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Toxicon 48 (2006) 1075–1082 www.elsevier.com/locate/toxicon
Ultrastructural evidences of growth inhibitory effects of a novel biocide, Akacidsplus, on an aflatoxigenic Aspergillus parasiticus Mehdi Razzaghi-Abyaneha,, Masoomeh Shams-Ghahfarokhib, Masanobu Kawachic, Ali Eslamifard, Oskar J. Schmidte, Andreas Schmidte, Abdolamir Allamehb, Tomoya Yoshinarif a Department of Mycology, Pasteur Institute of Iran, Tehran 13164, Iran Faculty of Medical Sciences, Tarbiat Modares University, Tehran 14115-111, Iran c National Institute for Environmental Studies, 16-2 Onagawa, Tsukuba 305-8506, Japan d Electron Microscopy Unit, Pasteur Institute of Iran, Tehran 13164, Iran e POC Polymer Productions GmbH, Strohg, 14c, Vienna, A-1030, Austria f Department of Applied Biological Chemistry, Graduate School of Agriculture and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan b
Received 18 July 2006; received in revised form 1 September 2006; accepted 1 September 2006 Available online 10 September 2006
Abstract The effects of Akacidsplus, a novel member of guanidine-based polymeric compounds recently introduced as a potent inhibitor of fungal growth and aflatoxin biosynthesis were studied on Aspergillus parasiticus by transmission electron microscopy (TEM). The toxigenic fungus was cultured on yeast extract-sucrose broth in presence of serial two-fold concentrations of Akacidsplus (1.5–96 mL/50 mL medium) for 96 h at 28 1C with shaking. Mycelial samples exposed to fungistatic concentrations of compound (1.5–48 mL) were processed for TEM. Corresponding to the growth inhibition, TEM observations revealed morphological anomalies in fungal compartments. The results demonstrated that Akacidsplus targets the plasma membrane of the hyphae by its breaking down at variable intervals with the formation of small membrane-bound vesicles inside the cytoplasm, while no obvious damage was observed on the cell wall. A marked depletion of cytoplasmic contents of hyphae accompanied with lysis and disruption of membranes of major organelles such as nuclei, mitochondria and endoplasmic reticulum indicates that in high fungistatic concentrations, Akacidsplus passes not only through the cell wall but also through the plasma membrane and then interact with membranous structures of the cytoplasmic organelles. Ultrastructural changes of fungal compartments exposed to Akacidsplus in relation to the fungal growth and aflatoxin biosynthesis are discussed. r 2006 Elsevier Ltd. All rights reserved. Keywords: Akacidsplus; Aspergillus parasiticus; Ultrastructure; Aflatoxin; Growth inhibition; Guanidine-based polymers
1. Introduction Corresponding author. Tel.: +98 21 66953311 20; fax: +98 21 66465132. E-mail address:
[email protected] (M. Razzaghi-Abyaneh).
