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Drosophila cecropin as an antifungal agent
Insect Biochemistry and Molecular Biology 29 (1999) 965–972 www.elsevier.com/locate/ibmb
Drosophila cecropin as an antifungal agent Sophia Ekengren b
a,b
, Dan Hultmark
a,*
a Umea˚ Center for Molecular Pathogenesis, Umea˚ University, S-90 187 Umea˚, Sweden Department of Developmental Biology, The Wenner-Gren Institute, Stockholm University, S-106 91 Stockholm, Sweden
Received 3 March 1999; received in revised form 14 June 1999; accepted 16 June 1999
Abstract The effects of Drosophila and Hyalophora cecropins were tested against different fungi, both insect pathogens and fungi from the normal environment of Drosophila. The fungi were generally found to be as susceptible to the cecropins as most bacteria, the only exception being the insect pathogen Beauveria bassiana which is completely resistant. This is also the only fungus tested which is virulent to Drosophila, giving 100% lethality within 5 days after injection. Lethal concentrations of cecropins against other fungi tested ranged between 0.4 and 4 µM. Andropin is less fungicidal than the cecropins, and Drosophila cecropin A is somewhat more potent than cecropin B. Even dense cultures of Saccharomyces cerevisiae can be cleared by micromolar concentrations of cecropin, whereas Geotrichum candidum is unaffected by cecropin when tested in a dense culture. 1999 Elsevier Science Ltd. All rights reserved. Keywords: Cecropin; Fungi; Insect immunity; Antifungal peptides
1. Introduction The fruitfly, Drosophila melanogaster, lives and feeds on decaying plants and fruits. An essential source for nutrients are yeasts that grow in this environment. Yeasts are both utilized and spread by the insect, a relationship that seems to be kept in balance on a basis of mutualism (Baumberger, 1919; Giglioli, 1897). Whereas Drosophila is normally feeding on microbes, tissues of insects can on the other hand also be a rich source for nutrients for the microbes. Entomopathogens such as the fungi Metarhizium anisopliae and Beauveria bassiana can infect a wide variety of insects through the cuticle, and they have the capacity to invade and kill insects by interfering with the host’s immune defense (Tanada and Kaya, 1993). Therefore, the growth of both noxious and beneficial microbes has to be controlled by the insect, and it is not surprising that a number of antimicrobial effector molecules have been found in insects. In Drosophila, they include antibacterial proteins and peptides such as the cecropins, attacins, drosocin, defensin and
* Corresponding author. Tel.: +46-90-785-67-78; fax: +46-90-7780-07. E-mail address: [email protected] (D. Hultmark)
lysozyme, as well as antifungal peptides such as drosomycin and metchnikowin (reviewed by Hetru et al., 1998). The cecropins are a well characterized family of antibacterial peptides (for reviews, see Faye and Hultmark, 1993; Boman, 1995; Hetru et al., 1998; Shai, 1998). They were first found in the cecropia moth, Hyalophora cecropia (Hultmark et al., 1980; Steiner et al., 1981), and later also isolated from several lepidopteran and dipteran species, including Drosophila (Kylsten et al., 1990; Samakovlis et al., 1990). Cecropins have not yet been recorded from other insect orders, but similar peptides have been found in tunicates and mammals (Zhao et al., 1997; Lee et al., 1989). Mature cecropins are highly basic 35–39 amino acid residue peptides that can fold into two amphipathic α-helices, separated by a more flexible hinge. The peptides are thought to integrate into the acidic cell membranes of bacteria, either forming voltage-dependent ion channels (Christensen et al., 1988), or causing a general disruption of the membrane by a ‘carpet-like’ mechanism (Steiner et al., 1988; Shai, 1998). Cecropins are rapidly induced upon infection and secreted into the hemolymph. In the hemolymph of infected insects, cecropins may reach concentrations of 25 to perhaps 100 µM (Samakovlis et al., 1990; Gud-
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mundsson et al., 1991), and at this concentration they show a very broad spectrum of activity against both Gram-positive and Gram-negative bacteria. The lethal concentration for most bacterial species is in the 0.2–20 µM range (Hultmark et al., 1982; Samakovlis et al., 1990). In contrast, most eukaryotic cells are either completely resistant, or are affected only at very high concentrations. Significant effects have been observed against some protozoan parasites, such as Plasmodium and Leishmania (Boman et al., 1989; Gwadz et al., 1989; Akuffo et al., 1998), but only at concentrations around 100 µM or higher. Given the resistance of most eukaryotic cells against cecropin (Steiner et al. 1981, 1988), we were surprised to find that Drosophila cecropins have a strong antifungal activity. However, this finding is not entirely unprecedented; DeLucca et al. (1997, 1998) recently described potent activity of cecropin from H. cecropia against several Fusarium and Aspergillus species, and synthetic cecropin-like peptides have been shown to have good activity against various fungal plant pathogens (Cavallarin et al., 1998). Park et al. (1997) also noted that a cecropin from Hyphantrea is able to kill Candida tropicalis. We have now followed up on these findings, and quantitated the effect of Drosophila cecropin A, cecropin B and andropin as well as cecropin A from H. cecropia, against some fungi that the insects are likely to encounter in their normal environment. Metarhizium anisopliae and Beauveria bassiana are entomopathogenic strains, and Dipodascopsis uninucleata and Geotrichum candidum are yeast-like fungi, that grow in the normal environment of Drosophila. For the latter two species we used strains which were in fact isolated from Drosophila. We also tested the effect on two strains of Saccharomyces cerevisiae, a major food source of Drosophila in nature (Begon, 1982), as well as in the laboratory. Here we show that cecropins have a strong inhibitory effect on fungal growth at micromolar concentrations. We can also show a correlation between the antifungal potential of the cecropins and the ability of the fly to survive a fungal infection.
