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Antimicrobial Agents and Chemotherapy, November 2007, p. 3948-3959, Vol. 51, No. 11
0066-4804/07/$08.00+0     doi:10.1128/AAC.01007-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Global Phenotype Screening and Transcript Analysis Outlines the Inhibitory Mode(s) of Action of Two Amphibian-Derived, {alpha}-Helical, Cationic Peptides on Saccharomyces cerevisiae{triangledown} ,{dagger}

C. Oliver Morton,1 Andrew Hayes,2 Michael Wilson,2,{ddagger} Bharat M. Rash,2 Stephen G. Oliver,2 and Peter Coote1*

Centre for Biomolecular Sciences, School of Biology, University of St. Andrews, The North Haugh, St. Andrews KY16 9ST, United Kingdom,1 Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, United Kingdom2

Received 1 August 2007/ Returned for modification 27 August 2007/ Accepted 29 August 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dermaseptin S3(1-16) [DsS3(1-16)] and magainin 2 (Mag 2) are two unrelated, amphibian-derived cationic peptides that adopt an {alpha}-helical structure within microbial membranes and have been proposed to kill target organisms via membrane disruption. Using a combination of global deletion mutant library phenotypic screening, expression profiling, and physical techniques, we have carried out a comprehensive in vitro analysis of the inhibitory action of these two peptides on the model fungus Saccharomyces cerevisiae. Gene ontology profiling (of biological processes) was used to identify both common and unique effects of each peptide. Resistance to both peptides was conferred by genes involved in telomere maintenance, chromosome organization, and double-strand break repair, implicating a common inhibitory action of DNA damage. Crucially, each peptide also required unique genes for maintaining resistance; for example, DsS3(1-16) required genes involved in protein targeting to the vacuole, and Mag 2 required genes involved in DNA-dependent DNA replication and DNA repair. Thus, DsS3(1-16) and Mag 2 have both common and unique antifungal actions that are not simply due to membrane disruption. Physical techniques revealed that both peptides interacted with DNA in vitro but in subtly different ways, and this observation was supported by the functional genomics experiments that provided evidence that both peptides also interfered with DNA integrity differently in vivo. This implies that both peptides are able to pass through the cytoplasmic membrane of yeast cells and damage DNA, an inhibitory action that has not been previously attributed to either of these peptides.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Antimicrobial peptides have now been discovered from the microbial, animal, and plant kingdoms and are classified on the basis of their secondary structures (reviewed in reference 12). There is a perception that antimicrobial peptides kill microorganisms nonspecifically by disrupting the plasma membrane of target cells with a detergent-like action. However, increasing evidence indicates that the inhibitory actions of many peptides are complex and involve targets interior to the cytoplasmic membrane (51). Evidence from a range of studies has shown that an important target for cationic peptides could be interference with the synthesis or function of critical intracellular macromolecules, such as DNA and proteins.

Magainin 2 (Mag 2) from Xenopus laevis (53) and dermaseptin S3(1-16) [DsS3(1-16)] (31), a truncated derivative of DsS3 from Phyllomedusa sauvagii (32) with full activity, are unrelated, cationic, {alpha}-helical antimicrobial peptides that have been extensively investigated. Studies of Mag 2 have clearly shown that the peptide binds to negatively charged bacterial membranes and induces the formation of a toroid pore that permeabilizes the membrane (29, 51). Similarly, studies on DsS3 have shown that this peptide also binds to, and disrupts, membranes (15). Therefore, it is not surprising that the principal mechanism of cell death induced by these cationic peptides has been attributed to membrane disruption. However, membrane disruption alone may not be sufficient to kill microorganisms (reviewed in reference 51). Thus, in this work, we used functional genomic techniques to compare in detail the inhibitory actions of Mag 2 and DsS3(1-16).

Genome-wide expression profiling has been employed extensively to determine the inhibitory effects of antimicrobials. The rationale for this approach is that the mRNA profiles generated in response to a particular antimicrobial represent the transcriptional response of the cell to the inhibition of particular vital cellular functions, in effect, a signature of the type of stress induced. Research examining the response to antifungals includes studies of the response of Candida albicans upon exposure to itraconazole (11), amphotericin B and nystatin (54), and, collectively, ketoconazole, amphotericin B, caspofungin, and flucytosine (25). More-sophisticated approaches have included the construction of a reference database or compendium of gene expression profiles occurring in Saccharomyces cerevisiae as a consequence of either specific mutations or a range of chemical treatments (19). In addition, Bammert and Fostel (6) used expression profiling to identify patterns of gene expression convergent between drug-treated cells and cells with genetic alterations in the same pathway targeted by the drug and were able to assign a mode of action to a novel azole antifungal.

Although this approach has been successful, problems with this approach include the assumption that a single interaction between a drug and a target accounts for the inhibitory action of the drug and that drug treatment also changes the expression of many genes that are not directly related to the drug target and are thus functionally irrelevant. Thus, for more-precise mechanism-of-action studies, it would be beneficial to combine and complement expression profiling with functional screening at the level of the entire genome. In this work, we combined a genome-wide phenotypic screen, using the S. cerevisiae deletion mutant library, with expression profiling to characterize the inhibitory action of two unrelated cationic, {alpha}-helical peptides whose principal inhibitory action is believed to be disruption of microbial cell membranes. The data imply that the inhibitory actions of these two peptides are very different and far more complex than simple disruption of the cellular membrane.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antimicrobial peptides. DsS3(1-16)-NH2 was synthesized according to the published sequence ALWKNMLKGIGKLAGK (31) and Mag 2 according to the sequence GIGKFLHSAKKFGKAFVGEIMNS (53) by Peptide Protein Research Ltd., Wickham, United Kingdom, to >95% purity and verified by high-performance liquid chromatography and mass spectrometry. Experiments were carried out using a stock solution of 50 mg ml–1 in water.

