Peptides Plant Defensin NaD1 and Other Cationic ...

3 downloads 77290 Views 2MB Size Report
Apr 10, 2014 - processed in Illustrator CS6 (Adobe). Flow cytometry. S. cerevisiae strains were grown overnight in YPD medium and then diluted to an OD600 ...
Agp2p, the Plasma Membrane Transregulator of Polyamine Uptake, Regulates the Antifungal Activities of the Plant Defensin NaD1 and Other Cationic Peptides Mark R. Bleackley, Jennifer L. Wiltshire, Francine Perrine-Walker, Shaily Vasa, Rhiannon L. Burns, Nicole L. van der Weerden and Marilyn A. Anderson Antimicrob. Agents Chemother. 2014, 58(5):2688. DOI: 10.1128/AAC.02087-13. Published Ahead of Print 24 February 2014.

These include: REFERENCES

CONTENT ALERTS

This article cites 42 articles, 18 of which can be accessed free at: http://aac.asm.org/content/58/5/2688#ref-list-1 Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more»

Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/

Downloaded from http://aac.asm.org/ on April 10, 2014 by guest

Updated information and services can be found at: http://aac.asm.org/content/58/5/2688

Agp2p, the Plasma Membrane Transregulator of Polyamine Uptake, Regulates the Antifungal Activities of the Plant Defensin NaD1 and Other Cationic Peptides Mark R. Bleackley, Jennifer L. Wiltshire, Francine Perrine-Walker, Shaily Vasa, Rhiannon L. Burns, Nicole L. van der Weerden, Marilyn A. Anderson La Trobe Institute for Molecular Science, Melbourne, Victoria, Australia

F

ungal pathogens infect plant and animal species and are being increasingly recognized as a threat to human health, biodiversity, and agriculture (1). Present-day infections of major crops, including wheat, soybeans, corn, and bananas, are leading to significant yield losses (1, 2). Thus, the development of novel systems for the prevention of fungal infection in plants has been identified as a key point in the effort to protect global food security (3). One way to combat the damaging effects of fungal pathogens is to identify the most potent antifungal molecules that have evolved in nature. The elucidation of the mechanisms by which they inhibit infection can in turn be used to facilitate their development as tools to combat fungal pathogens. Of particular interest are geneencoded innate immunity peptides that are produced by a wide range of organisms, including animals, plants, insects, fungi, and bacteria (4). Plants lack an adaptive immune system and have therefore evolved a variety of innate immune responses to protect against infection (5). A major component of the plant innate immune system is the production of antimicrobial peptides (6), many of which are active against a variety of fungi (4). Gene-encoded peptides are of particular interest, as once their sequence is known, the encoding DNA can be used to produce the peptides recombinantly for characterization and for the construction of transgenic plants with increased antifungal resistance. Gaining an understanding of the mechanisms by which these proteins act against fungal pathogens is crucial for the development of improved antifungal molecules and treatment regimens. One of the largest families of plant antimicrobial peptides is the defensins (7–9). Plant defensins are small (45 to 54 amino acids) basic proteins with four or five disulfide bonds (7). Although there is structural conservation among plant defensins, there is substantial variability in their amino acid sequences, which leads to a

2688

aac.asm.org

variety of biological functions and mechanisms of action (4, 9). The mechanisms of antifungal activity also vary between defensins, and although not well defined, they include features such as sphingolipid binding, generation of reactive oxygen species (ROS), cell wall stress, septin mislocalization, programmed cell death, blocking of calcium channels, and cell cycle arrest (10–16). NaD1 is a defensin from the ornamental tobacco Nicotiana alata (17) that kills filamentous fungi, such as Fusarium graminearum and Fusarium oxysporum (18), the human fungal pathogens Candida albicans and Cryptococcus neoformans, and the model yeast Saccharomyces cerevisiae (19). Transgenic cotton plants expressing NaD1 are resistant to F. oxysporum and Verticillium dahliae in the field (20). The killing of the fungal cell is proposed to occur via a three-step process that is dependent on the presence of the fungal cell wall. The exposure of fungi to NaD1 results in ROS and nitric oxide (NO) production, as well as permeabilization of the fungal membrane (18, 19, 21), and has been proposed to involve dimerization of the defensin (22). Studies on the activity of NaD1 in C. albicans have revealed that the Hog1 pathway plays a role in protecting the cell from the damaging effects of NaD1, and S. cerevisiae studies have provided evidence for the involvement of active mitochondria in ROS production

Antimicrobial Agents and Chemotherapy

Received 24 September 2013 Returned for modification 17 October 2013 Accepted 15 February 2014 Published ahead of print 24 February 2014 Address correspondence to Mark R. Bleackley, [email protected], or Marilyn A. Anderson, [email protected]. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/AAC.02087-13

p. 2688 –2698

May 2014 Volume 58 Number 5

Downloaded from http://aac.asm.org/ on April 10, 2014 by guest

Cationic antifungal peptides (AFPs) act through a variety of mechanisms but share the common feature of interacting with the fungal cell surface. NaD1, a defensin from Nicotiana alata, has potent antifungal activity against a variety of fungi of both hyphal and yeast morphologies. The mechanism of action of NaD1 occurs via three steps: binding to the fungal cell surface, permeabilization of the plasma membrane, and internalization and interaction with intracellular targets to induce fungal cell death. The targets at each of these three stages have yet to be defined. In this study, the screening of a Saccharomyces cerevisiae deletion collection led to the identification of Agp2p as a regulator of the potency of NaD1. Agp2p is a plasma membrane protein that regulates the transport of polyamines and other molecules, many of which carry a positive charge. Cells lacking the agp2 gene were more resistant to NaD1, and this resistance was accompanied by a decreased uptake of defensin. Agp2p senses and regulates the uptake of the polyamine spermidine, and competitive inhibition of the antifungal activity of NaD1 by spermidine was observed in both S. cerevisiae and the plant pathogen Fusarium oxysporum. The resistance of agp2⌬ cells to other cationic antifungal peptides and decreased binding of the cationic protein cytochrome c to agp2⌬ cells compared to that of wild-type cells have led to a proposed mechanism of resistance whereby the deletion of agp2 leads to an increase in positively charged molecules at the cell surface that repels cationic antifungal peptides.

Agp2 and NaD1 Activity

(19). However, the cell surface receptor and intracellular targets of NaD1 have yet to be elucidated. With the aim of identifying these components, we screened the nonessential S. cerevisiae deletion collection for strains that are resistant to NaD1. This led to the identification of a set of genes involved in polyamine transport that have a role in the antifungal activity of NaD1. Of particular interest was the cell membrane regulator of polyamine and carnitine transport, Agp2p. Deletion of the agp2 gene imparted resistance to NaD1 via a mechanism that includes diminished uptake of the defensin. Further analysis of the antifungal peptide sensitivity in agp2⌬ cells revealed a link between polyamine uptake and the activities of cationic antifungal peptides as a whole. MATERIALS AND METHODS