cultures (approximately 2 mm×2 mm) of B. bassiana and M. anisopliae, were inoculated in 1×MEA (2% malt extract, 0.1% Peptone, 2% glucose) and grown on a shaker (100 rev/min) at 30°C, in the dark. After 3 days, the cultures were filtered through three layers of sterile gauze. Spores were concentrated by centrifuging the filtrate at 10 000g for 15 min, washed and resuspended in sterile Drosophila ringer, pH 7.2 (182 mM KCl, 46 mM NaCl, 3 mM CaCl2, 10 mM Tris–HCl). Cell concentration was determined and adjusted after counting in a Bu¨rker chamber. D. uninucleata, G. candidum and the two Saccharomyces strains were inoculated in 1×MEA and grown as above, except that G. candidum and S. cerevisiae were grown for 24 h and D. uninucleata for 48 h before harvest. 2.2. Synthetic cecropins Drosophila cecropin A, cecropin B and andropin were ˚ ke synthesized by automated solid-phase synthesis by A Engstro¨m, Uppsala. Hyalophora cecropin A was synthesized by D. Andreu and was a gift from Prof. Hans Boman. The synthesis of the Drosophila peptides has been described, including purification by reverse-phase chromatography, purity control by mass spectroscopy and characterization of their antibacterial activity (Samakovlis et al. 1990, 1991; Akuffo et al., 1998). 2.3. Inhibition zone assay Antifungal activity was determined by inhibition zone assay. A solution of 0.8% agarose in 1×MEA was autoclaved and cooled to 45°C. Aliquots of 5 ml were inoculated with 500 µl spore suspension (2×106 spores/ml) and poured into Petri dishes (6.7 or 9.0 cm diameter, Falcon). Holes with a diameter of 2 mm were punched into the agar, filled with 2 µl of each cecropin dilution and incubated in the dark at 30°C until fungal growth was visible (16–48 h). The diameters of the growth inhibition zones were determined with a caliper, and the lethal concentrations calculated as described (Hultmark, 1998). 2.4. Microplate assay
2. Materials and methods 2.1. Fungi Beauveria bassiana (strain 118.30), Metarhizium anisopliae (strain 464.70), Dipodascopsis uninucleata (strain 741.74) and Geotrichum candidum (strain 606.85) were obtained from the Centraalbureau voor Schimmelcultures (Baarn, the Netherlands), and Saccharomyces cerevisiae (strains W308 and S289C) were ˚ stro¨m, Umea˚. The fungi were cultia gift from Stefan A vated on Sabouraud agar with 2% glucose. Pieces of agar
Samples of approximately 100 cells each of M. anisopliae, G. candidum, S. cerevisiae and B. bassiana were inoculated in 1×MEA to a final volume of 100 µl, in a flat bottomed 96-well microplate. One-microliter aliquots of Hyalophora cecropia cecropin A were then added from a twofold dilution series, starting at a concentration of 17.1 µM in the well. Similarly, Drosophila melanogaster cecropin A was tested at a single concentration of 18 µM. Cultures were kept in the dark at 30°C. Fungal growth was analyzed both under the microscope and by a microplate reader at 620 nm. In another set
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of experiments the fungi were allowed to grow to an absorbance of approximately 0.1 before addition of cecropin. Further growth was then assayed as previously. 2.5. Injection of spores into flies Drosophila melanogaster, Canton S, was reared at 25°C on standard yeast agar media. Flies (3–5 days old) were anesthetized with CO2 and injected in the thorax with freshly made suspensions of fungal spores from B. bassiana, S. cerevisiae W308, M. anisopliae or G. candidum (1×106 spores/ml in Drosophila ringer). Injected flies were kept at 25°C. Viability was scored every 24 h. Surviving flies were transferred to fresh vials every third day. 2.6. Northern blot Drosophila melanogaster, Canton S flies (3–5 days old) were injected in the thorax with freshly made suspensions of fungal spores from B. bassiana, D. uninucleata, M. anisopliae or G. candidum (1×106 spores/ml in Drosophila ringer). Injected flies were kept at 25°C. Surviving flies were frozen in liquid nitrogen 12 h, 24 h, 50 h or 9 days after injection. RNA was extracted with Trizol (GIBCO) according to the manufacturer’s protocol. An amount of 15 µg RNA per sample was separated on a 1% denaturing agarose gel and blotted onto a Hybond N filter. Actin 5C (Fyrberg et al., 1980) and Cecropin A1 (Kylsten et al., 1990) probes were synthesized using RediPrime II labeling reaction (Amersham) following the manufacturer’s protocol. Hybridization was performed under high stringency conditions (50% formamide, 42°C).