Yeast strains and media. The yeast strain used in this study was S. cerevisiae BY4741 (MATa his-{Delta}31 leu-{Delta}0 met-{Delta}150 ura-{Delta}30) from Research Genetics (Huntsville, AL). Gene deletion mutants were from the Research Genetics BY4741 MATa haploid genome deletion mutant set. Malt extract broth (MEB; 0.6% malt extract, 0.6% dextrose, 0.18% maltose, 0.12% yeast extract), pH 7.0 (Difco), was used for all growth assays.

Screening of the yeast deletion mutant library. The S. cerevisiae genome gene deletion mutant set was screened for sensitivity and resistance to DsS3(1-16) and Mag 2. By use of a 48-pin replica plater, individual yeast mutant strains were inoculated from the strain collection stored in 96-well plates into sterile 96-well plates containing 200 µl MEB, pH 7.0, with G418 sulfate (1.5 µg ml–1). The cultures were incubated overnight at 30°C until they had grown to an optical density at 600 nm (OD600) of 0.3, and these were transferred into 96-well plates containing MEB, pH 7.0, with 6 µg ml–1 DsS3(1-16) or 15 µg ml–1 Mag 2 by using the replica plater as described above. The cultures were agitated to resuspend the cells, and the OD600 was measured by using a Powerwave XS universal microplate spectrophotometer (BIO-TEK Instruments Inc., Winooski, VT). Deletion mutants showing an OD600 significantly higher or lower than that of the isogenic parent were selected for detailed screening by monitoring growth turbidometrically with the Powerwave XS spectrophotometer in the presence or absence of antimicrobial peptide. Growth was measured in 48-well microtiter plates (Greiner Bio-One) with wells containing 300 µl MEB, pH 7.0, with or without 6 µg ml–1 DsS3(1-16) or 15 µg ml–1 Mag 2; S. cerevisiae culture was added to each sample well to give a final starting inoculum of 5 x 103 cells ml–1. Growth was measured by determining the change in OD600 every 15 min over 24 h at 30°C with continuous shaking. The data generated were imported into Excel software (Microsoft, Seattle, WA), and growth curves were generated.

RNA preparation. Yeast cultures were grown in 150 ml MEB, pH 7.0, in 250-ml flasks overnight at 30°C to an OD600 of 0.25. The cells were treated with either 7 µg ml–1 DsS3(1-16) or 15 µg ml–1 Mag 2 for 20 and 40 min. Cells were harvested and resuspended in 200 µl sterile water, and the suspension was dropped directly into liquid nitrogen to form cell pellets. The cell pellets were ground with a mortar and pestle under liquid nitrogen to form a fine powder which was taken up in 2 ml TRIzol (Invitrogen). The cell paste was subjected to a vortex for 1 min, incubated for 5 min at room temperature, and then centrifuged at 12,000 x g for 10 min. The clear supernatant was mixed with 0.4 volumes of chloroform, shaken for 15 s, and incubated for 10 min at room temperature. The mixture was centrifuged at 12,000 x g for 10 min, and the supernatant was combined with 0.5 volumes of isopropanol to precipitate the RNA. This was incubated for 10 min at room temperature and centrifuged at 12,000 x g for 10 min to pellet the RNA. The isopropanol was taken off, and the pellet was washed with 70% ethanol. The pellet was air dried and resuspended in 500 µl diethyl pyrocarbonate-treated water.

Microarray hybridization and analysis. Microarray hybridization was performed using the Affymetrix GeneChip Yeast Genome S98 array as described previously (49). Microarray scanner output (in the form of .cel files) was normalized by the robust multiarray average (RMA) method (22), using default parameters in R/Bioconductor software (www.bioconductor.org). Results were expressed in log2 form. The results were trimmed to remove genes where the values in the paired control chips differed from each other by more than 25%.

Ontology profiling of gene expression changes occurring in response to peptide treatments was performed using the Gene Ontology (GO) Term Finder tool (http://db.yeastgenome.org/cgi-bin/GO/goTermFinder) in the Saccharomyces Genome Database (http://www.yeastgenome.org/). Only the top 10 most significant GO terms are shown (P < 0.05).

Peptide-DNA interactions. The plasmid DNA pRS313 was extracted from an Escherichia coli culture by using a QIAprep Spin Miniprep kit from QIAGEN. DNA was resuspended in 10 mM sodium phosphate buffer, pH 7.0, and concentrations were determined by measuring the OD260 in a Varian spectrophotometer. All gels were visualized using a UV Transilluminator from UVP and photographed with the BioDoc-It imaging system.

Gel retardation experiments. The DNA binding assay was performed as described in the study of Park et al. (40) except 100 ng of pRS313 (previously digested overnight with BamHI) was used.