May 2014 Volume 58 Number 5

aac.asm.org 2689

Downloaded from http://aac.asm.org/ on April 10, 2014 by guest

Yeast strains and media. The S. cerevisiae nonessential deletion collection was purchased from Thermo Scientific and is in the S. cerevisiae strain BY4741 (MAT␣ his3⌬0 leu2⌬0 met15⌬0 ura3⌬0) background. The mutant strains were retrieved from the deletion collection and compared to BY4741. Double mutants were made by amplifying the URA3 gene from the pRS426 plasmid using primers containing 40-bp regions corresponding to the 5= and 3= ends of the coding region upstream of the pRS426 binding sequence (bases shown below in italics). The primer pairs were Dur3F (ATGGGAGAATTTAAACCTCCGCTACCTCAAGGCGCTGG GTCTGTGCGGTATTTCACACCG) and Dur3R (TTAAATTATTTCAT CAACTTGTCCGAAATGTGATGATTGTCGATTGTACTGAGAGTGCAC), and Sam3F (ATGGATATACTCAAGAGGGGAAATGAATCGGACAAG TTTACTGTGCGGTATTTCACACCG), and Sam3R (TAACACCAAAAT CTGTAGATTTTGTAATAGAATGGCTTAGGATTGTACTGAGAGT GCAC). PCR products were purified using a Wizard PCR cleanup kit (Promega) and transformed into yeast cells via electroporation. The mutant colonies were selected on synthetic defined medium with uracil dropout (SD-Ura) (0.67% yeast nitrogen base without amino acids [Sigma], 0.077% uracil dropout (Ura DO) supplement [Clontech]) agar plates. Overnight cultures for all S. cerevisiae experiments were grown in YPD medium (1% yeast extract, 2% peptone, 2% dextrose). All mutants were confirmed by genotyping. Fusarium oxysporum f. sp. vasinfectum (Australian isolate VCG01111 isolated from cotton; Farming Systems Institute, Department of Primary Industries (DPI), Queensland, Australia; a gift from Wayne O’Neill) overnight cultures were grown in half-strength potato dextrose broth (1/2 PDB) (BD Difco). All experiments were conducted in 1/2 PDB except where otherwise noted. All chemicals were purchased from Sigma unless otherwise noted. NaD1 was purified from N. alata flowers as outlined by van der Weerden et al. (18). HBD2 was expressed in PichiaPink (Life Technologies) using the protocol outlined by Hayes et al. (19). Other antifungal peptides were purchased from GenScript (Hong Kong China) or GL Biochem (Shanghai, China). Yeast deletion screen. The nonessential S. cerevisiae deletion collection (Thermo Scientific) (23, 24) was grown overnight at 30°C in 96-well plates in YPD medium supplemented with G418 (200 ␮g/ml) (Amresco). Each strain was diluted 1:100 with 1/2 PDB, and diluted culture (5 ␮l) was added to 95 ␮l of 1/2 PDB containing 4 ␮M NaD1. This concentration of NaD1 was chosen, as it was the lowest concentration for which ⬎90% inhibition was regularly observed in preliminary experiments on wildtype yeast conducted under the same conditions as the screen (data not shown). Half-strength PDB with no added protein was used as a control. Liquid handling was performed with a Bravo automated liquid handling platform (Agilent Technologies). Growth was measured as the increase in absorbance at 595 nm after incubation for 20 h at 30°C. Absorbance at 595 nm was measured using a SpectraMax M5e plate reader (Molecular Devices). The percent growth inhibition [(growth in control medium ⫺ growth in NaD1 medium)/(growth in control medium ⫻ 100)] was calculated for each strain, and resistant strains were defined as those with a percent growth inhibition that was more than two standard deviations

below the mean. Strains that did not grow in 1/2 PDB were eliminated from the analysis, and the remaining resistant strains were rescreened in triplicate in 4 ␮M NaD1 to ensure reproducibility. Strains exhibiting ⬍60% growth inhibition in the triplicate screen were then considered the NaD1-resistant subset. P values were calculated using the embedded function in Microsoft Excel. Antifungal assays. Antifungal protein stock solutions were prepared at 10⫻ the desired final concentration in sterile Milli-Q water. Ten microliters of each solution was added to the wells of a 96-well microtiter plate along with 90 ␮l of overnight culture that had been diluted to an optical density at 600 nm (OD600) of 0.01 in 1/2 PDB. Growth was measured as the increase in absorbance at 595 nm after incubation for 20 h at 30°C. The absorbance at 595 nm was measured using a SpectraMax M5e plate reader (Molecular Devices). The percent growth inhibition was calculated as shown above. All experiments were replicated at least three times. Survival assays. Antifungal protein stocks were prepared as described above and added to 90-␮l aliquots of overnight cultures that had been diluted to an OD600 of 0.2. For experiments on the effect of spermidine, spermidine was prepared at 100⫻ the final concentration, and 1 ␮l was added to the diluted cells together with 10 ␮l of the antifungal protein. The cells were incubated for 1 h at 30°C. After incubation, the cells were serially diluted three times, and each dilution was plated (4 ␮l) on a YPD plate and incubated at 30°C for 48 h. All experiments were repeated a minimum of three times, and the results were consistent across the replicates. Membrane permeabilization assays. Membrane permeabilization assays were performed using a protocol modified from that of van der Weerden et al. (21). Briefly, antifungal protein solutions were prepared as 10⫻ concentrates in Milli-Q water as described for the antifungal assays except for experiments also involving polyamines. In those experiments, NaD1 was made up at 20⫻ the final concentration, and the polyamine (putrescine or spermidine) was made up at 10⫻ the final concentration. The NaD1 solution (5 ␮l) and the polyamine solution (10 ␮l) were transferred to the wells of a black 96-well plate (Nunc). S. cerevisiae overnight cultures (85 ␮l) that had been diluted to an OD600 of 0.1 in 1/2 PDB with 1 ␮M Sytox green (Molecular Probes) were then added to the wells prior to analysis. Sytox green uptake was measured using a SpectraMax M5e plate reader (Molecular Devices) with excitation and emission wavelengths of 488 nm and 538 nm, respectively. All data presented were consistent across at least three independent replicates. Microscopy. Yeast cells were grown overnight in YPD liquid medium and diluted to an OD600 of 0.5 to 0.6 (BioPhotometer; Eppendorf) in 1/2 PBD. The yeast cell suspension (300 ␮l) was then placed in ␮-slide 8 wells (ibidi) for live-cell imaging. All live-cell imaging experiments were maintained at 30°C within a Zeiss clear Perspex incubator equipped with a heater and temperature controller (Tempcontrol 37-2 digital). NaD1 was labeled with the fluorophore BODIPY-FL EDA (Molecular Probes) as outlined by van der Weerden et al. (21). The samples were treated with 0 or 25 ␮l of 2.5 mg/ml BODIPY-labeled NaD1 peptide stock solution. The yeast cells were then visualized by confocal microscopy using a Zeiss LSM 510 laser scanning Axiovert 200M inverted confocal microscope with a Plan Apochromat 100⫻/1.4 oil differential interference contrast (DIC) objective. For live-cell imaging, the cells were excited at 488 nm with an argon laser using band-pass filters BP505-530 or BP505-550 to detect BODIPY fluorescence (green). Time series images at 30-s and 60-s intervals over 36 min were acquired using the Zen 2009 image acquisition software (Carl Zeiss MicroImaging GmbH). Control experiments were performed using the same parameters. The experiments were conducted in triplicate. The images were further analyzed using Fiji software (ImageJ 1.47h version) (see http://imagej.nih.gov/ij/) and Zeiss LSM Image Browser version 4.2.0.121 (Carl Zeiss MicroImaging GmbH) and were processed in Illustrator CS6 (Adobe). Flow cytometry. S. cerevisiae strains were grown overnight in YPD medium and then diluted to an OD600 of 0.1 in 1/2 PDB before 100-␮l aliquots were treated with a given concentration (0, 5, 10, or 20 ␮M) of

Bleackley et al.