3. Results 3.1. Effect of cecropins on fungal growth Large zones of growth inhibition were observed when cecropins were applied on fungal plates (Fig. 1), allowing us to calculate the corresponding lethal concentrations. As shown in Table 1, cecropins have a strong activity against different classes of higher fungi. The lethal concentrations vary slightly for different fungi and among the different cecropins, but are in most cases in the micromolar or submicromolar range. However, none of the antimicrobial substances tested show any effect against the entomopathogen B. bassiana. Drosophila cecropin A is somewhat more potent than cecropin B, and comparable to that of the Hyalophora cecropin. We also tested the activity of andropin, a cecropin-like peptide from the male reproductive tract of Drosophila (Samakovlis et al., 1991), but this peptide is considerably less active than the cecropins.
Fig. 1. Inhibition zone assay. The figure shows the effect of three concentrations from a longer dilution series of Drosophila cecropin A, against the fungi S. cerevisiae W308, D. uninucleata, G. candidum, M. anisopliae and B. bassiana. As a comparison we also show the growth inhibition against the bacterial strain E. cloacae β12.
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The inhibition zone assay is a simple way to estimate lethal concentrations, and it requires modest amounts of the active peptides (Hultmark, 1998). However, with some of the filamentous fungi the diameters of the inhibition zones were poorly defined (Fig. 1), and for this reason we repeated some of our results with the Hyalophora cecropin, using a microplate assay (MIC value, Table 1). This also allowed us to observe the effects of the cecropins under the microscope. As shown in Table 1, there is good agreement between the two assays, although the microplate assay tended to give slightly higher values. Again, fungal growth is completely blocked by cecropin at micromolar concentrations, except for B. bassiana which is unaffected. High concentrations of cecropin A causes total cell lysis for S. cerevisiae and G. candidum, as observed under the microscope (data not shown). In contrast, hyphae of M. anisopliae remain intact, although their growth is inhibited. The assays discussed so far show the effect of cecropins on single cells at low density. We also looked at the effect when cecropin is added directly to a dense culture (Fig. 2). Drosophila and Hyalophora cecropins are able to clear even late log phase or early stationary phase culture of S. cerevisiae within a few hours. In contrast, there is no effect of cecropin on dense cultures of G. candidum, either in stationary phase or in a moderately dense growing fungal culture. 3.2. The resistance of flies against injected fungi We tested the actual resistance of Drosophila to the different fungi, in order to correlate it to the antifungal effect of the cecropins (Fig. 3). The flies show good resistance to injection of S. cerevisiae or G. candidum. Surprisingly, a similar survival is also seen after injection of the broad host-range entomopathogen M. anisopliae, and more than 60% of the flies are still alive after 5 days. Clearly, the immune defense is quite efficient
against these fungi. In contrast, B. bassiana, the other insect pathogen, causes 100% lethality within this period of time. Cecropins are normally not present in the flies unless the immune system is stimulated, for instance by a bacterial infection. We therefore tested if fungal infections can induce cecropin gene expression under the conditions used here. Fig. 4 shows that significant levels of Cecropin A1 mRNA can be detected in the flies within 12 h after infection, and that the mRNA concentration increases further during the following days. In particular, the insect pathogen M. anisopliae, to which Drosophila is relatively resistant, gives a very strong induction of cecropin.
4. Discussion Our data show that cecropins have the capacity to inhibit fungal growth at concentrations far below the natural concentrations found in the hemolymph of a challenged fly, and that insect pathogens as well as more harmless fungi are susceptible. The cecropins are induced by fungi, and they efficiently kill fungal cells and repress the growth of hyphae from five out of six tested fungal species, at concentrations as low as 0.5–3 µM. Thus, the cecropins can now be added to a growing list of fungicidal effector molecules in Drosophila. Previously, the inducible peptides drosomycin and metchnikowin have been shown to be fungicidal (Fehlbaum et al., 1994; Levashina et al., 1998), and antifungal activity has also been demonstrated for lysozyme from Galleria mellonella (Vilcinskas and Matha, 1997). Work by Lemaitre et al. (1995, 1997) has indicated that the antifungal effector molecules in Drosophila may be induced by a specific signal pathway, independent from that of the antibacterial peptides. This is most clearly shown for drosomycin. This antifungal peptide is preferentially induced by fungal infections, and its
Table 1 Lethal concentrations (µM) of different cecropins. Values were derived from the inhibition zone assay (Hultmark, 1998). For comparison, we also tested the activity of Hyalophora cecropin A against four fungi in a minimal inhibitory concentration (MIC) assay Drosophila
Saccharomyces cerevisiae W308 Saccharomyces cerevisiae S289C Dipodascopsis uninucleata Geotrichum candidum Metarhizium anisopliae Beauveria bassianaa Enterobacter cloacae β12
Hyalophora
Cecropin A
Cecropin B
Andropin
Cecropin A
(MIC value)
0.7 1.2 0.5 0.9 1.8 ⬎44 0.3
3.3 0.7 1.2 4.0 2.7 ⬎59 0.3
36 59 12 14 68 ⬎65 42
1.0 1.3 0.4 1.4 1.1 ⬎10 0.2
(1.1–2.1)
(2.1–4.3) (2.1–4.3) (⬎17)
a No inhibition zones were detected for B. bassiana. In this case the limit of detection was estimated from the assumption that zones larger than 4 mm would have been recognized.