DNA strand break assay. The DNA strand break assay was performed by measurement of relaxation of supercoiled plasmid DNA (pRS313) to an open circular form (41). The reaction mixtures were prepared as described for the retardation experiments except that the plasmid DNA was not digested with BamHI.

DNase I protection assay. The DNase I protection assay was performed according to the methods of Niidome et al. (37). A total of 500 ng of pRS313 (previously digested overnight with BamHI) was mixed with increasing concentrations of Mag 2 in 20 µl of binding buffer.

Measurement of DNA damage by quantitative PCR (qPCR) assay. The assay was exactly as described by Morton et al. (34). We arbitrarily selected the S. cerevisiae gene IZH3. A 10-kb genomic sequence upstream and downstream of IZH3 was obtained from the Saccharomyces Genome Database, and forward and reverse primers were designed using software made available by Integrated DNA Technologies and synthesized by MWG Biotech, Germany.

CD spectroscopy. Circular dichroism (CD) spectroscopy was conducted on combinations of the plasmid pRS313 (50 µg ml–1) and antimicrobial peptide DsS3(1-16) or Mag 2 (250 µg ml–1). CD spectra were measured in the far-UV (260- to 180-nm) range using a Jasco J-810 spectropolarimeter. pRS313 and the peptides were dissolved in 10 mM sodium phosphate buffer, pH 7.0. For each experiment, a buffer-blank spectrum was subtracted from the spectrum of the peptide plus the buffer, and a plasmid-blank spectrum was subtracted from the spectrum from the peptide-and-plasmid experiments. Data were also analyzed using the CDSSTR algorithm (http://public-1.cryst.bbk.ac.uk/cdweb/html/) (47) to show the secondary structural motifs for each peptide.

Microarray data accession number. In compliance with MIAME guidelines (http://www.mged.org/Workgroups/MIAME/miame.html), the data from this study have been submitted to the ArrayExpress repository at the European Bioinformatics Institute website (http://www.ebi.ac.uk/arrayexpress/) under accession no. E-MEXP-1060.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Phenotype screening identified many gene deletions conferring resistance and sensitivity to DsS3(1-16) and Mag 2. To gain a detailed understanding of the antifungal modes of action of DsS3(1-16) and Mag 2, we carried out a phenotypic screening of mutants with the 4,847 nonessential gene deletions in S. cerevisiae BY4741 MATa.

Deletion mutants displaying sensitivity. Of the 4,847 gene deletion mutants tested, 32 (0.7%) showed hypersensitivity to both DsS3(1-16) and Mag 2, 82 (1.7%) were sensitive to DsS3(1-16) alone, and 21 (0.4%) were sensitive to Mag 2 alone (Table 1). We used the Saccharomyces Genome Database GO Term Finder to identify significantly (P < 0.05) associated gene groups in terms of biological process ontology. Hence, we conducted searches on the 32 gene deletion mutants showing common sensitivity to both peptides, thus representing common mediators of resistance to {alpha}-helical, cationic antifungal peptides. The most significant hits were represented by the terms "telomere organization and maintenance" (9 out of 32 genes [29%] [P = 1.38E–6]; VPS54, NUP60, RTF1, ARV1, RSC2, SRB5, CTK1, SNF7, and LDB7), "chromosome organization and biogenesis" (11 out of 32 genes [35.4%] [P = 2.52E–6]; SGF29, VPS54, NUP60, RTF1, ARV1, RSC1, RSC2, SRB5, CTK1, SNF7, and LDB7), "lipid transport" (3 out of 32 genes [9.6%] [P = 0.00037]; OSH3, CDC50, and ARV1), and "double-strand break repair" (3 out of 32 genes [9.6%] [P = 0.00125]; MMS22, RSC1, and RSC2). The observation that a number of deletions in genes involved in maintenance of chromosome integrity and DNA repair induce sensitivity to both peptides implies that a common inhibitory action of these molecules could be due to the induction of DNA damage. Examples included the deletions of MMS22, which acts in a double-strand break repair pathway that may be involved in resolving replication intermediates or preventing the damage caused by blocked replication forks (7); RSC1, RSC2, and LDB7, which are constituents of the RSC (remodel the structure of chromatin) complex involved in chromatin structure remodeling and double-strand break repair via nonhomologous end joining (43, 48); and IMP2', a transcriptional activator believed to be involved in regulating ion homeostasis and DNA repair in response to DNA-damaging agents (28).


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  		TABLE 1. Screening of the yeast disruptome revealed genes whose deletions confer sensitivity to the antimicrobial peptides DsS3(1-16) and Mag 2

 
Next, we studied the gene deletions that conferred sensitivity exclusively to either DsS3(1-16) or Mag 2 alone, thus representing indicators of unique, peptide-specific effects on the cells. Thus, ontology profiling (biological processes) of the gene deletions conferring sensitivity to DsS3(1-16) alone revealed significant hits for "vacuolar transport" (9 out of 82 genes [10.9%] [P = 1.14E–5]; VPS8, VPS61, VAC8, VAM3, VPS69, VMA6, VMA5, CUP5, and GGA1), "protein targeting to the vacuole" (5 out of 82 genes [6.0%] [P = 0.00099]; VPS61,VAC8, VPS69, CUP5, and GGA1), and "vacuolar acidification" (3 out 82 genes [3.6%] [P = 0.0026]; VMA6, VMA5, and CUP5). This suggests that optimal function of the vacuolar H+-ATPase is required specifically for resistance to the inhibitory effects of DsS3(1-16); perhaps to enable sequestration, and ultimately destruction of the inhibitory peptide in the vacuole. However, other gene deletions that conferred specific sensitivity to DsS3(1-16) included the deletions of MRE11, encoding a protein vital for double-strand break repair and telomere maintenance (24); DPB3, encoding the third-largest subunit of DNA polymerase II, capable of mismatch and nucleotide-excision repair, and required to maintain the fidelity of chromosomal replication (21); and CTF4, encoding a chromatin-associated protein, required for sister chromatid cohesion (16).