RESULTS

Yeast deletion screen. Screening of the S. cerevisiae haploid nonessential deletion collection for strains resistant to 4 ␮M NaD1 revealed the expected normal distribution of phenotypes across the collection (Fig. 1A). Strains were selected as resistant if the percent growth inhibition was more than two standard deviations below the mean (⬍89%). Eighty-one strains were resistant to NaD1, and these strains were rearrayed in a microtiter plate and rescreened against 4 ␮M NaD1 in triplicate. Strains that exhibited an average percent growth inhibition of ⬍60% were then considered the set of resistant deletion strains (Table 1 and Fig. 1B). No growth was observed for strain BY4741 in the triplicate rescreen. A functional cluster analysis of the resistant strains was performed using FunSpec (26). Minimal clustering was observed for any Gene Ontology (GO) or Munich Information Center for Protein Sequences (MIPS) classifications. However, there was some indication of enrichment for genes with functions in the mitochondria and in polyamine transport. A manual examination of the annotated locations and functions of the genes deleted in the NaD1-resistant set

2690

aac.asm.org

FIG 1 Screening of the nonessential S. cerevisiae deletion collection for resistance to 4 ␮M NaD1. (A) Distribution of percent growth inhibition phenotypes across the collection. Extreme outliers were removed from this graph. (B) Growth phenotypes of the 81 strains initially identified as sensitive when rescreened in triplicate against 4 ␮M NaD1. No growth was observed for the wild type (BY4741) at this concentration. Strains with a growth inhibition of ⬍60% were considered resistant. The error bars are the standard deviation for each set of triplicates.

using the Saccharomyces genome database (www.yeastgenome .org) gave further support for the roles of mitochondria (cbp1, ccs1, tuf1, and mrp7) and polyamine transport (agp2, brp1, ptk2, and sky1) (27) in the antifungal activity of NaD1. A link between functional mitochondria and NaD1 activity has been reported previously (19), so we chose to pursue the role of polyamine transport in NaD1 activity as the focus for subsequent experiments. Resistant strains with deletions in genes with known functions in polyamine transport (agp2, brp1, ptk2, and sky1) (27), all of which had a P value for resistance of ⬍1 ⫻ 10⫺4 from the triplicate rescreen, were retrieved from the deletion collection and reassessed across a range of NaD1 concentrations to determine the 50% inhibitory concentration (IC50) of NaD1 against these strains. All four strains showed significant resistance to NaD1, with IC50s between 2.75 and 4 ␮M (relative to 2 ␮M for the wild type) (Fig. 2A). The most resistant strain was agp2⌬, which encodes a regulator of high-affinity polyamine transport (28). It is important to note that the antifungal assays were performed at a lower cell density (OD600, 0.01) than the experiments outlined in the following sections (OD600, 0.1 to 0.5). Higher concentrations of NaD1 are required to achieve the same level of growth inhibition when higher cell densities are used. Link between polyamine transport and NaD1-induced membrane permeabilization. To further investigate NaD1 resistance

Antimicrobial Agents and Chemotherapy

Downloaded from http://aac.asm.org/ on April 10, 2014 by guest

BODIPY-labeled NaD1 for 30 min or 3 h in the absence of light. The cells were then washed two times with 100 ␮l of phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4 [pH 7.4]) before they were resuspended in 100 ␮l PBS. The samples were then transferred to a U-bottom 96-well plate (Greiner) and analyzed using the fluorescein isothiocyanate (FITC) channel on a fluorescence-activated cell sorter (FACS) Canto II fitted with a high-throughput sequencing (HTS) sampler (BD Biosciences). FACS analysis was performed using Weasel (Walter and Elisa Hall Institute for Medical Research). NaD1 pulldown and Western blot. BY4741, sam3 dur3, and agp2⌬ cells were grown overnight at 30°C in liquid YPD medium. One milliliter of cell culture was pelleted and the cells washed two times with 1 ml of Milli-Q H2O in 1.5-ml microcentrifuge tubes. The cells were then resuspended in 1/2 PDB at an OD600 of 0.5. BY4741 cells were resuspended with and without the addition of 100 ␮M spermidine or putrescine. NaD1 was added to a final concentration of 20 ␮M in a final volume of 100 ␮l. Each treatment was then rotated at room temperature on a Ratek RSM6 wheel for 30 min. The cells were then pelleted at maximum speed in a microcentrifuge, washed with 100 ␮l of Milli-Q water, and resuspended in 20 ␮l of 0.01% SDS. NuPAGE lithium dodecyl sulfate (LDS) sample buffer (Life Technologies) and Bond-Breaker TCEP solution (Thermo Scientific) (5 ␮l total of a 9:1 solution of NuPAGE LDS buffer to Bond-Breaker) was added to each sample and incubated at 90°C for 20 min. Total cell suspensions were run on SDS-PAGE. The proteins were transferred to nitrocellulose using a Trans-Blot Turbo system (Bio-Rad). The NaD1 levels were determined by Western blotting using an anti-NaD1 primary antibody (18) and donkey anti-rabbit (GE Healthcare) secondary antibody. Immunoreactive NaD1 was visualized using the GE Healthcare ECL Western blotting detection reagents kit, and densitometry of the resulting bands was performed using the Image Lab software package (Bio-Rad). Cytochrome c binding assay. The cytochrome c binding assay was modified from the protocol outlined by Peschel et al. (25). Yeast cells were grown overnight at 30°C in liquid YPD medium and the cells were pelleted and resuspended at an OD600 of 13. In a 1-ml aliquot for each strain, the cells were washed two times with PBS and resuspended in 1 ml of PBS. The cells (100 ␮l) were then added to 300 ␮l of a cytochrome c solution (0.5 mg/ml of equine cytochrome c [Sigma] in PBS) and incubated at room temperature on a Ratek RSM6 wheel for 20 min. The cells were then pelleted and the supernatant (3 ⫻ 100 ␮l) was transferred to the wells of a 96-well microtiter plate. Cytochrome c remaining in the supernatant was quantified spectrophotometrically at 530 nm, the absorption maximum of the protein resulting from the heme prosthetic group.

Agp2 and NaD1 Activity

TABLE 1 Genes deleted in S. cerevisiae strains that were found to be resistant when screened in triplicate against 4 ␮M NaD1 % Growth inhibitiona ⬍20 20–40

40–60 a

Genes deleted PAC10, SLS1, LPX1, ORM2, EMC6, PET494, TVP18, PTK2, YDL034W, TRP2, YNL109W, VMA11, SPO7, MSN5, YSA1, SKY1, TUF1, KEX1, YJL120W, UPF3, MTH1, YOR200W, FUN19, AAT2, YGP1, UBP14, TCO89, YOR379C, SUR2, YBL028C, OPI9, YGL214W, IRC21 CCS1, SEM1, FAR7, AGP2, PTC3, RPL26B, YGR269W, LDB19, RAS1, NCL1, RXT2, URE2, BRP1, RIB4, YFR012W, CBP1, RSM27, SAP185, YDL062W, FMP48, YPL182C, CTI6, YGR069W, YVC1, ARP8, YHR039C-B, DOA1, HSV2, YGL149W, MAP1, EMI2, RTK1, MRPL6, YDR290W, NAP1, CPR7, GZF3, CUE3, NAM7 YJL027C, TDH3, HIS7, YOR199W, MTC7, RGD2, UBC4, YDL063C, MRPS35

Only strains where all three replicates had ⱕ60% growth inhibition were included in the resistant set.