0041-0101/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2006.09.002
Fungal contamination of food and feeds is a public health problem throughout the world (Payne, 1998; Bennett and Klich, 2003). The members of
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Aspergillus section Flavi specially three species Aspergillus flavus Link, A. parasiticus Speare and A. nomius Kurtzman have received major consideration regard to their potential for producing carcinogenic aflatoxins (AFs) (Pitt and Hocking 1999; Klich, 2002). Crop contamination with AFs as the most economically relevant mycotoxins is sometimes an unavoidable event that considered as a economic and public health hazard specially in third-world countries. According to the reports of Agricultural Research Service (ARS) of the United State Department of Agriculture (USDA), economic losses of AFaffected agricultural industries reach to some millions of dollars every year (Cleveland et al., 2003). Therefore, considerable efforts have made to control of pre-harvest AF contamination of susceptible crops using different biochemical and molecular strategies which are mainly focused on inhibition of either growth or AF production by producing fungi (Cleveland and Bhatnagar, 1992; Yu et al., 2005; Cleveland et al., 2005). Based on previous studies, many substances were successfully used for inhibition of growth and AF biosynthesis by producing fungi (Zaik and Buchanan, 1987). Despite that and the recent data from fungal biochemistry to fungal genomics and proteomics which led to the identification of potent fungal invation and/or AF inhibitory compounds such as aflastatins and Blasticidin A (Sakuda et al., 2000), walnut tannins-derived gallic acid (Molyneux et al., 2004), a maize ribosome inactivating protein (Payne et al., 2004), some maize chitinases (Payne et al., 2004), pyrrocidines A and B (Wicklow et al., 2005) and anticalmodulin agent, triflouperazine (Juvvadi and Chivukula, 2006), very little has been documented about the exact mechanism of action of these interesting inhibitors at cellular and molecular levels. Our previous data showed that some medicinal plants i.e. Neem (Azadirachta indica A. Juss), Thymus x-prolock and Thymus eriocalyx were capable of inhibiting growth of A. parasiticus and its AF production in a dose-dependent manner by an unknown mechanism (Allameh et al., 2001; Rasooli and Razzaghi-Abyaneh., 2004; Razzaghi-Abyaneh et al., 2005). In continuing our research on AF inhibitors, Akacidsplus, a new member of the guanidine-based polymeric disinfectants was recently introduced for the first time as a potent inhibitor of A. parasiticus growth and its AF productivity (Razzaghi-Abyaneh et al., 2006). Akacidsplus was developed by POC
Polymer Productions GmbH, Vienna, Austria as a new member with enhanced broad antimicrobial activity while significantly less toxicity comparing to the former compounds of this class. Akacidsplus is a water soluble, nonflammable, nonexplosive, colourless and odorless formulation, which is composed of a mixture of two different polymeric guanidine compounds (Chemical Abstracts Service registry no. 374572-91-5 and 57028-96-3). It was accepted as a biocide according the new EU-guidelines and it has in vitro antimicrobial activity against some important pathogenic bacteria and fungi (Kratzer et al., 2006a, b; Buxbaum et al., 2006). Recent toxicological studies in experimental animals have revealed that Akacidsplus is a safe compound with low oral and dermal toxicity (Buxbaum et al., 2006). It has been shown that it can be also used as an antitumor (Neuwirt et al., 2006) and room disinfectant agent (Kratzer et al., 2006a). Despite the broad antimicrobial activity of Akacidsplus specially against aflatoxigenic A. parasiticus which makes it a promising tool for field application in prevention of pre-harvested AF contamination of crops, its exact mechanism of action has not been studied yet. In this communication, an electron microscopic study of the A. parasiticus NRRL 2999, a potent producer of major AFs (B1, B2, G1 & G2) was carried out in order to find out the mode of action of this interesting novel polymeric biocide. 2. Materials and methods 2.1. Chemicals A stock solution of Akacidsplus containing a 3:1 mixture of poly-(hexamethylen-guanidinium-chloride; CAS No.: 57028-96-3) and poly-[2-(2-ethoxy)ethoxyethyl)-guanidinium-chloride; CAS No.: 374572-91-5) as a 25% aqueous solution (Ch. 1007, Polymers of Creativity, Vienna, Austria) was used in different concentrations. Epon-812, glutaraldehyde, osmium tetroxide and 2,4,6-tri(dimethylaminomethyl) phenol (DMP-30) was purchased from Taab Laboratories, UK. All other solvents and reagents were of analytical grade obtained from E. Merck, Germany. 2.2. Fungal strain and growth conditions A standard aflatoxigenic A. parasiticus NRRL 2999 was used in this study. The fungus was
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2.3. Sample processing for transmission electron microscopy (TEM)
1.6 Total fungal growth (g)
cultured in Yeast extract-sucrose (YES) broth medium in presence of two-fold serial dilutions of Akacidsplus (1.5–96 mL final concentrations) as described previously (Razzaghi-Abyaneh et al., 2006). Fungal dry weight was calculated according to Rasooli and Razzaghi-Abyaneh (2004) and it was considered as growth index.