In contrast to profiling of the gene deletions conferring sensitivity to DsS3(1-16), ontology profiling (biological processes) of the gene deletions conferring specific sensitivity to Mag 2 revealed top hits for "second-messenger-mediated signaling" (3 out of 21 genes [14.2%] [P = 8.77E–05]; RCN1, SAC1, and SOK1), "endosome transport" (2 out of 21 genes [9.5%] [P = 0.00648]; VPS53 and ENT5), and "cell wall organization and biogenesis" (3 out of 21 genes [14.2%] [P = 0.00994]; ROM1, SPR3, and CWH41).

Notably, mutants with deletions of genes known to be involved in fungal drug resistance were not identified in large numbers, indicating that the inhibitory mode of action of these peptides is different to well-known and characterized antifungal agents. Exceptions that induced sensitivity to DsS3(1-16) alone included deletions of RDS1, a transcriptional activator known to confer resistance to cycloheximide (1), and BFR1, a multicopy suppressor of brefeldin A sensitivity. The deletion of a gene already known to be involved in drug resistance mechanisms, RCY1, conferred sensitivity to both peptides. RCY1 encodes an F-box protein involved in recycling plasma membrane proteins internalized by endocytosis and confers resistance to the antifungals brefeldin A and monensin (36).

Intriguingly, the deletion of two genes, KTI12 and FYV12, known to mediate resistance to yeast killer toxins, conferred sensitivity on DsS3(1-16) alone. KTI12 encodes a protein that associates with a histone acetyltransferase complex that functions during transcription and was first identified in a screening for mutants insensitive to the Kluyveromyces lactis toxin zymocin, which causes cell cycle arrest in G1 phase (5), and FYV12, a protein of unknown function, is required for survival upon exposure to the K1 killer toxin (39).

Deletion mutants displaying resistance. Study of gene deletions that conferred resistance revealed that no deletions conferred resistance to both DsS3(1-16) and Mag 2, but 33 mutants (0.5%) were resistant to DsS3(1-16) alone and only 4 (0.08%) were resistant to Mag 2 alone (Table 2). Notably, the deletion of a number of genes known to be involved in the induction of programmed cell death in yeast (including IZH2, IZH3, STM1, and AIF1) led to resistance to DsS3(1-16). Subsequently, we recently reported that DsS3(1-16) induced caspase-independent but AIF-dependent apoptosis in yeast (34), and thus, we do not discuss the role of these genes further here.


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  		TABLE 2. Screening of the yeast disruptome revealed genes whose deletions confer increased resistance to the antimicrobial peptides DsS3(1-16) and Mag 2a

 
Notably, deletion of different genes involved in telomere maintenance resulted in resistance to DsS3(1-16) and Mag 2. Resistance to DsS3(1-16) was induced by deletion of HST3, which encodes a member of the Sir2p family of NAD+-dependent protein deacetylases that are involved in telomeric silencing, radiation resistance, genomic stability, and short-chain fatty acid metabolism (8), and NNT1, a putative nicotinamide N-methyltransferase gene that plays a role in ribosomal DNA silencing and in life span determination, deletion of which results in decreased telomeric silencing (3). Deletion of NMD2 induced resistance to Mag 2. NMD2 is also involved in telomere maintenance, and its deletion confers increased resistance to the anticancer drug cisplatin (4). Also, deletion of three genes involved in chromosome/chromatid segregation and cohesion, GLC8, SRC1, and SPO13, induced resistance to DsS3(1-16).

Related to a common theme of possible peptide interference with DNA, deletion of TOP1 induced specific resistance to Mag 2. TOP1 encodes the type IB topoisomerase that cleaves one DNA strand and relaxes both positively and negatively supercoiled DNA. Top1p is the target of the antitumor drug camptothecin that induces double-strand breaks during DNA replication (5).

Our findings from the genome deletion mutant screen analysis suggest, but do not explicitly define, the relative importance of each deletion phenotype observed. Neither necessity nor sufficiency of any candidate gene has yet been validated by complementation; thus, the potential cause-versus-effect relationships hypothesized have not yet been confirmed. This will form the basis of future more-detailed study of the precise role key genes play in mediating resistance to the individual peptides.

In summary, the deletion mutant screen has indicated that DsS3(1-16) and Mag 2 have common inhibitory effects that may involve interference with DNA, chromosome integrity, and telomere maintenance. However, despite both peptides being cationic, with similar {alpha}-helical structures, they also induce very different resistance/sensitivity patterns in the global gene deletion mutant screen. Thus, it is also clear that each peptide has unique, independent antifungal effects.