FIG 2 NaD1 resistance in strains lacking components of the high-affinity spermidine pathway. (A) Growth of deletion strains across a range of NaD1 concentrations. Less growth inhibition occurred in strains lacking a component of the polyamine uptake system, sky1⌬ (dotted black line), brp1⌬ (dotted gray line), ptk2⌬ (dashed black line), or agp2⌬ (solid gray), than the wild type (solid black line). (B) NaD1-induced (20 ␮M) membrane permeabilization of the agp2⌬ mutant (solid gray line) was dramatically decreased compared to the wild type (solid black line), and the membrane permeabilization kinetics of sky1⌬ (dotted black line), brp1⌬ (dotted gray line), and ptk2⌬ (dashed black line) are intermediate compared to the agp2⌬ mutant and wild type. Error bars are standard deviation of three biological replicates. The trend was consistent over a minimum of three independent experiments. RFU, relative fluorescence units.

May 2014 Volume 58 Number 5

mary mode of action (21). Consistent with the growth assays, the set of NaD1-resistant strains was permeabilized more slowly by NaD1 than the wild type (Fig. 2B), with agp2⌬ mutants exhibiting a lower (around 20% wild type) level of Sytox green uptake than the other mutants (60 to 90% wild type). Agp2p is a plasma membrane protein that senses the polycation spermidine and regulates the expression of proteins that transport spermidine into the cell (28). Since NaD1 is a positively charged protein, we considered whether it binds to the cell by exploiting polyamine binding molecules on the surface of the cell. This was investigated by measuring NaD1-mediated membrane permeabilization in the presence of increasing concentrations of spermidine or putrescine. Spermidine protected S. cerevisiae from membrane permeabilization by NaD1 in a concentration-dependent manner (Fig. 3A). In contrast, putrescine had little effect on NaD1-induced membrane permeabilization (Fig. 3B). The effect of spermidine on NaD1induced cell death was also measured (Fig. 4). The addition of spermidine enhanced the survival of BY4741 cells. The level of survival of cells treated with 100 ␮M spermidine was similar to that of agp2⌬ cells without added spermidine. As with the membrane permeabilization assay, spermidine protected cells against the activity of NaD1 in a concentration-dependent manner. The addition of exogenous putrescine had no observable effect on cell survival (data not shown). To confirm that the link between polyamines and NaD1 is conserved from the model yeast to plant pathogens, membrane permeabilization assays in the presence of polyamines were repeated using F. oxysporum f. sp. vasinfectum. Consistent with the results in S. cerevisiae, spermidine inhibited NaD1-induced membrane permeabilization in a concentration-dependent manner, and putrescine was less effective than spermidine (Fig. 3C). Cells lacking Agp2p have reduced uptake of NaD1. In previous studies with F. oxysporum and C. albicans, we demonstrated that NaD1 is transported into the fungal cytoplasm as part of the mechanism of fungal cell killing (18, 19). Confocal microscopy using NaD1 labeled with the fluorophore BODIPY-FL EDA revealed that uptake of the defensin was lower in agp2⌬ cells than in wild-type cells over a time course of 30 min (Fig. 5A). The uptake of BODIPY-NaD1 was also measured by flow cytometry. As observed using fluorescence microscopy, the uptake of BODIPY-NaD1 over 30 min was lower in agp2⌬ cells than in wild-type BY4741 cells at all NaD1 concentrations tested (Fig. 5B and C). The uptake of BODIPY-NaD1 was concentration dependent in both strains and was not fully dependent on the presence of Agp2p. Seeing as spermidine decreased the membrane-permeabilizing activity of NaD1, the effects of spermidine on NaD1 binding and uptake were assayed using a whole-cell pulldown, followed by Western blotting, to detect levels of NaD1 associated

aac.asm.org 2691

Downloaded from http://aac.asm.org/ on April 10, 2014 by guest

in yeast strains with deletions of genes that function in polyamine uptake, the kinetics of membrane permeabilization were monitored using a Sytox green-mediated assay. Membrane permeabilization is likely a downstream effect of the activity of NaD1, and as such, membrane permeabilization is delayed compared to other antifungal peptides for which membrane disruption is the pri-

Bleackley et al.

FIG 3 Exogenous spermidine protects against membrane permeabilization by NaD1 in S. cerevisiae. (A) Treatment with increasing concentrations of spermidine, the preferential substrate for Agp2p, decreases membrane permeabilization by 20 ␮M NaD1 in a concentration-dependent manner; spermidine concentrations were 100 ␮M (solid gray line), 25 ␮M (dotted gray line), 12.5 ␮M (dotted black line), and 0 ␮M (solid black line). (B) Treatment with putrescine does not provide the same level of protection against membrane permeabilization by NaD1; putrescine concentrations were 100 ␮M (solid gray line), 25 ␮M (dotted gray line), 12.5 ␮M (dotted black line), and 0 ␮M (solid black line). (C) Permeabilization of the F. oxysporum plasma membrane by 20 ␮M NaD1 in the presence of 100 ␮M spermidine (solid gray line), 12.5 ␮M spermidine (dotted gray line), 100 ␮M putrescine (dashed black line), 12.5 ␮M putrescine (dotted black line), and no added polyamine (solid black line). As with S. cerevisiae, permeabilization of the F. oxysporum membrane by NaD1 was inhibited better by spermidine than putrescine. The data are representative of and consistent over a minimum of three independent replicates.

2692

aac.asm.org

with the yeast cells. Cells treated with 100 ␮M spermidine pulled down 47.3% of the amount of NaD1 that was pulled down by untreated wild-type cells. Similarly, the agp2⌬ cells pulled down only 29.7% of the NaD1 that was associated with the untreated wild-type cells (Fig. 5D to F). Analysis of other polyamine transport mutants. To determine whether the resistance observed in agp2⌬ cells was specific to the lack of Agp2 protein in the membrane or a defect in cellular polyamine transport, the phenotype of yeast strains lacking two other polyamine transport proteins, dur3⌬ and sam3⌬ (29), as well as the double mutants dur3⌬ agp2⌬ and sam3⌬ agp2⌬, was investigated. The single mutants exhibited the same sensitivity to NaD1 as the wild type, and the double mutants had the same resistance as agp2⌬ mutants (Fig. 6). That is, the deletion of other polyamine transporters did not enhance resistance to NaD1 in a wild-type or agp2⌬ background. To further this line of investigation, the phenotype of a sam3⌬ dur3⌬ double mutant was also examined. No resistance to NaD1 was observed in the sam3⌬ dur3⌬ double mutant compared to the wild type (Fig. 7B). Interestingly, the double mutant bound less NaD1 in the whole-cell pulldown assay despite the lack of phenotype (Fig. 5E and F). Resistance of agp2⌬ to other antifungal peptides. We hypothesized that the mechanism for the involvement of Agp2p in the activity of NaD1 was providing a binding partner through a cation binding site normally utilized for the polyamine spermidine. To determine whether the interaction was specific for NaD1, we assayed agp2⌬ cells for resistance to other positively charged antifungal proteins: CP29, a cecropin-bee melittin hybrid peptide variant that permeabilizes microbial membranes (30); BMAP28, a cathelicidin variant with membrane-permeabilizing activity (31); Bac2a, a bactenecin variant that enters the cytoplasm of microbial cells (32); and HBD2, a human beta defensin (33). In addition, hydrogen peroxide was used as a control. The agp2⌬ mutant was more resistant than BY4741 for every antifungal peptide tested (Fig. 8) but not hydrogen peroxide, implicating Agp2p in the general sensitivity of S. cerevisiae to cationic antifungal peptides. The lack of resistance to hydrogen peroxide indicates that this resistance is specific to cationic antifungal proteins and is not merely due to an increase in general fitness in the agp2⌬ mutant. As we had already constructed the sam3⌬ dur3⌬ double mutant, we also

Antimicrobial Agents and Chemotherapy

Downloaded from http://aac.asm.org/ on April 10, 2014 by guest

FIG 4 Spermidine (SPD) protects S. cerevisiae against the antifungal activity of NaD1. BY4741 cells were treated with 20 ␮M NaD1 in the presence of 2-fold serial dilutions of spermidine from a top concentration of 100 ␮M. Spermidine protected BY4741 cells against the activity of NaD1 in a concentrationdependent manner. The activity of NaD1 on BY4741 in the presence of 100 ␮M spermidine is similar to the activity on agp2⌬ cells.