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1.4 1.2 1 0.8 0.6 0.4 0.2 0 0
1.5
3
6
12
24
48
96
Akacid final concentration (uL)
Fungal materials obtained from 4-day-old cultures grown in either Akacidsplus (1.5–48 mL final concentrations) or no biocide (controls) were processed for TEM (Bozzola and Russel, 1999). The samples were pre-fixed in 3% (w/v) glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.2 for 3 h at room temperature followed by thoroughly washing with phosphate buffer. Fixed materials were then post-fixed in 1% aqueous osmium tetroxide for 3 h at room temperature after infiltrating with 2% molten agar. Dehydration of samples was achieved by transferring to vials containing a graded water–acetone series (10% steps for 30–90% each of 60 min, 100% for 180 min and finally 100% overnight). Dehydrated specimens were embedded with Epon 812 and then polymerized in spurr’s resin (Epon 812 with 1.5% hardening agent, DMP-30) at 45 1C for 24 h and 65 1C for 72 h. Thin sections (80 nm thickness) were prepared using a Leica Ultracut UCP on 100-mesh grids and then examined under a JEOL JEM-2010 transmission electron microscope after staining with uranyl acetate for 20 min and with lead citrate for 5 min. 3. Results 3.1. Growth kinetics of normal and Akacid-treated mycelia As shown in Fig. 1, exposure of aflatoxigenic A. parasiticus to various concentrations of Akacidsplus resulted in the inhibition of fungal growth in a dose-dependent manner. The lowest and the highest growth inhibition was estimated as 9.62% and 100% in presence of 1.5 and 96 mL final concentrations of Akacidsplus, respectively. This inhibition was found to be significant for all concentrations except 1.5 mL in comparison with control (untreated) group (ANOVA; Po0.05). As reported previously, a dose-dependent inhibition of
Fig. 1. Inhibitory effects of Akacid splus on growth of toxigenic A. parasiticus in 4-day-old submerged cultures. Siginificant differences were reported for 3.0–96 mL Akacid compared to the control (ANOVA; Po0.05).
AF synthesis was also observed for Akacid-treated mycelia (data not shown in details). 3.2. Effects of Akacidsplus on fungal ultrastructure Fig. 2 illustrates the morphological changes of fungal compartments in Akacid-treated samples in comparison with their normal counterparts in untreated controls. Interestingly, A. parasiticus growth inhibition induced by Akacid in 4-day-old cultures was found to be well correlated with correspondence morphological changes of the fungus exposed to different fungistatic concentrations of the biocide (1.5–48 mL in total cultures). In untreated normal samples (Fig. 2a and b), the cell wall was uniform and thoroughly surrounded by an intact fibrilar layer. Plasma membrane was unfolded with a uniform shape in all parts. All the organelles, such as nuclei, mitochondria, endoplasmic reticulum, vacuoles, electron dense granules and septum, were appeared normal. In Akacid-treated samples (Fig. 2c-r), ultrastructural changes were noticed at cell wall, plasmalemma and cytoplasmic levels. The major pathologic changes were found to occur on endomembrane system mainly affecting plasma membrane and membranous organelles specially nuclei and mitochondria. As shown in Fig. 2c and d, early changes in fungal compartments in presence of the lowest concentration of Akacid (1.5 mL) were noticed by initial cell depression signs including abnormal shaped and swelled hyphae, increased vacuolation of cytoplasm accompanied with vacuole fusion, swelling of septum and early degradation of electron-dense granules.