Analysis of up-regulated genes reveals both common and distinct gene expression profiles upon exposure to DsS3(1-16) or Mag 2. To obtain gene expression profiles occurring as a consequence of sublethal exposure to DsS3(1-16) and Mag 2, concentrations of each peptide, 7 and 15 µg ml–1, respectively, that partially inhibited growth in shake-flask cultures and had only a minor detrimental effect on cell viability were selected (data not shown).

Up-regulated genes common to both peptides. A total of 164 common genes were found to be up-regulated (>1.5-fold increase in expression) upon treatment with DsS3(1-16) (7 µg ml–1) or Mag 2 (15 µg ml–1) at both exposure times tested, 20 and 40 min postaddition (see Table SA in the supplemental material). As described above, these gene expression changes represent a response to common effects induced by both peptides. GO profiling revealed that the most significant gene clusters identified belonged to the biological process terms "cellular catabolism," "ubiquitin-dependent protein catabolism," "modification-dependent protein catabolism," and "proteolysis during cellular protein catabolism" (Table 3). Thus, exposure to both peptides induces pathways resulting in ubiquitin-dependent protein breakdown and proteolysis. This response could be to remove excess inhibitory peptide directly or could be occurring as a consequence of the stress response induced by the peptides affecting endogenous cellular functions.


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  		TABLE 3. GO analysis of biological process genes >1.5-fold upregulated by treatment with DsS3(1-16) and Mag 2a

 
Comparison of genes up-regulated by both peptides with the gene deletion mutant sensitivity data revealed only one gene, HAL5, whose deletion conferred sensitivity to both peptides and whose expression was up-regulated by both peptides at both time points tested [for DsS3(1-16), 5.41-fold at 20 min and 6.41-fold at 40 min; for Mag 2, 5.32-fold at 20 min and 4.8-fold at 40 min). HAL5 encodes a protein kinase that activates the Trk1-Trk2p potassium transporters, increasing the influx of potassium and decreasing the membrane potential. The resulting loss in electrical driving force reduces the uptake of toxic cations and increases salt tolerance. Indeed, overexpression of HAL5 enhances the tolerance of yeast cells to sodium and lithium, whereas gene disruption results in greater cation sensitivity (35). Notably, because HAL5 regulates the membrane potential, it is known to be a pleiotropic determinant of sensitivity to other chemotherapeutic agents (45). Thus, it could be that the membrane potential is a significant mediator of the efficacies of both peptides. This conclusion is further supported by the observation that other genes involved in cation homeostasis, for example, HAL9, CUP2, PTK2, ENA2, BSD2, ENA5, MNR2, and PPZ1, are up-regulated by both peptides (see Table SA in the supplemental material).

A number of genes were notable for their strong up-regulation by both peptides (defined as an average increase in expression greater than sevenfold over both time points tested, 20 and 40 min), for example, SPG4 (encoding a protein with unknown function but required for survival at high temperature during stationary phase) (27), YER185W (encoding a transmembrane protein with unknown function), FMP48 (encoding a protein kinase with unknown function and localized to the mitochondria), PUT1 (encoding proline oxidase, a nuclear-encoded mitochondrial protein involved in utilization of proline as the sole nitrogen source) (18), YDR034W-B (encoding a protein of unknown function whose green fluorescent protein-fusion localizes to the cell periphery) (20).

Genes specifically up-regulated by DsS3(1-16). A total of 70 genes were found to be uniquely up-regulated (>1.5-fold increase in expression) upon treatment with DsS3(1-16) (7 µg ml–1) alone at both exposure times tested, 20 and 40 min postaddition (see Table SB in the supplemental material). These gene expression changes represent a response to specific effects induced by DsS3(1-16). GO profiling revealed that the most significant gene clusters identified belonged to the biological process terms "neutral amino acid transport," "response to stimulus," "response to reactive oxygen species," "actin cytoskeleton organization and biogenesis," and "response to drug" (Table 4). The "response to stimulus" cluster includes genes induced by general stress as well as genes induced by oxidative stress but also includes genes such as PHR1, encoding a photolyase that repairs pyrimidine dimers in DNA in the presence of visible light; HEX3, involved in genome stability and induced by DNA damage; and SIN3, involved in chromosome integrity. The drug treatment cluster includes three genes encoding ATP-binding cassette (ABC) transporter proteins involved in multidrug resistance; SNQ2, which also confers resistance to singlet oxygen species (46); PDR5, which actively exports various drugs across the membrane and is also involved in steroid transport, cation resistance, and cellular detoxification during exponential growth (26); and finally, PDR10, encoding a protein similar to that encoded by PDR5, also involved in the pleiotropic drug resistance network.


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  		TABLE 4. GO analysis of biological process genes upregulated >1.5-fold by DsS3(1-16) treatmenta

 
Comparison of genes up-regulated by exposure (at both time points tested) to DsS3(1-16) alone with genes whose deletion has conferred specific sensitivity to this peptide revealed three linked genes. MSC1 encodes a protein of unknown function, but deletion of the gene results in a defect in directing meiotic recombination events to homologous chromatids (44). The other two genes, YGR122W and YPR115W, encode proteins of unknown function.