Agp2 and NaD1 Activity

BODIPY-NaD1 on wild-type and agp2⌬ cells. The images are overlays of the green and DIC channels. The time points in seconds are in the top left of each panel. The uptake of labeled defensin was retarded in agp2⌬ compared to wild-type cells at each time point up to 36 min (2,160 s). Bars, 20 ␮m. (B) Representative FACS histogram of BODIPY-NaD1 uptake after 30 min; the BODIPY-NaD1 concentrations are 0 ␮M (gray), 5 ␮M (red), 10 ␮M (green), and 20 ␮M (blue). Less defensin was taken up by agp2⌬ cells than by wild-type cells at all concentrations. (C) Relative uptake of BODIPY-NaD1 in wild-type and agp2⌬ cells from FACS histograms; BY4741 (gray), agp2⌬ (black). Labeled defensin uptake was concentration dependent. The data were averaged across three independent replicates, and the values for BY4741 and agp2⌬ cells were significantly different (P value of ⬍0.05) at all concentrations. (D) Anti-NaD1 Western blot of pulldown experiments comparing the binding of NaD1 to BY4741 in the presence of 100 ␮M spermidine (SPD), 100 ␮M putrescine (PUT), and with no added polyamine. (E) Anti-NaD1 Western blot of pulldown experiments comparing the binding of NaD1 to the agp2⌬ mutant and the sam3⌬ dur3⌬ double mutant to the wild-type BY4741. (F) Densitometry of bands from Western blot pulldown. Less NaD1 was pulled down by agp2⌬ and the spermidine-treated cells than wild-type cells, double mutants, or the putrescine-treated cells. The data are averaged across three independent experiments.

tested other antifungal peptides (AFPs) against the double mutant. No resistance was observed in the double mutant compared to the wild type for the panel of AFPs we tested (Fig. 7). In fact, the double mutant was slightly more sensitive to cationic AFPs than the wild type. Cytochrome c binding in agp2⌬. As the agp2⌬ mutant was resistant to a range of cationic AFPs, we hypothesized that the

May 2014 Volume 58 Number 5

mechanism of resistance was an increase in the positive charge on the cell surface of the mutant cells, which decreased binding of cationic proteins to the cell surface. To test this hypothesis of an increase in positive charge, we measured the binding activity of cytochrome c. Cytochrome c is a cationic protein (pI, 10 to 10.5) with a heme prosthetic group that absorbs at 530 nm. Thus, the levels of cytochrome c remaining in the supernatant were mea-

aac.asm.org 2693

Downloaded from http://aac.asm.org/ on April 10, 2014 by guest

FIG 5 Cells lacking the polyamine transporter Agp2p have reduced uptake of NaD1 compared to wild type. (A) Time course confocal microscopy of 40 ␮M

Bleackley et al.

DISCUSSION

percentages of growth inhibition of the single and double mutants across a range of NaD1 concentrations. Single mutants dur3⌬ () and sam3⌬ (gray triangle) have similar growth phenotypes to the wild-type BY4741 (gray diamond). The double mutants dur3⌬ agp2⌬ (*) and sam3⌬ agp2⌬ (gray circle) have similar growth phenotypes to the single mutant agp2⌬ (X). This indicates that Dur3 and Sam3 do not play a role in the antifungal activity of NaD1. The error bars are the standard deviation of three biological replicates. The trend was consistent over a minimum of three independent experiments.

sured after the cells were incubated with cytochrome c by measuring the absorbance at 530 nm. Wild-type (BY4741) cells bound most of the added cytochrome c, whereas a negligible amount of cytochrome c bound to the agp2⌬ cells over the 20-min time course (Fig. 9).

FIG 7 Susceptibility of sam3⌬ dur3⌬ double mutant to cationic antifungal peptides. Shown are the percentages of growth inhibition of the wild-type BY4741 (black) and sam3⌬ dur3⌬ double mutant (gray) when incubated with increasing concentrations of H2O2 (A), NaD1 (B), BMAP28 (C), and CP29 (D). The double mutant is slightly sensitive to NaD1, CP29, and H2O2, and there is no difference in susceptibility to BMAP28.

2694

aac.asm.org

Antimicrobial Agents and Chemotherapy

Downloaded from http://aac.asm.org/ on April 10, 2014 by guest

FIG 6 Phenotypes of polyamine transporter double mutants. Shown are the

In this paper, we describe a genetic approach for identifying the target molecules of the antifungal defensin NaD1. The S. cerevisiae nonessential deletion collection was screened to identify strains that were resistant to NaD1. An enrichment of mitochondrial genes, many of which yield a petite phenotype when deleted, was found in the set of NaD1-resistant deletions. This is in agreement with a previous study in which roles for respiratory-competent mitochondria and reactive oxygen species in the antifungal activity of NaD1 were described (19). In this study, a role for the plasma membrane regulator of spermidine transport, Agp2p, was also identified and confirmed via growth assays, membrane permeabilization assays, microscopy, and flow cytometry. Cells lacking Agp2 were resistant to NaD1 and showed decreased uptake of the labeled defensin. This decreased uptake led to the conclusion that the moiety that NaD1 binds to on the cell surface is absent, downregulated, or modified in the absence of Agp2p or that the lack of Agp2p caused an increase in the concentration of molecules that interfere with NaD1 binding to the cell surface. The resistance of agp2⌬ mutants to NaD1 was more robust at earlier time points, which indicates that the defensin was able to partially overcome the resistance mechanism with prolonged exposure to the fungus. The application of exogenous spermidine, one of the key molecules sensed by Agp2p, provided protection against the antifungal activity of NaD1 and decreased the uptake of the defensin into yeast cells, further linking Agp2p to the activity of NaD1. Spermidine also protected F. oxysporum against NaD1, which indicates that this mechanism may be conserved in pathogenic fungi. The

Agp2 and NaD1 Activity

Downloaded from http://aac.asm.org/ on April 10, 2014 by guest FIG 8 Resistance of agp2⌬ mutant to a range of cationic antifungal proteins. Growth inhibition of wild-type BY4741 (black) and agp2⌬ mutant (gray) when incubated with increasing concentrations of H2O2 (A), NaD1 (B), Bac2a (C), BMAP28 (D), CP29 (E), and HBD2 (F). Resistance in the agp2⌬ mutant was observed for all cationic peptides but not for H2O2.

