puroindoline-b) and non specific lipid transfer protein (ns-LTP1e1) of Triticum aestivum seeds: relationships with their in vitro antifungal properties. Plant Sci 138(2):121–135.

    CAS  Article  Google Scholar 

  24. 24.

    Capparelli R, Amoroso MG, Palumbo D, Iannaccone M, Faleri C, Cresti M (2005) Two plant puroindolines colocalize in wheat seed and in vitro synergistically fight against pathogens. Plant Mol Biol 58(6):857–867.

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Shagaghi N, Alfred RL, Clayton AHA, Palombo EA, Bhave M (2016) Anti-biofilm and sporicidal activity of peptides based on wheat puroindoline and barley hordoindoline proteins. J Pept Sci 22(7):492–500.

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Capparelli R, Ventimiglia I, Palumbo D, Nicodemo D, Salvatore P, Amoroso MG, Iannaccone M (2007) Expression of recombinant puroindolines for the treatment of staphylococcal skin infections (acne vulgaris). J Biotechnol 128(3):606–614.

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Capparelli R, Palumbo D, Iannaccone M, Ventimiglia I, Di Salle E, Capuano F, Salvatore P, Amoroso MG (2006) Cloning and expression of two plant proteins: similar antimicrobial activity of native and recombinant form. Biotechnol Lett 28(13):943–949.

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Caporale C, Di Berardino I, Leonardi L, Bertini L, Cascone A, Buonocore V, Caruso C (2004) Wheat pathogenesis-related proteins of class 4 have ribonuclease activity. FEBS Lett 575:71–76.

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Baneyx F, Mujacic M (2004) Recombinant protein folding and misfolding in Escherichia coli. Nat Biotechnol 22(11):1399–1408.

    CAS  Article  PubMed  Google Scholar 

  30. 30.

    Maxwell KL, Bona D, Liu C, Arrowsmith CH, Edwards AM (2003) Refolding out of guanidine hydrochloride is an effective approach for high-throughput structural studies of small proteins. Protein Sci 12(9):2073–2080.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Clifton LA, Green RJ, Frazier RA (2007) Puroindoline-b mutations control the lipid binding interactions in mixed puroindoline-a:puroindoline-b systems. Biochemistry 46(48):13929–13937.

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Wang QY, Lin QL, Peng K, Cao JZ, Yang C, Xu D (2017) Surfactin variants from Bacillus subtilis natto CSUF5 and their antifungal properities against Aspergillus niger. J Biobased Mater Bioenergy 11(3):210–215.

    CAS  Article  Google Scholar 

  33. 33.

    Moon TM, D'Andréa ÉD, Lee CW, Soares A, Jakoncic J, Desbonnet C, Garcia-Solache M, Rice LB, Page R, Peti W (2018) The structures of penicillin-binding protein 4 (PBP4) and PBP5 from Enterococci provide structural insights into β-lactam resistance. J Biol Chem 293(48):18574–18584.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Bernardo-García N, Mahasenan KV, Batuecas MT, Lee M, Hesek D, Petráčková D, Doubravová L, Branny P, Mobashery S, Hermoso JA (2018) Allostery, recognition of nascent peptidoglycan, and cross-linking of the cell wall by the essential penicillin-binding protein 2x of Streptococcus pneumoniae. ACS Chem Biol 13(3):694–702.

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Ytzhak S, Ehrenberg B (2014) The effect of photodynamic action on leakage of ions through liposomal membranes that contain oxidatively modified lipids. Photochem Photobiol 90(4):796–800.

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    Gurtovenko AA, Vattulainen I (2007) Ion leakage through transient water pores in protein-free lipid membranes driven by transmembrane ionic charge imbalance. Biophys J 92(6):1878–1890.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Lee MT, Yang PY, Charron NE, Hsieh MH, Chang YY, Huang HW (2018) Comparison of the effects of daptomycin on bacterial and model membranes. Biochemistry 57(38):5629–5639.

    CAS  Article  PubMed  Google Scholar 

  38. 38.

    Wojda I, Alonso-Monge R, Bebelman JP, Mager WH, Siderius M (2003) Response to high osmotic conditions and elevated temperature in Saccharomyces cerevisiae is controlled by intracellular glycerol and involves coordinate activity of MAP kinase pathways. Microbiology 149(5):1193–1204.

    CAS  Article  PubMed  Google Scholar 

  39. 39.

    Czövek P, Király I (2011) Inducible trehalase enzyme activity of Morchella conica Persoon mycelium. Acta Microbiol Immunol Hung 58(1):1–11.

    CAS  Article  PubMed  Google Scholar 

  40. 40.

    Cox SD, Mann CM, Markham JL (2001) Interactions between components of the essential oil of Melaleuca alternifolia. J Appl Microbiol 91(3):492–497.

    CAS  Article  PubMed  Google Scholar 

  41. 41.

    Akram M (2014) Citric acid cycle and role of its intermediates in metabolism. Cell Biochem Biophys 68(3):475–478.

    CAS  Article  PubMed  Google Scholar 

  42. 42.