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Fig. 2. Transmission electron micrographs of Akacid-treated A. parasiticus 4-day-old cultures. Control mycelia (a,b): hyphae with normal septum (S), woronin bodies (Wo), cell wall (CW), plasma membrane (PM), nucleus (N), mitochondria (M), endoplasmic reticulum (ER) and electron-dense granules (EDGs); Akacid-treated mycelia (c-r): 1.5 mL Akacid (c,d): initial cell depression signs including abnormal shaped and swelled hyphae, vacuolation of cytoplasm accompanied with vacuole fusion, swelling of septum and early degradation of EDGs; 3.0 mL Akacid (e–g): swelled and malshaped hyphae, progressed depletion and destruction of EDGs, change of cell permeability led to disruption of plasma membrane with the formation of small vesicles in site of hypha wall (black arrows in f and g) and septum wall (white arrow in f); 6.0 mL Akacid (h–j): detachment of fibrilar layer (FL) of cell wall (as shown in h), complete loss of cell permeability led to formation of membrane-bounded vesicles (arrows in i), destruction and lysis of hyphae membranous organelles including nucleus and mitochondria (as shown in i) and massive destruction and dysorganisation of a fungal conidium (as indicated in j); 12 mL Akacid (k,l): cell depression (arrow in k), massive vacuolation of cytoplasm with vacuole fusion (as shown in k), and disorganization of cytoplasmic contents accompanied with intensive degradation and lysis of nucleus and mitochondria (as shown in l); 24 & 48 mL Akacid (m–r): complete autolysis and disorganization of hyphae cytoplasm characterized by disrupted membranes (DMs) (m–o) accompanied with destruction and breaking down of plasma membrane with massive formation of membrane-bounded vesicles (arrow heads in m, p & r), destruction and lysis of membranous organelles including nuclei, endoplasmic reticulum and mitochondria (as shown in m–r) which finally led to cell dead. Magnifications: c, e ( 4000); d ( 5000); m ( 6000); k ( 8000); a, h, n, o ( 12,000); f, p ( 15,000); b, i, j, l, q ( 20,000); r ( 30,000); g ( 50,000).
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Fig. 2. (Continued)
In presence of 3.0, 6.0 & 12 mL final concentrations of Akacid where the fungal growth was inhibited 35%, moderate pathologic changes were noticed in both mycelia and conidia (Fig. 2e-l). In this stage, the major changes appeared to be resulted from disrupting of plasma membrane accompanied with the formation of small vesicles in site of hypha walls. Subsequent events were loss of normal conidia and hyphae shape, progressed depletion and destruction of EDGs, abnormal
accumulation of polysaccharids inside the hypha, detachment of fibrilar layer of cell wall, destruction of hypha memberanous organelles including nucleus and mitochondria, and finally disorganization of cytoplasmic contents accompanied with intensive degradation and lysis of nucleus and mitochondria. The most remarkable changes in fungal compartments were observed in fungus treated with the highest fungistatic concentrations of Akacid i.e. 24 and 48 mL per total culture where more that 90%
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Fig. 2. (Continued)
inhibition in fungal growth occurred (Fig. 2m-r). Destruction and breaking down of plasma membrane at variable sites accompanied with complete autolysis and disorganization and leak out of hyphae cytoplasm were appeared to be the final event. Following destruction and complete lysis of membranous organelles including nuclei, endoplasmic reticulum and mitochondria seemed to be finally led to cell dead.