resistance of agp2⌬ mutants to other cationic antifungal peptides was also observed. These observations have led us to the conclusion that polyamine uptake and metabolism are linked to the susceptibility of fungi to cationic antifungal proteins. Spermidine is a polycationic molecule belonging to a family called polyamines, which have a number of biological functions, including roles as plant growth factors and stress response molecules in a range of organisms (34), as well as in the induction of mycotoxin production in F. graminearum (35). The positive charge allows polyamines to associate with polyanions, such as nucleic acids and lipids, as well as proteins. Furthermore, interactions with cellular components allow

May 2014 Volume 58 Number 5

polyamines to mediate a variety of intracellular processes by modulating gene expression (36). An example of polyamine-mediated gene expression has been well described in Pseudomonas aeruginosa PAO1. In this system, exogenous spermidine induces the oprHphoPQ operon, which encodes a membrane porin and a two-component regulatory system. It also induces the PA3552-PA3559 operon, which functions in lipopolysaccharide (LPS) modification. The result of this altered gene expression is resistance to cationic peptide antibiotics, such as polymyxin B, and to aminoglycoside and quinolone antibiotics (37). As this mechanism centers on LPS, which is not present in fungi, it was eliminated as

aac.asm.org 2695

Bleackley et al.

a possible mechanism of resistance to NaD1, as reported here. Interestingly, polyamines are reported to have the opposite effect on other antibiotics, including ␤-lactams, for which the application of exogenous polyamine increases the sensitivity of the bacteria (38). Three hypotheses for the role of Agp2 and polyamines in the activity of NaD1 were initially considered. First, as Agp2p is a plasma membrane protein, we considered that it may be the cell surface receptor for NaD1. The implication of plasma membrane polyamine binding proteins in the mechanism of antifungal proteins is not unique. Histatin 5, an antifungal peptide from human saliva, kills C. albicans cells via a mechanism that involves binding to Dur3p and Dur31p, which are both polyamine transporters (39). In addition, histatin 5-resistant Candida glabrata strains can be made sensitive to histatin 5 by expressing the C. albicans DUR3 and DUR31 proteins (40). Thus, the first hypothesis was based on similar rationale as proposed for histatin 5, that is, polyamines are positively charged molecules and the proteins that bind them would possess a cation binding region. NaD1 also carries a positive charge and thus might bind to Agp2p on the cell surface by exploiting the cation binding sites in the protein. This model fits the decreased activity of NaD1 in the presence of increased spermidine, as spermidine would compete with NaD1 for binding to Agp2p in the plasma membrane. However, when we also considered the broad-spectrum resistance of agp2⌬ cells to cationic antifungal peptides, this model did not seem applicable. The sequences and structures of the other AFPs tested vary significantly from each other and from NaD1. NaD1 acts through a complex mechanism that involves binding to the cell wall and translocation into the fungal cytoplasm. CP29 and BMAP28 are short membrane-permeabilizing peptides with little secondary structure. Bac2a is a short linear peptide that enters the cytoplasm and kills fungal cells through a yet-to-be-described mechanism. HBD2 adopts the characteristic defensin fold and also acts through a complex mechanism. It is unlikely that a group of proteins as diverse as the ones tested here have a common binding partner on the cell surface. Agp2p was recently shown to regulate the expression of a large number of genes, many of which encode plasma membrane trans-

2696

aac.asm.org

Antimicrobial Agents and Chemotherapy

Downloaded from http://aac.asm.org/ on April 10, 2014 by guest

FIG 9 Cytochrome c (Cyt c) binding to BY4741 and agp2⌬ cells. Cytochrome c is a cationic protein with an absorption maximum at 530 nm. The cells were incubated with 0.5 mg/ml cytochrome c for 20 min and pelleted. The levels of cytochrome c remaining in the supernatant were estimated spectrophotometrically at 530 nm. The supernatant from the BY4741 cells contained less cytochrome c than the supernatant from agp2⌬ cells, which had a similar amount of cytochrome c as the buffer control with no cells.

porters, including those for polyamine uptake (28). This formed the basis of our second model for the role of Agp2p in the activities of cationic antimicrobial peptides. Here, we hypothesized that different cationic AFPs bind to different plasma membrane proteins, all of which are regulated by Agp2p. This fits with the agp2⌬ phenotype, as in the absence of Agp2p, these genes are not expressed to such a high extent. That is, a decreased level of receptor protein on the cell surface would underlie the resistance to cationic AFPs observed in agp2⌬ cells. Along the same lines, the application of exogenous spermidine to wild-type BY4741 would cause Agp2p to signal that there was adequate polyamine availability and decrease the expression of the membrane binding partners. Sam3p and Dur3p are the plasma membrane proteins responsible for polyamine uptake in S. cerevisiae (29) and are known to be regulated by Agp2p (28) and polyamine concentration (29). As a link between polyamines and the activity of cationic AFPs had been established, we examined whether the resistant phenotype was observed in sam3⌬ and dur3⌬ single mutants, as these proteins are potential membrane binding partners. sam3⌬ and dur3⌬ single mutants exhibited the same sensitivity to NaD1 as the wild type, and the double mutants sam3⌬ agp2⌬ and dur3⌬ agp2⌬ had the same level of resistance as the agp2⌬ single mutant. As Sam3p and Dur3p have a redundant function in polyamine transport, we also looked at the sam3⌬ dur3⌬ double mutant and found the same NaD1 sensitivity as observed for the wild-type BY4741. This extended to the other cationic AFPs we tested. Sam3p and Dur3p were eliminated as potential membrane binding targets of NaD1 and other cationic AFPs. However, it is possible that one or more of the other membrane proteins with levels regulated by Agp2p on the fungal cell surface has a function in binding to cationic AFPs. An agp2⌬ strain was previously reported to have decreased levels of spermidine uptake (27) resulting from the decreased expression of polyamine transporters (28). Thus, we propose a third alternate model whereby spermidine competes with cationic AFPs for negatively charged binding sites on the cell surface. In the case of the agp2⌬ mutant, the lack of spermidine transport might lead to an accumulation of spermidine on the cell surface, effectively increasing the local concentration and occupying the binding sites as would occur with the application of exogenous spermidine. Polyamines have long been known to associate with negatively charged moieties in membranes, shield surface charges, and in some cases, provide stability to the membrane (41). This shielding of binding sites by spermidine fits with the observation that agp2⌬ cells are resistant to cationic AFPs, which bind to negatively charged sites on the cell surface but not hydrogen peroxide, which does not need to bind to charged surfaces to exert its cell-killing activity. The observation that most of the other AFPs tested are still active at higher concentrations suggests that there is competitive binding for the cell surface sites and that higher AFP concentrations outcompete spermidine for these binding sites. This model is also supported by the decreased binding of cytochrome c to agp2⌬ cells. The observation that decreased binding extends beyond AFPs to other cationic peptides indicates that the mechanism of resistance does not relate to a characteristic that is specific to AFPs but is due to an increase in positive charge on the cell surface that repels cationic proteins. Further investigation into the mechanism by which Agp2p and polyamines influence the activities of cationic AFPs is required to fully understand the biological phenomena involved. Perhaps, fungal cells have evolved to maintain a prophylactic shield made up of polyamines and

Agp2 and NaD1 Activity

11.

12.

13.

14.

15.

16.

17. 18. 19.

ACKNOWLEDGMENTS This work was supported by a discovery project from the Australian Research Council (ARC) (grant DP120102694 to M.A.A. and N.L.V.D.W.) and a La Trobe University Early Career Research grant (to M.R.B.).

20.