    Tian J, Ban XQ, Zeng H, He JS, Chen YX, Wang YW (2012) The mechanism of antifungal action of essential oil from dill (Anethum graveolens L.) on Aspergillus flavus. PLoS One 7(1):e30147.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Ma WB, Zhao LL, Zhao WH, Xie YL (2019) (E)-2-Hexenal, as a potential natural antifungal compound, inhibits Aspergillus flavus spore germination by disrupting mitochondrial energy metabolism. J Agric Food Chem 67(4):1138–1145.

    CAS  Article  PubMed  Google Scholar 

  44. 44.

    Wang CH, Wu SB, Wu YT, Wei YH (2013) Oxidative stress response elicited by mitochondrial dysfunction: implication in the pathophysiology of aging. Exp Biol Med 238(5):450–460.

    CAS  Article  Google Scholar 

  45. 45.

    Saidana D, Boussaada O, Ayed F, Mahjoub MA, Mighri Z, Helal AN (2013) The in vitro free radical-scavenging and antifungal activities of the medicinal herb Limonium echioides L. growing wild in Tunisia. J Food Process Pres 37(5):533–540.

    CAS  Article  Google Scholar 

  46. 46.

    Long DD, Fu RR, Han JR (2017) Tolerance and stress response of sclerotiogenic Aspergillus oryzae G15 to copper and lead. Folia Microbiol 62(4):295–304.

    CAS  Article  Google Scholar 

  47. 47.

    Cavalcanti Luna MA, Vieira ER, Okada K, Campos-Takaki GM, Nascimento AE (2015) Copper-induced adaptation, oxidative stress and its tolerance in Aspergillus niger UCP1261. Electron J Biotechnol 18(6):418–427.

    CAS  Article  Google Scholar 

  48. 48.

    Lanubile A, Maschietto V, De Leonardis S, Battilani P, Paciolla C, Marocco A (2015) Defense responses to mycotoxin-producing fungi Fusarium proliferatum, F. subglutinans, and Aspergillus flavus in kernels of susceptible and resistant maize genotypes. Mol Plant Microbe In 28(5):546–557.

    CAS  Article  Google Scholar 

  49. 49.

    Wang Y, Feng K, Yang H, Yuan Y, Yue T (2018) Antifungal mechanism of cinnamaldehyde and citral combination against Penicillium expansum based on FT-IR fingerprint, plasma membrane, oxidative stress and volatile profile. RSC Adv 8(11):5806–5815.

    CAS  Article  Google Scholar 

  50. 50.

    Sporer AJ, Kahl LJ, Price-Whelan A, Dietrich LEP (2017) Redox-based regulation of bacterial development and behavior. Annu Rev Biochem 86:777–797.

    CAS  Article  PubMed  Google Scholar 

  51. 51.

    Kumar KJS, Chu F-H, Hsieh H-W, Liao J-W, Li W-H, Lin JC-C, Shaw J-F, Wang S-Y (2011) Antroquinonol from ethanolic extract of mycelium of Antrodia cinnamomea protects hepatic cells from ethanol-induced oxidative stress through Nrf-2 activation. J Ethnopharmacol 136(1):168–177.

    CAS  Article  PubMed  Google Scholar 

  52. 52.

    Grintzalis K, Vernardis SI, Klapa MI, Georgiou CD (2014) Role of oxidative stress in sclerotial differentiation and aflatoxin B1 biosynthesis in Aspergillus flavus. Appl Environ Microbiol 80(18):5561–5571.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Zaccaria M, Ludovici M, Sanzani SM, Ippolito A, Cigliano RA, Sanseverino W, Scarpari M, Scala V, Fanelli C, Reverberi M (2015) Menadione-induced oxidative stress re-shapes the oxylipin profile of Aspergillus flavus and its lifestyle. Toxins 7(10):4315–4329.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Zhang F, Xu GP, Geng LP, Lu XY, Yang KL, Yuan J, Nie XY, Zhuang ZH, Wang SH (2016) The stress response regulator AflSkn7 influences morphological development, stress response, and pathogenicity in the fungus Aspergillus flavus. Toxins 8(7):202.

    CAS  Article  PubMed Central  Google Scholar 

  55. 55.

    Marchetti P, Decaudin D, Macho A, Zamzami N, Hirsch T, Susin SA, Kroemer G (1997) Redox regulation of apoptosis: impact of thiol oxidation status on mitochondrial function. Eur J Immunol 27(1):289–296.

    CAS  Article  PubMed  Google Scholar 

Download references


This study was supported by the National Science Foundation of China (grand numbers 31871852, 31501575) and Natural Science Foundation of Henan Province (grand number 162300410047).

Author information



Corresponding author

Correspondence to Yuan-Sen Hu.

Ethics declarations

Conflict of Interest

The authors declare that there is no conflict of interest.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Tian, P., Lv, Y., Lv, A. et al. Antifungal Effects of Fusion Puroindoline B on the Surface and Intracellular Environment of Aspergillus flavus. Probiotics & Antimicro. Prot. (2020).

Download citation


  • Aspergillus flavus
  • Puroindoline B
  • Energy metabolism
  • Oxidative stress
  • The nuclear damage