Notably, the genes showing the highest levels of induction specifically by DsS3(1-16) at both time points, GPA2 (4.5- and 3.2-fold induction at 20 min and 40 min, respectively), encoding the nucleotide-binding alpha subunit of a heterotrimeric G protein that interacts with the receptor Gpr1p and plays a role in signaling changes in nutrient levels (13), and YAK1 (4.8- and 3.7-fold induction at 20 min and 40 min, respectively), encoding a protein kinase that is part of a glucose-sensing system involved in growth control in response to glucose availability (33), are both involved in signaling nutrient availability.

Genes specifically up-regulated by Mag 2. A total of 315 genes were found to be uniquely up-regulated (>1.5-fold increase in expression) upon treatment with Mag 2 (15 µg ml–1) alone at both exposure times tested, 20 and 40 min postaddition (see Table SC in the supplemental material). These gene expression changes represent a response to specific effects induced by Mag 2. GO profiling revealed that the most significant gene clusters belonged to the biological process terms "DNA replication," "DNA-dependent DNA replication," "DNA strand elongation," "DNA repair," and "maintenance of fidelity during DNA-dependent DNA replication" (Table 5). Clearly, these related GO terms imply that Mag 2 has a specific effect on the synthesis of new strands of DNA, using parental DNA as a template for the DNA-dependent DNA polymerases that synthesize the new strands. The GO term "maintenance of fidelity during DNA-dependent DNA replication" indicates that Mag 2 may be directly interfering with replication, causing DNA damage that has to be repaired by the genes present in the categories for the other significant GO terms "mismatch repair," "nucleotide-excision repair," and "double-strand break repair." Thus, these results imply that Mag 2 may have a more specific and potent DNA-damaging action than DsS3(1-16).


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  		TABLE 5. GO analysis of biological process genes upregulated >1.5-fold by Mag 2 treatmenta

 
Analysis of genes up-regulated by exposure (at both time points tested) to Mag 2 alone and gene deletions conferring specific sensitivity to this peptide revealed two genes: MRM2, encoding a mitochondrial 21S rRNA methyltransferase involved in telomere maintenance and required for methylation of 21S rRNA (42), and ROM1, encoding a GDP/GTP exchange protein that negatively regulates Rho1p, a protein that itself regulates protein kinase C and the cell wall-synthesizing enzyme 1,3-beta-glucan synthase (38).

Notable genes showing the highest level of induction by Mag 2 alone included MSH2 (3.1- and 2.0-fold induction at 20 min and 40 min, respectively), a mitochondrial DNA-binding protein involved in mismatch repair of mitochondrial DNA (30); SPG1 (2.1- and 3.89-fold induction at 20 min and 40 min, respectively), encoding a protein with unknown function that is required for survival at high temperatures during stationary phase (27); TPO3 (3.15- and 2.07-fold induction at 20 min and 40 min, respectively), encoding a plasma membrane polyamine transport protein specific for spermine (2); and SSH4 (3.54- and 2.31-fold induction at 20 min and 40 min, respectively), encoding a protein with unknown function whose overexpression confers resistance to the anti-inflammatory drug leflunomide (14).

Analysis of down-regulated genes upon exposure to DsS3(1-16) and Mag 2. (i) Down-regulated genes common to both peptides. A total of 64 common genes were found to be down-regulated (>1.5-fold decrease in expression) upon treatment with DsS3(1-16) (7 µg ml–1) or Mag 2 (15 µg ml–1) at both exposure times tested, 20 and 40 min postaddition (see Table SD in the supplemental material). As described above, these expression changes represent a response to common effects induced by both peptides. GO profiling revealed that the most significant gene clusters identified belonged to the categories for the biological process terms "ribosome biogenesis and assembly" (P = 5.65E–20), "rRNA processing" (P = 2.25E–10), and "organelle organization and biogenesis" (P = 2.85E–09) (see Table SE in the supplemental material). This profiling indicates that both peptides have a general effect of shutting down protein synthesis in response to stress via loss of ribosomes. Genes that encode proteins with functions in ribosome structure, function, or biogenesis have been shown previously to be coordinately and transiently repressed by multiple environmental stress factors (9). Ribosome synthesis is a major cellular biosynthetic activity, and the repression of these ribosomal components under stress conditions has been proposed to free significant energy resources for other cellular processes.

LDB7 was the only gene that was down-regulated by both peptides (at 20 and 40 min exposure), and the corresponding deletion strain also displayed sensitivity to both peptides. LDB7 is a component of the RSC complex involved in chromatin structure remodeling and double-strand break repair via nonhomologous end joining (48). Notably, in a screen for mutants conferring a low-dye-binding phenotype for the cationic dye Alcian Blue, LDB7 was isolated (10). While the molecular function of LDB7 remains unknown, our demonstration that both peptides down-regulate the expression of the gene could be explained by the ability of LDB7 to enhance binding of basic, positively charged compounds such as cationic peptides, which would clearly be detrimental. However, this does not explain why deletion of LDB7 confers sensitivity to both peptides.