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Fig. 2. Effect of cecropin on dense fungal cultures. Drosophila cecropin A, or Hyalophora cecropin A was added directly to late cultures of S. cerevisiae W308 or G. candidum in microplate wells, and the cell density was recorded. For the Hyalophora cecropin, a range of concentrations was tested but we only show two concentrations for Saccharomyces and the highest for Geotrichum. Cecropin gives a rapid clearance of the Saccharomyces culture, even at the low concentration, whereas there is no effect on Geotrichum at any concentration tested.
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Fig. 3. Survival of Drosophila after injection of fungi. For each of the indicated fungal strains, 60 adult flies were injected with approximately 100 cells per fly. There was some initial lethality due to the injection procedure itself, and the number of living flies at 17 h was therefore set to 100%.
induction requires the gene products of the Toll pathway. In contrast, antibacterial peptides such as diptericin are best induced by bacteria, via a signal transduction pathway which is defined by the imd gene. The fact that cecropins are potent antifungal as well as antibacterial agents fits nicely into this scheme, since the induction of the cecropins is affected by both pathways (Rosetto et al., 1995; Lemaitre et al. 1995, 1997). The cecropin molecules do not have an ordered structure in solution, but they are thought to be stabilized in an alpha-helical conformation after interaction with the hydrophobic lipids in the bacterial cell membrane (Steiner, 1982). This interaction between cecropins and the cell membrane must have a high degree of specificity as cecropins do not have any lethal effect on insect or mammalian cells. However, the molecular mechanism
behind this specificity remains an open question. The selective killing of bacteria has previously been attributed to the absence of cholesterol in the bacterial cell membrane (Nakajima et al., 1987; Christensen et al., 1988), to the presence of acidic molecules such as lipopolysaccharides or teichoic acids (Shai, 1998), or to differences in membrane potential. The latter factor has been shown to affect the lytic activity of magainins (Cruciani et al., 1991). However, neither of these models are entirely satisfactory to explain why fungi are more susceptible than other eukaryotes to the action of cecropins. The target of the cecropins is believed to be the cell membrane. However, it is worth noting that we could in several cases observe full lysis of the fungal cells, implicating a breakdown of the entire cell wall. It is possible that endogenous lytic enzymes are released as a result of the cecropin action. Of the fungi investigated here, only the entomopathogen Beauveria bassiana is entirely resistant to cecropin. B. bassiana is a broad range insect pathogen that can attach to the cuticle of the insect and grow through the integument and into the hemocoel. The resistance of this pathogen may reflect an adaptation to the defense reactions of the insect host. Protection from lysis may come from a passive resistance of the cell wall or cell membrane, or may depend on the secretion of proteases or similar substances that inactivate or destroy the cecropins. Secreted proteases have been shown to be important for cecropin resistance in several other microbes (Go¨tz et al., 1981; Dalhammar and Steiner, 1984; DeLucca et al., 1997). Secretion of proteases may also be a reason why dense cultures of G. candidum are unaffected by cecropin. It is also possible that the
Fig. 4. Expression of cecropin in Drosophila after injection of fungi. Total RNA from infected flies was separated by electrophoresis, blotted, and probed for cecropin expression using a CecA1 cDNA clone as a probe. The filter was also probed with an actin probe to check for equal loading (not shown). No samples were taken before injection in this particular experiment, but cecropin expression is normally very low in untreated animals. Since the flies are eventually killed by the B. bassiana infection, only early time points are shown for this fungus.
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properties of the cellular envelope is different in the growing hyphae of the dense cultures, compared with the spores tested in the other assays. In the hemolymph, the cecropins undoubtedly act in concert with several other peptides but, as we show here, they can also act as powerful defense molecules on their own. There is indeed a correlation between the ability of the fly to survive a fungal challenge and the effect of cecropin on the fungi. The potent effect against yeasts, which are very abundant in the normal environment of Drosophila, is likely to be important for the protection against accidental infections. Our data support the importance of cecropins as antifungal as well as antibacterial effector molecules in the natural defense of Drosophila melanogaster.
Acknowledgements ˚ stro¨m. The yeast strains used were a gift from Stefan A ˚ Cecropins were synthesized by Ake Engstro¨m and David Andreu. We are grateful to Ha˚kan Steiner for help and discussions. This work was supported by grants from the Swedish Natural Science Research Council, the Swedish Medical Research Council, and by the Go¨ran Gustafsson Foundation for Scientific Research.