REFERENCES 1. Fisher MC, Henk DA, Briggs CJ, Brownstein JS, Madoff LC, McCraw SL, Gurr SJ. 2012. Emerging fungal threats to animal, plant and ecosystem health. Nature 484:186 –194. http://dx.doi.org/10.1038/nature10947. 2. Pennisi E. 2010. Armed and dangerous. Science 327:804 – 805. http://dx .doi.org/10.1126/science.327.5967.804. 3. Strange RN, Scott PR. 2005. Plant disease: a threat to global food security. Annu. Rev. Phytopathol. 43:83–116. http://dx.doi.org/10.1146/annurev .phyto.43.113004.133839. 4. van der Weerden NL, Bleackley MR, Anderson MA. 2013. Properties and mechanisms of action of naturally occurring antifungal peptides. Cell. Mol. Life Sci. http://dx.doi.org/10.1007/s00018-013-1260-1. 5. Jones JDG, Dangl JL. 2006. The plant immune system. Nature 444:323– 329. http://dx.doi.org/10.1038/nature05286. 6. Castro MS, Fontes W. 2005. Plant defense and antimicrobial peptides. Prot. Pept. Lett. 12:13–18. http://dx.doi.org/10.2174/0929866053405832. 7. Thomma BP, Cammue BP, Thevissen K. 2002. Plant defensins. Planta 216:193–202. http://dx.doi.org/10.1007/s00425-002-0902-6. 8. Lay FT, Anderson MA. 2005. Defensins– components of the innate immune system in plants. Curr. Protein Pept. Sci. 6:85–101. http://dx.doi .org/10.2174/1389203053027575. 9. van der Weerden NL, Anderson MA. 2013. Plant defensins: common fold, multiple functions. Fungal Biol. Rev. 26:121–131. http://dx.doi.org /10.1016/j.fbr.2012.08.004. 10. Aerts AM, François IE, Meert EM, Li QT, Cammue BP, Thevissen K. 2007. The antifungal activity of RsAFP2, a plant defensin from Raphanus sativus, involves the induction of reactive oxygen species in Candida albi-

May 2014 Volume 58 Number 5

21.

22.

23.

24.

cans. J. Mol. Microbiol. Biotechnol. 13:243–247. http://dx.doi.org/10 .1159/000104753. Aerts AM, Carmona-Gutierrez D, Lefevre S, Govaert G, François IE, Madeo F, Santos R, Cammue BP, Thevissen K. 2009. The antifungal plant defensin RsAFP2 from radish induces apoptosis in a metacaspase independent way in Candida albicans. FEBS Lett. 583:2513–2516. http: //dx.doi.org/10.1016/j.febslet.2009.07.004. Thevissen K, de Mello Tavares P, Xu D, Blankenship J, Vandenbosch D, Idkowiak-Baldys J, Govaert G, Bink A, Rozental S, de Groot PW, Davis TR, Kumamoto CA, Vargas G, Nimrichter L, Coenye T, Mitchell A, Roemer T, Hannun YA, Cammue BP. 2012. The plant defensin RsAFP2 induces cell wall stress, septin mislocalization and accumulation of ceramides in Candida albicans. Mol. Microbiol. http://dx.doi.org/10.1111/j .1365-2958.2012.08017.x. Aerts AM, Bammens L, Govaert G, Carmona-Gutierrez D, Madeo F, Cammue BPA, Thevissen K. 2011. The antifungal plant defensin HsAFP1 from Heuchera sanguinea induces apoptosis in Candida albicans. Front. Microbriol. 2:47. http://dx.doi.org/10.3389/fmicb.2011.00047. Thevissen K, François IE, Takemoto JY, Ferket KKA, Meert EMK, Cammue BP. 2003. DmAMP1, an antifungal plant defensin from dahlia (Dahlia merckii), interacts with sphingolipids from Saccharomyces cerevisiae. FEMS Microbiol. Lett. 226:169 –173. http://dx.doi.org/10.1016 /S0378-1097(03)00590-1. Spelbrink RG, Dilmac N, Allen A, Smith TJ, Shah DM, Hockerman GH. 2004. Differential antifungal and calcium channel-blocking activity among structurally related plant defensins. Plant Physiol. 135:2055–2067. http://dx.doi.org/10.1104/pp.104.040873. Lobo DS, Pereira IB, Fragel-Madeira L, Medeiros LN, Cabral LM, Faria J, Bellio M, Campos RC, Linden R, Kurtenbach E. 2007. Antifungal Pisum sativum defensin 1 interacts with Neurospora crassa cyclin F related to the cell cycle. Biochemistry 46:987–996. http://dx.doi .org/10.1021/bi061441j. Lay FT, Brugliera F, Anderson MA. 2003. Isolation and properties of floral defensins from ornamental tobacco and petunia. Plant Physiol. 131: 1283–1293. http://dx.doi.org/10.1104/pp.102.016626. van der Weerden NL, Lay FT, Anderson MA. 2008. The plant defensin, NaD1, enters the cytoplasm of Fusarium oxysporum hyphae. J. Biol. Chem. 283:14445–14452. http://dx.doi.org/10.1074/jbc.M709867200. Hayes BM, Bleackley MR, Wilshire JL, Anderson MA, Traven A, van der Weerden NL. 2013. Identification and mechanism of action of the plant defensin NaD1 as a new member of the antifungal drug arsenal against Candida albicans. Antimicrob. Agents Chemother. 57:3667–3675. http://dx.doi.org/10.1128/AAC.00365-13. Gaspar YM, McKenna JA, McGinness BS, Hinch J, Poon S, Connelly AA, Anderson MA, Heath RL. 6 February 2014. Field resistance to Fusarium oxysporum and Verticillium dahliae in transgenic cotton expressing the plant defensin NaD1. J. Exp. Bot. http://dx.doi.org/10.1093/jxb /eru021. van der Weerden NL, Hancock RE, Anderson MA. 2010. Permeabilization of fungal hyphae by the plant defensin NaD1 occurs through a cell wall-dependent process. J. Biol. Chem. 285:37513–37520. http://dx.doi .org/10.1074/jbc.M110.134882. Lay FT, Mills GD, Poon IK, Cowieson NP, Kirby N, Baxter AA, van der Weerden NL, Dogovski C, Perugini MA, Anderson MA, Kvansakul M, Hulett MD. 2012. Dimerization of plant defensin NaD1 enhances its antifungal activity. J. Biol. Chem. 287:19961–19972. http://dx.doi.org/10 .1074/jbc.M111.331009. Winzeler EA, Shoemaker DD, Astromoff A, Liang H, Anderson K, Andre B, Bangham R, Benito R, Boeke JD, Bussey H, Chu AM, Connelly C, Davis K, Dietrich F, Dow SW, El Bakkoury M, Foury F, Friend SH, Gentalen E, Giaever G, Hegemann JH, Jones T, Laub M, Liao H, Liebundguth N, Lockhart DJ, Lucau-Danila A, Lussier M, M’Rabet N, Menard P, Mittmann M, Pai C, Rebischung C, Revuelta JL, Riles L, Roberts CJ, Ross-MacDonald P, Scherens B, Snyder M, Sookhai-Mahadeo S, Storms RK, Véronneau S, Voet M, Volckaert G, Ward TR, Wysocki R, Yen GS, Yu K, Zimmermann K, Philippsen P, et al. 1999. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285:901–906. http://dx.doi.org/10 .1126/science.285.5429.901. Giaever G, Chu AM, Ni L, Connelly C, Riles L, Véronneau S, Dow S, Lucau-Danila A, Anderson K, André B, Arkin AP, Astromoff A, ElBakkoury M, Bangham R, Benito R, Brachat S, Campanaro S, Curtiss M, Davis K, Deutschbauer A, Entian KD, Flaherty P, Foury F, Garfinkel DJ,