4. Discussion Although it has been clearly shown that secondary metabolism i.e. mycotoxin biosynthesis is closely related to fungal differentiation and development, many aspects of this correlation remain to be discovered (Calvo et al., 2002; Keller et al., 2005). In aflatoxigenic fungi, production of AFs is usually reported to occur after sporulation. Therefore,
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certain compounds that inhibit growth and sporulation have also been shown to inhibit AFs production (Reib, 1982). Despite that, exact relationship between fungal growth and AF production in toxigenic fungi is now a matter of controversy. The results from our studies on aflatoxigenic fungi have shown that inhibitory compounds are able to inhibit fungal growth, AF biosynthesis or both after adding to the culture media (Allameh et al., 2001; Rasooli and Razzaghi-Abyaneh., 2004; RazzaghiAbyaneh et al., 2005, 2006). Greene-McDowelle et al. (1999) studied the morphological effects of selected cotton-leaf volatiles on A. parasiticus in relation to fungal growth and AF production. They showed that morphological alterations such as reducing radial growth, loss of mycelial pigmentation, decrease in sporulation and induction of uniquely aerial hyphae may be correlated with inhibition in either fungal growth or AF biosynthesis. Results from the works of Ono et al. (1997) revealed that aflastatin A, a natural bacterial metabolite, inhibits AF biosynthesis in A. parasiticus without any obvious effect on growth and morphology of producing fungus. Our previous data showed that exposure of toxigenic A. parasiticus to Neem (Azadirachta indica A. Juss) leaf aqueous extract resulted in the inhibition of AF production not fungal growth, while exposure of fungus to essential oils from Thymus species caused inhibition in both fungal growth and AF synthesis (Allameh et al., 2001; Rasooli and Razzaghi-Abyaneh, 2004; RazzaghiAbyaneh et al., 2005). Despite that and the large data exist now about fungal growth and AF inhibitors (Sakuda et al., 2000; Molyneux et al., 2004; Payne et al., 2004; Wicklow et al., 2005; Juvvadi and Chivukula, 2006), very little has been documented regarding their mechanism of action. Recently, we discovered the inhibitory effects of a novel biocide, Akacidsplus, on both A. parasiticus growth and its AF productivity (Razzaghi-Abyaneh et al., 2006). Respect to the strong inhibitory activity (2000 folds of Neem as a known inhibitor of AF biosynthesis in equal amounts) and unique physico-chemical properties of this chemical which make it a promising candidate for use in AF prevention programmes, its mechanism of action on growth of aflatoxigenic A. parasiticus at ultrastructure level was investigated for the first time in this study. The TEM of Akacid-treated fungus in comparison with untreated samples clearly showed dose-dependent pathologic changes of fungal cells especially on membranous structures. The results
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precisely showed that Akacidsplus exerts its effect directly on plasma membrane without any obvious damage on cell wall. It seems that alteration in cell permeability due to breaking down of plasma membrane at variable intervals resulted in the loss of normal shape of fungal mycelia and formation of membrane bound vesicles inside the cells. Moreover, a marked depletion of cytoplasmic contents of hyphae accompanied with lysis and disruption of membranes of major organelles such as nuclei, mitochondria and endoplasmic reticulum indicate that in high fungistatic concentrations, Akacidsplus passes not only through the cell wall but also through the plasma membrane and then interact with membranous structures of the cytoplasmic organelles. Similar results have been reported for growth inhibitory effects of a lipopeptide antibiotic, iturin A, on yeast cells of Candida albicans and Saccharomyces cerevisiae (Thimon et al., 1995). Based on our previous data on the mechanism of action of onion-induced growth inhibition of pathogenic dermatophytes (Shams-Ghahfarokhi et al., 2004), as well as morphological changes of aflatoxigenic A. parasiticus exposed to Neem leaf extract (Razzaghi-Abyaneh et al., 2005), the present results clearly show that compounds will be able to inhibit fungal growth which they can change the cell uniformity via direct interaction with either cell wall or cytoplasmic membranes. Whether Akacid-mediated inhibition of AF synthesis is a consequence of its mechanical damages to fungal compartments or it clearly targets a specific site in AF biosynthetic pathway remains to be further studied. Acknowledgements The present work was financially supported by Pasteur Institute of Iran. The authors wish to thank Prof. H.G. Raj, Patel Chest Institute, Delhi, India for kind providing A. parasiticus NRRL 2999. We also appreciate Shizuko Kinoshita from National Institute for Environmental Studies, Tsukuba, Japan and Jaleh Taeb and Manijeh Deljoodokht from Electron microscopy Unit of Pasteur Institute of Iran for excellent EM technical assistance. References Allameh, A., Razzaghi abyaneh, M., Shams, M., Rezaee, M.B., Jaimand, K., 2001. Effects of neem leaf extract on production of aflatoxins and activities of fatty acid synthetase, isocitrate
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