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Drosophila cecropin as an antifungal agent Sophia Ekengren b
a,b
, Dan Hultmark
a,*
a Umea˚ Center for Molecular Pathogenesis, Umea˚ University, S-90 187 Umea˚, Sweden Department of Developmental Biology, The Wenner-Gren Institute, Stockholm University, S-106 91 Stockholm, Sweden
Received 3 March 1999; received in revised form 14 June 1999; accepted 16 June 1999
Abstract The effects of Drosophila and Hyalophora cecropins were tested against different fungi, both insect pathogens and fungi from the normal environment of Drosophila. The fungi were generally found to be as susceptible to the cecropins as most bacteria, the only exception being the insect pathogen Beauveria bassiana which is completely resistant. This is also the only fungus tested which is virulent to Drosophila, giving 100% lethality within 5 days after injection. Lethal concentrations of cecropins against other fungi tested ranged between 0.4 and 4 µM. Andropin is less fungicidal than the cecropins, and Drosophila cecropin A is somewhat more potent than cecropin B. Even dense cultures of Saccharomyces cerevisiae can be cleared by micromolar concentrations of cecropin, whereas Geotrichum candidum is unaffected by cecropin when tested in a dense culture. 1999 Elsevier Science Ltd. All rights reserved. Keywords: Cecropin; Fungi; Insect immunity; Antifungal peptides
1. Introduction The fruitfly, Drosophila melanogaster, lives and feeds on decaying plants and fruits. An essential source for nutrients are yeasts that grow in this environment. Yeasts are both utilized and spread by the insect, a relationship that seems to be kept in balance on a basis of mutualism (Baumberger, 1919; Giglioli, 1897). Whereas Drosophila is normally feeding on microbes, tissues of insects can on the other hand also be a rich source for nutrients for the microbes. Entomopathogens such as the fungi Metarhizium anisopliae and Beauveria bassiana can infect a wide variety of insects through the cuticle, and they have the capacity to invade and kill insects by interfering with the host’s immune defense (Tanada and Kaya, 1993). Therefore, the growth of both noxious and beneficial microbes has to be controlled by the insect, and it is not surprising that a number of antimicrobial effector molecules have been found in insects. In Drosophila, they include antibacterial proteins and peptides such as the cecropins, attacins, drosocin, defensin and
* Corresponding author. Tel.: +46-90-785-67-78; fax: +46-90-7780-07. E-mail address: [email protected] (D. Hultmark)
lysozyme, as well as antifungal peptides such as drosomycin and metchnikowin (reviewed by Hetru et al., 1998). The cecropins are a well characterized family of antibacterial peptides (for reviews, see Faye and Hultmark, 1993; Boman, 1995; Hetru et al., 1998; Shai, 1998). They were first found in the cecropia moth, Hyalophora cecropia (Hultmark et al., 1980; Steiner et al., 1981), and later also isolated from several lepidopteran and dipteran species, including Drosophila (Kylsten et al., 1990; Samakovlis et al., 1990). Cecropins have not yet been recorded from other insect orders, but similar peptides have been found in tunicates and mammals (Zhao et al., 1997; Lee et al., 1989). Mature cecropins are highly basic 35–39 amino acid residue peptides that can fold into two amphipathic α-helices, separated by a more flexible hinge. The peptides are thought to integrate into the acidic cell membranes of bacteria, either forming voltage-dependent ion channels (Christensen et al., 1988), or causing a general disruption of the membrane by a ‘carpet-like’ mechanism (Steiner et al., 1988; Shai, 1998). Cecropins are rapidly induced upon infection and secreted into the hemolymph. In the hemolymph of infected insects, cecropins may reach concentrations of 25 to perhaps 100 µM (Samakovlis et al., 1990; Gud-
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mundsson et al., 1991), and at this concentration they show a very broad spectrum of activity against both Gram-positive and Gram-negative bacteria. The lethal concentration for most bacterial species is in the 0.2–20 µM range (Hultmark et al., 1982; Samakovlis et al., 1990). In contrast, most eukaryotic cells are either completely resistant, or are affected only at very high concentrations. Significant effects have been observed against some protozoan parasites, such as Plasmodium and Leishmania (Boman et al., 1989; Gwadz et al., 1989; Akuffo et al., 1998), but only at concentrations around 100 µM or higher. Given the resistance of most eukaryotic cells against cecropin (Steiner et al. 1981, 1988), we were surprised to find that Drosophila cecropins have a strong antifungal activity. However, this finding is not entirely unprecedented; DeLucca et al. (1997, 1998) recently described potent activity of cecropin from H. cecropia against several Fusarium and Aspergillus species, and synthetic cecropin-like peptides have been shown to have good activity against various fungal plant pathogens (Cavallarin et al., 1998). Park et al. (1997) also noted that a cecropin from Hyphantrea is able to kill Candida tropicalis. We have now followed up on these findings, and quantitated the effect of Drosophila cecropin A, cecropin B and andropin as well as cecropin A from H. cecropia, against some fungi that the insects are likely to encounter in their normal environment. Metarhizium anisopliae and Beauveria bassiana are entomopathogenic strains, and Dipodascopsis uninucleata and Geotrichum candidum are yeast-like fungi, that grow in the normal environment of Drosophila. For the latter two species we used strains which were in fact isolated from Drosophila. We also tested the effect on two strains of Saccharomyces cerevisiae, a major food source of Drosophila in nature (Begon, 1982), as well as in the laboratory. Here we show that cecropins have a strong inhibitory effect on fungal growth at micromolar concentrations. We can also show a correlation between the antifungal potential of the cecropins and the ability of the fly to survive a fungal infection.