aac.asm.org 2697

Downloaded from http://aac.asm.org/ on April 10, 2014 by guest

other positively charged molecules to protect against the effects of cationic antifungal peptides, and the lack of expression of genes controlled by Agp2p leads to increased thickness and/or stability of this layer. Current methods to control fungal pathogens have encountered hurdles associated with emerging resistance in fungi as well as undesirable off-target toxicity. The identification of novel agents for the control of pathogenic fungi is necessary to minimize the catastrophic effect these microorganisms might have on the world. One particularly attractive class of molecules for the control of fungal pathogens is plant defensins. The results from this study are encouraging for the development of antifungal proteins, such as NaD1, as molecules for combating fungal pathogens in agriculture and the clinic. Although we did identify a set of resistant strains, the level of resistance was not insurmountable, as full inhibition of all strains analyzed was achieved by increasing the concentration of protein ⬍3-fold. In addition, polyamine uptake and metabolism are essential for the viability of yeast and other fungi. Strains in nature that may develop mutations that mimic those investigated here would be considerably less fit, as evidenced by the decreased competitive fitness observed in an agp2⌬ strain (42). The likelihood of mutations causing resistance via the mechanisms discussed here arising in a natural population is low. Using the model yeast S. cerevisiae, a link between polyamine uptake and the antifungal mechanism of the plant defensin NaD1 and other cationic antifungal peptides has been established. We anticipate that this also operates in other fungal pathogens, and further investigation is required to confirm this prediction. The elucidation of the interplay between polyamines and antifungal peptides will provide information that is be crucial for the design of novel antifungal molecules.

Bleackley et al.

25.

26. 27.

28.

30. 31.

32.

2698

aac.asm.org

33. Schröder JM, Harder J. 1999. Human beta-defensin-2. Int. J. Biochem. Cell Biol. 31:645– 651. 34. Takahashi T, Kakehi JI. 2010. Polyamines: ubiquitous polycations with unique roles in growth and stress responses. Ann. Bot. 105:1– 6. http://dx .doi.org/10.1093/aob/mcp259. 35. Gardiner DM, Kazan K, Praud S, Torney FJ, Rusu A, Manners JM. 2010. Early activation of wheat polyamine biosynthesis during Fusarium head blight implicates putrescine as an inducer of trichothecene mycotoxin production. BMC Plant Biol. 10:289. http://dx.doi.org/10.1186 /1471-2229-10-289. 36. Childs AC, Mehta DJ, Gerner EW. 2003. Polyamine-dependent gene expression. Cell. Mol. Life Sci. 60:1394 –1406. http://dx.doi.org/10.1007 /s00018-003-2332-4. 37. Kwon DH, Lu CD. 2006. Polyamines induce resistance to cationic peptide, aminoglycoside, and quinolone antibiotics in Pseudomonas aeruginosa PAO1. Antimicrob. Agents Chemother. 50:1615–1622. http://dx.doi .org/10.1128/AAC.50.5.1615-1622.2006. 38. Kwon DH, Lu CD. 2007. Polyamine effects on antibiotic susceptibility in bacteria. Antimicrob. Agents Chemother. 51:2070 –2077. http://dx.doi .org/10.1128/AAC.01472-06. 39. Kumar R, Chadha S, Saraswat D, Bajwa JS, Li RA, Conti HR, Edgerton M. 2011. Histatin 5 uptake by Candida albicans utilizes polyamine transporters Dur3 and Dur31 proteins. J. Biol. Chem. 286:43748 – 43758. http: //dx.doi.org/10.1074/jbc.M111.311175. 40. Tati S, Jang WS, Li R, Kumar R, Puri S, Edgerton M. 2013. Histatin 5 resistance of Candida glabrata can be reversed by insertion of Candida albicans polyamine transporter-encoding genes DUR3 and DUR31. PLoS One 8:61480. http://dx.doi.org/10.1371/journal.pone.0061480. 41. Schuber F. 1989. Influence of polyamines on membrane functions. Biochem. J. 260:1–10. 42. Breslow DK, Cameron DM, Collins SR, Schuldiner M, StewartOrnstein J, Newman HW, Braun S, Madhani HD, Krogan NJ, Weissman JS. 2008. A comprehensive strategy enabling high-resolution functional analysis of the yeast genome. Nat. Methods 5:711–718. http://dx.doi .org/10.1038/nmeth.1234.

Antimicrobial Agents and Chemotherapy

Downloaded from http://aac.asm.org/ on April 10, 2014 by guest

29.

Gerstein M, Gotte D, Güldener U, Hegemann JH, Hempel S, Herman Z, Jaramillo DF, Kelly DE, Kelly SL, Kötter P, LaBonte D, Lamb DC, Lan N, Liang H, Liao H, Liu L, Luo C, Lussier M, Mao R, Menard P, Ooi SL, Revuelta JL, Roberts CJ, Rose M, Ross-Macdonald P, Scherens B, et al. 2002. Functional profiling of the Saccharomyces cerevisiae genome. Nature 418:387–391. http://dx.doi.org/10.1038/nature00935. Peschel A, Otto M, Jack RW, Kalbacher H, Jung G, Götz F. 1999. Inactivation of the dlt operon in Staphylococcus aureus confers sensitivity to defensins, protegrins, and other antimicrobial peptides. J. Biol. Chem. 274:8405– 8410. http://dx.doi.org/10.1074/jbc.274.13.8405. Robinson MD, Grigull J, Mohammad N, Hughes TR. 2002. FunSpec: a web-based cluster interpreter for yeast. BMC Bioinformatics 3:35. http: //dx.doi.org/10.1186/1471-2105-3-35. Aouida M, Leduc A, Poulin R, Ramotar D. 2005. AGP2 encodes the major permease for high affinity polyamine import in Saccharomyces cerevisiae. J. Biol. Chem. 280:24267–24276. http://dx.doi.org/10.1074/jbc .M503071200. Aouida M, Texeira MR, Thevelein JM, Poulin R, Ramotar D. 2013. Agp2, a member of the yeast amino acid permease family, positively regulates polyamine transport at the transcriptional level. PLoS One 8:e65717. http://dx.doi.org/10.1371/journal.pone.0065717. Uemura T, Kashiwagi K, Igarashi K. 2007. Polyamine uptake by DUR3 and SAM3 in Saccharomyces cerevisiae. J. Biol. Chem. 282:7733–7741. http://dx.doi.org/10.1074/jbc.M611105200. Friedrich C, Scott MG, Karunaratne N, Yan H, Hancock RE. 1999. Salt-resistant alpha-helical cationic antimicrobial peptides. Antimicrob. Agents Chemother. 43:1542–1548. Skerlavaj B, Gennaro R, Bagella L, Merluzzi L, Risso A, Zanetti M. 1996. Biological characterization of two novel cathelicidin-derived peptides and identification of structural requirements for their antimicrobial and cell lytic activities. J. Biol. Chem. 271:28375–28381. http://dx.doi.org/10.1074 /jbc.271.45.28375. Wu M, Hancock REW. 1999. Improved derivatives of bactenecin, a cyclic dodecameric antimicrobial cationic peptide. Antimicrob. Agents Chemother. 43:1274 –1276.