(ii) Genes specifically down-regulated by DsS3(1-16) or Mag 2. Space constraints do not allow detailed discussion or presentation of these results. Ontology profiling of genes specifically down-regulated by DsS3(1-16) and by Mag 2 at both time points is shown in Tables SF and SG in the supplemental material, respectively. Confirming the observations made with up-regulated transcripts, the expression profiles for specifically down-regulated genes were very different for each peptide. The top biological process hits for DsS3(1-16) were "rRNA metabolism" (P = 1.53E–26), "ribosome biogenesis" (P = 4.51E–16), and "cytoplasm organization and biogenesis" (P = 5.59E–16). This profile indicated specific effects of DsS3(1-16) similar to those that were identified as common to both peptides, e.g., shut-down of ribosome biosynthesis. In contrast, the top process GO hits for Mag 2 were "regulation of metabolism" (P = 4.26E–05), "glycolysis" (P = 0.00011), and "external encapsulating structure organization and biogenesis" (P = 0.00013). These results indicated a general shut-down of cellular metabolism and physiological processes in the presence of Mag 2. Thus, these data support our previous observations that the individual peptides have very different effects on cells.

Mag 2 and DsS3(1-16) interact differently with DNA in vitro. The deletion mutant screen indicated that genes involved in mediating chromosome integrity and DNA damage repair were important common mediators of sensitivity to both peptides. Furthermore, ontology profiling revealed that the genes most significantly specifically up-regulated by Mag 2 alone included genes involved in DNA replication and repair. Thus, we explored the interaction of DsS3(1-16) and Mag 2 with DNA in more detail.

Recently, we reported that DsS3(1-16), but not Mag 2, induces programmed cell death in yeast (34). In vitro, it was shown that DsS3(1-16) retarded the electrophoretic mobility of DNA in an agarose gel and protected DNA from DNase I digestion but did not induce direct DNA damage via double-stand breaks. Thus, we concluded that DsS3(1-16) was capable of interacting with DNA in vitro. To determine if exposure to DsS3(1-16) could induce DNA damage in vivo, we measured DNA damage by a qPCR assay (40). This assay revealed that DsS3(1-16) did induce some DNA damage in vivo, probably due to the onset of programmed cell death and associated chromosome breakdown (measured by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay) (34). This may explain why, in this study, we identified many deletions of genes involved in DNA repair and DNA integrity that were sensitive to the peptide and why the expression of some genes with similar functions was induced upon exposure to DsS3(1-16).

To explain why Mag 2 induced many more genes involved with DNA repair and DNA replication than DsS3(1-16) did, we carried out the same assays described above with Mag 2 (Fig. 1). In vitro, we observed that Mag 2 retarded the migration of DNA in an agarose gel (Fig. 1a) in a fashion similar to that of DsS3(1-16) (34). Significantly, Mag 2 was able to completely retard the migration of pRS313 into the gel at a lower DNA-to-peptide ratio (1:100) than DsS3(1-16) (34), indicating that the interaction of Mag 2 with DNA was stronger than DsS3(1-16). The presence of Mag 2 also resulted in plasmid DNA remaining in the loading wells, apparently unable to move into the gel (Fig. 1a). To study this interaction further, we also performed a DNase I protection assay with the plasmid in the presence of Mag 2 (37). Thus, if the peptide binds to the plasmid DNA, it would be expected that the digestion of the DNA by DNase I would be inhibited. After adding Mag 2 to the plasmid DNA at various ratios, the mixtures were then incubated with DNase I (Fig. 1b). Consistent with the previous observation, DNA in the presence of Mag 2 at a ratio of 1:50 or higher resulted in the complete protection of pRS313 from digestion. Also, Mag 2 was able to protect pRS313 from DNase at a lower DNA-to-peptide ratio (1:100) (Fig. 1b) than DsS3(1-16) (34), confirming that the interaction of Mag 2 with DNA was stronger than DsS3(1-16).


Figure 1
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  		FIG. 1. Mag 2 interacts with DNA in vitro and induces DNA damage. (a) Results from a gel retardation assay showing the effect of increasing concentrations of Mag 2 on the electrophoretic mobility of plasmid DNA (pRS313). Ratios indicate increasing peptide-to-plasmid concentrations. M, molecular weight markers (1-kb ladder). "pDNA" indicates migration of pRS313 without the presence of Mag 2. (b) The left panel shows results from a DNase protection assay showing the protection of pRS313 from digestion by 0.5 µg ml–1 DNase I in the presence of increasing concentrations of Mag 2. Ratios indicate increasing peptide-to-plasmid concentrations. The right panel shows results from a control experiment, showing the digestion of pRS313 after exposure to 0.1 and 0.01 U µl–1 DNase I. M, molecular weight markers. "pDNA" indicates the electrophoresis of pRS313 without the presence of Mag 2 or DNase I. (c) DNA strand break assay results showing the effect of exposure to increasing concentrations of Mag 2 on the relative abundance of supercoiled-form (SF) and relaxed-form (RF) bands of pRS313. In each case, representative results are shown.

 
To explore whether the interaction of Mag 2 with DNA could result in direct damage, we performed DNA strand break assays (41). Strand breaks were assayed by measuring the relaxation of supercoiled plasmid to an open circular form. Exposure of plasmid DNA to increasing concentrations of Mag 2 resulted in the gradual loss of supercoiled bands at DNA-to-peptide ratios of 1:12 and above (Fig. 1c). Therefore, in contrast to the interaction of DsS3(1-16) with DNA (34), interaction of Mag 2 with DNA in vitro is strong enough to induce direct DNA damage in the form of strand breaks.