cultures (approximately 2 mm×2 mm) of B. bassiana and M. anisopliae, were inoculated in 1×MEA (2% malt extract, 0.1% Peptone, 2% glucose) and grown on a shaker (100 rev/min) at 30°C, in the dark. After 3 days, the cultures were filtered through three layers of sterile gauze. Spores were concentrated by centrifuging the filtrate at 10 000g for 15 min, washed and resuspended in sterile Drosophila ringer, pH 7.2 (182 mM KCl, 46 mM NaCl, 3 mM CaCl2, 10 mM Tris–HCl). Cell concentration was determined and adjusted after counting in a Bu¨rker chamber. D. uninucleata, G. candidum and the two Saccharomyces strains were inoculated in 1×MEA and grown as above, except that G. candidum and S. cerevisiae were grown for 24 h and D. uninucleata for 48 h before harvest. 2.2. Synthetic cecropins Drosophila cecropin A, cecropin B and andropin were ˚ ke synthesized by automated solid-phase synthesis by A Engstro¨m, Uppsala. Hyalophora cecropin A was synthesized by D. Andreu and was a gift from Prof. Hans Boman. The synthesis of the Drosophila peptides has been described, including purification by reverse-phase chromatography, purity control by mass spectroscopy and characterization of their antibacterial activity (Samakovlis et al. 1990, 1991; Akuffo et al., 1998). 2.3. Inhibition zone assay Antifungal activity was determined by inhibition zone assay. A solution of 0.8% agarose in 1×MEA was autoclaved and cooled to 45°C. Aliquots of 5 ml were inoculated with 500 µl spore suspension (2×106 spores/ml) and poured into Petri dishes (6.7 or 9.0 cm diameter, Falcon). Holes with a diameter of 2 mm were punched into the agar, filled with 2 µl of each cecropin dilution and incubated in the dark at 30°C until fungal growth was visible (16–48 h). The diameters of the growth inhibition zones were determined with a caliper, and the lethal concentrations calculated as described (Hultmark, 1998). 2.4. Microplate assay
2. Materials and methods 2.1. Fungi Beauveria bassiana (strain 118.30), Metarhizium anisopliae (strain 464.70), Dipodascopsis uninucleata (strain 741.74) and Geotrichum candidum (strain 606.85) were obtained from the Centraalbureau voor Schimmelcultures (Baarn, the Netherlands), and Saccharomyces cerevisiae (strains W308 and S289C) were ˚ stro¨m, Umea˚. The fungi were cultia gift from Stefan A vated on Sabouraud agar with 2% glucose. Pieces of agar
Samples of approximately 100 cells each of M. anisopliae, G. candidum, S. cerevisiae and B. bassiana were inoculated in 1×MEA to a final volume of 100 µl, in a flat bottomed 96-well microplate. One-microliter aliquots of Hyalophora cecropia cecropin A were then added from a twofold dilution series, starting at a concentration of 17.1 µM in the well. Similarly, Drosophila melanogaster cecropin A was tested at a single concentration of 18 µM. Cultures were kept in the dark at 30°C. Fungal growth was analyzed both under the microscope and by a microplate reader at 620 nm. In another set
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of experiments the fungi were allowed to grow to an absorbance of approximately 0.1 before addition of cecropin. Further growth was then assayed as previously. 2.5. Injection of spores into flies Drosophila melanogaster, Canton S, was reared at 25°C on standard yeast agar media. Flies (3–5 days old) were anesthetized with CO2 and injected in the thorax with freshly made suspensions of fungal spores from B. bassiana, S. cerevisiae W308, M. anisopliae or G. candidum (1×106 spores/ml in Drosophila ringer). Injected flies were kept at 25°C. Viability was scored every 24 h. Surviving flies were transferred to fresh vials every third day. 2.6. Northern blot Drosophila melanogaster, Canton S flies (3–5 days old) were injected in the thorax with freshly made suspensions of fungal spores from B. bassiana, D. uninucleata, M. anisopliae or G. candidum (1×106 spores/ml in Drosophila ringer). Injected flies were kept at 25°C. Surviving flies were frozen in liquid nitrogen 12 h, 24 h, 50 h or 9 days after injection. RNA was extracted with Trizol (GIBCO) according to the manufacturer’s protocol. An amount of 15 µg RNA per sample was separated on a 1% denaturing agarose gel and blotted onto a Hybond N filter. Actin 5C (Fyrberg et al., 1980) and Cecropin A1 (Kylsten et al., 1990) probes were synthesized using RediPrime II labeling reaction (Amersham) following the manufacturer’s protocol. Hybridization was performed under high stringency conditions (50% formamide, 42°C).
3. Results 3.1. Effect of cecropins on fungal growth Large zones of growth inhibition were observed when cecropins were applied on fungal plates (Fig. 1), allowing us to calculate the corresponding lethal concentrations. As shown in Table 1, cecropins have a strong activity against different classes of higher fungi. The lethal concentrations vary slightly for different fungi and among the different cecropins, but are in most cases in the micromolar or submicromolar range. However, none of the antimicrobial substances tested show any effect against the entomopathogen B. bassiana. Drosophila cecropin A is somewhat more potent than cecropin B, and comparable to that of the Hyalophora cecropin. We also tested the activity of andropin, a cecropin-like peptide from the male reproductive tract of Drosophila (Samakovlis et al., 1991), but this peptide is considerably less active than the cecropins.
Fig. 1. Inhibition zone assay. The figure shows the effect of three concentrations from a longer dilution series of Drosophila cecropin A, against the fungi S. cerevisiae W308, D. uninucleata, G. candidum, M. anisopliae and B. bassiana. As a comparison we also show the growth inhibition against the bacterial strain E. cloacae β12.
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The inhibition zone assay is a simple way to estimate lethal concentrations, and it requires modest amounts of the active peptides (Hultmark, 1998). However, with some of the filamentous fungi the diameters of the inhibition zones were poorly defined (Fig. 1), and for this reason we repeated some of our results with the Hyalophora cecropin, using a microplate assay (MIC value, Table 1). This also allowed us to observe the effects of the cecropins under the microscope. As shown in Table 1, there is good agreement between the two assays, although the microplate assay tended to give slightly higher values. Again, fungal growth is completely blocked by cecropin at micromolar concentrations, except for B. bassiana which is unaffected. High concentrations of cecropin A causes total cell lysis for S. cerevisiae and G. candidum, as observed under the microscope (data not shown). In contrast, hyphae of M. anisopliae remain intact, although their growth is inhibited. The assays discussed so far show the effect of cecropins on single cells at low density. We also looked at the effect when cecropin is added directly to a dense culture (Fig. 2). Drosophila and Hyalophora cecropins are able to clear even late log phase or early stationary phase culture of S. cerevisiae within a few hours. In contrast, there is no effect of cecropin on dense cultures of G. candidum, either in stationary phase or in a moderately dense growing fungal culture. 3.2. The resistance of flies against injected fungi We tested the actual resistance of Drosophila to the different fungi, in order to correlate it to the antifungal effect of the cecropins (Fig. 3). The flies show good resistance to injection of S. cerevisiae or G. candidum. Surprisingly, a similar survival is also seen after injection of the broad host-range entomopathogen M. anisopliae, and more than 60% of the flies are still alive after 5 days. Clearly, the immune defense is quite efficient
against these fungi. In contrast, B. bassiana, the other insect pathogen, causes 100% lethality within this period of time. Cecropins are normally not present in the flies unless the immune system is stimulated, for instance by a bacterial infection. We therefore tested if fungal infections can induce cecropin gene expression under the conditions used here. Fig. 4 shows that significant levels of Cecropin A1 mRNA can be detected in the flies within 12 h after infection, and that the mRNA concentration increases further during the following days. In particular, the insect pathogen M. anisopliae, to which Drosophila is relatively resistant, gives a very strong induction of cecropin.
4. Discussion Our data show that cecropins have the capacity to inhibit fungal growth at concentrations far below the natural concentrations found in the hemolymph of a challenged fly, and that insect pathogens as well as more harmless fungi are susceptible. The cecropins are induced by fungi, and they efficiently kill fungal cells and repress the growth of hyphae from five out of six tested fungal species, at concentrations as low as 0.5–3 µM. Thus, the cecropins can now be added to a growing list of fungicidal effector molecules in Drosophila. Previously, the inducible peptides drosomycin and metchnikowin have been shown to be fungicidal (Fehlbaum et al., 1994; Levashina et al., 1998), and antifungal activity has also been demonstrated for lysozyme from Galleria mellonella (Vilcinskas and Matha, 1997). Work by Lemaitre et al. (1995, 1997) has indicated that the antifungal effector molecules in Drosophila may be induced by a specific signal pathway, independent from that of the antibacterial peptides. This is most clearly shown for drosomycin. This antifungal peptide is preferentially induced by fungal infections, and its
Table 1 Lethal concentrations (µM) of different cecropins. Values were derived from the inhibition zone assay (Hultmark, 1998). For comparison, we also tested the activity of Hyalophora cecropin A against four fungi in a minimal inhibitory concentration (MIC) assay Drosophila
Saccharomyces cerevisiae W308 Saccharomyces cerevisiae S289C Dipodascopsis uninucleata Geotrichum candidum Metarhizium anisopliae Beauveria bassianaa Enterobacter cloacae β12
Hyalophora
Cecropin A
Cecropin B
Andropin
Cecropin A
(MIC value)
0.7 1.2 0.5 0.9 1.8 ⬎44 0.3
3.3 0.7 1.2 4.0 2.7 ⬎59 0.3
36 59 12 14 68 ⬎65 42
1.0 1.3 0.4 1.4 1.1 ⬎10 0.2
(1.1–2.1)
(2.1–4.3) (2.1–4.3) (⬎17)
a No inhibition zones were detected for B. bassiana. In this case the limit of detection was estimated from the assumption that zones larger than 4 mm would have been recognized.
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Fig. 2. Effect of cecropin on dense fungal cultures. Drosophila cecropin A, or Hyalophora cecropin A was added directly to late cultures of S. cerevisiae W308 or G. candidum in microplate wells, and the cell density was recorded. For the Hyalophora cecropin, a range of concentrations was tested but we only show two concentrations for Saccharomyces and the highest for Geotrichum. Cecropin gives a rapid clearance of the Saccharomyces culture, even at the low concentration, whereas there is no effect on Geotrichum at any concentration tested.
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Fig. 3. Survival of Drosophila after injection of fungi. For each of the indicated fungal strains, 60 adult flies were injected with approximately 100 cells per fly. There was some initial lethality due to the injection procedure itself, and the number of living flies at 17 h was therefore set to 100%.
induction requires the gene products of the Toll pathway. In contrast, antibacterial peptides such as diptericin are best induced by bacteria, via a signal transduction pathway which is defined by the imd gene. The fact that cecropins are potent antifungal as well as antibacterial agents fits nicely into this scheme, since the induction of the cecropins is affected by both pathways (Rosetto et al., 1995; Lemaitre et al. 1995, 1997). The cecropin molecules do not have an ordered structure in solution, but they are thought to be stabilized in an alpha-helical conformation after interaction with the hydrophobic lipids in the bacterial cell membrane (Steiner, 1982). This interaction between cecropins and the cell membrane must have a high degree of specificity as cecropins do not have any lethal effect on insect or mammalian cells. However, the molecular mechanism
behind this specificity remains an open question. The selective killing of bacteria has previously been attributed to the absence of cholesterol in the bacterial cell membrane (Nakajima et al., 1987; Christensen et al., 1988), to the presence of acidic molecules such as lipopolysaccharides or teichoic acids (Shai, 1998), or to differences in membrane potential. The latter factor has been shown to affect the lytic activity of magainins (Cruciani et al., 1991). However, neither of these models are entirely satisfactory to explain why fungi are more susceptible than other eukaryotes to the action of cecropins. The target of the cecropins is believed to be the cell membrane. However, it is worth noting that we could in several cases observe full lysis of the fungal cells, implicating a breakdown of the entire cell wall. It is possible that endogenous lytic enzymes are released as a result of the cecropin action. Of the fungi investigated here, only the entomopathogen Beauveria bassiana is entirely resistant to cecropin. B. bassiana is a broad range insect pathogen that can attach to the cuticle of the insect and grow through the integument and into the hemocoel. The resistance of this pathogen may reflect an adaptation to the defense reactions of the insect host. Protection from lysis may come from a passive resistance of the cell wall or cell membrane, or may depend on the secretion of proteases or similar substances that inactivate or destroy the cecropins. Secreted proteases have been shown to be important for cecropin resistance in several other microbes (Go¨tz et al., 1981; Dalhammar and Steiner, 1984; DeLucca et al., 1997). Secretion of proteases may also be a reason why dense cultures of G. candidum are unaffected by cecropin. It is also possible that the
Fig. 4. Expression of cecropin in Drosophila after injection of fungi. Total RNA from infected flies was separated by electrophoresis, blotted, and probed for cecropin expression using a CecA1 cDNA clone as a probe. The filter was also probed with an actin probe to check for equal loading (not shown). No samples were taken before injection in this particular experiment, but cecropin expression is normally very low in untreated animals. Since the flies are eventually killed by the B. bassiana infection, only early time points are shown for this fungus.
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properties of the cellular envelope is different in the growing hyphae of the dense cultures, compared with the spores tested in the other assays. In the hemolymph, the cecropins undoubtedly act in concert with several other peptides but, as we show here, they can also act as powerful defense molecules on their own. There is indeed a correlation between the ability of the fly to survive a fungal challenge and the effect of cecropin on the fungi. The potent effect against yeasts, which are very abundant in the normal environment of Drosophila, is likely to be important for the protection against accidental infections. Our data support the importance of cecropins as antifungal as well as antibacterial effector molecules in the natural defense of Drosophila melanogaster.
Acknowledgements ˚ stro¨m. The yeast strains used were a gift from Stefan A ˚ Cecropins were synthesized by Ake Engstro¨m and David Andreu. We are grateful to Ha˚kan Steiner for help and discussions. This work was supported by grants from the Swedish Natural Science Research Council, the Swedish Medical Research Council, and by the Go¨ran Gustafsson Foundation for Scientific Research.
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