To determine if exposure to Mag 2 could induce DNA damage in vivo, we measured DNA damage by use of a qPCR assay (41). This method detects and quantifies DNA damage because if damaged DNA is used as a template, this blocks the progression of the PCR DNA polymerase and results in the production of fewer PCR products than equivalent undamaged DNA (50). Thus, prior exposure of cells to Mag 2 resulted in no significant reduction in specific PCR products relative to an untreated control (data not shown). Exposure to 5 mM H2O2 resulted in the virtual abolition of PCR products. Therefore, despite Mag 2 inducing strand breaks in vitro, we were unable to detect DNA damage in vivo. Thus, it seems likely that the huge up-regulation of genes involved in DNA repair and DNA replication that we detected upon exposure to Mag 2 occurs in response to the presence of the peptide targeting the DNA and, in this case, allows the cells to adapt and prevent detectable Mag 2-induced DNA damage in vivo.

To gain further insight into the mechanism of interaction of DsS3(1-16) and Mag 2 with DNA, we used CD. Far-UV CD spectroscopy was performed with DsS3(1-16) and Mag 2 either alone or in the presence of pRS313 (Fig. 2a and b). Without DNA, application of the CDSSTR algorithm revealed that the two peptides have similar CD spectra that indicate they are largely in an unordered state with some ß-sheet (Fig. 2c). However, the addition of Mag 2 in the presence of DNA results in a modification of the CD spectrum that can be interpreted as a change in the secondary structure content of the protein (Fig. 2b). The CDSSTR algorithm revealed that in the presence of DNA, Mag 2 adopts an {alpha}-helical structure (Fig. 2c). In contrast, in the case with DsS3(1-16), there was little evidence of change in secondary structure upon exposure to DNA; the peptide remained largely unordered. Detailed examination of the interaction of the peptides with DNA was not the purpose of this study, but the CD analysis has shown that the two peptides interact with DNA in solution in different ways.


Figure 2
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  		FIG. 2. CD revealed that, unlike DsS3(1-16), Mag 2 adopts an {alpha}-helical conformation upon binding to DNA. CD spectroscopy of peptides (250 µg ml–1) binding to DNA (pRS313; 50 µg ml–1) is shown. Far-UV spectra of DsS3(1-16) and Mag 2 with 10 mM sodium phosphate buffer, pH 7.0 (the spectrum shown constitutes a buffer-blank spectrum subtracted from the spectrum of the peptide plus the buffer) (a), and pRS313 DNA in 10 mM sodium phosphate buffer, pH 7.0 (the spectrum shown constitutes a DNA-blank spectrum subtracted from the spectrum of the peptide plus the DNA) (b). Results are expressed as mean residue ellipticity relative to the molar concentration of peptide. Representative results are shown. (c) Output from the CDSSTR algorithm (http://public-1.cryst.bbk.ac.uk/cdweb/html/) (47), showing the calculated proportion of secondary structural motifs for each peptide. Peptide names in bold type had 50 µg ml–1 pRS313 added.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Despite having no sequence identity, DsS3(1-16) and Mag 2 have been shown to adopt amphipathic {alpha}-helices in hydrophobic media, and thus, it has been proposed that their principal inhibitory action is due to membrane disruption (31, 52). Less certain is whether the membrane disruption induced by this interaction is the mechanism that kills the target microorganism.

Here, we have presented evidence implying that telomere maintenance, chromosome organization, and double-strand break repair are vital functions for maintaining resistance to the inhibitory actions of both peptides. Furthermore, ubiquitin-dependent protein catabolism, perhaps to remove peptides from inside the cells, was also shown to be important. Thus, Mag 2 and DsS3(1-16) share common inhibitory actions, one of which appears to involve interference with the integrity of DNA. Interaction of other basic, cationic peptides with negatively charged DNA has been proposed previously; for example, buforin 2, from the Asian toad Bufo bufo gargarizans, rapidly enters cells without perturbing the integrity of the cytoplasmic membrane and subsequently binds to DNA and RNA (40).

Study of factors unique to each peptide gave more subtle insight into the unique inhibitory actions of Mag 2 and DsS3(1-16). With DsS3(1-16), genes involved in vacuolar transport, acidification, and protein targeting to the vacuole were vital in conferring resistance to this peptide. The vacuole plays a key role in the sequestration and degradation of proteins delivered from the plasma membrane via endocytosis (23), and this could represent an effective means of clearing excessive toxic peptide from inside the cell. DsS3(1-16) also induced genes mediating a response to stress, reactive oxygen species, and drug treatment, implying toxic intracellular effects of the peptide and induction of ABC transporters, perhaps to remove excess peptide from the cell. In contrast, Mag 2 specifically induced genes involved in both Ca2+- and cyclic AMP-mediated cell signaling and regulation of cell wall organization and biogenesis. The cell wall is an important protective barrier to the yeast cell that the peptide has to cross to reach the plasma membrane. We have preliminary data showing that differences in the cell wall structure mediate huge differences in the susceptibility of yeast to cationic peptides (unpublished data). However, the most notable unique effect of Mag 2 compared to DsS3(1-16) was the up-regulation of many genes involved in DNA-dependent DNA replication and DNA repair, implying that this peptide has a more significant detrimental effect on the integrity of DNA than DsS3(1-16) does.

The deletion screen and expression analysis indicated that a common inhibitory mechanism of both peptides could be their ability to interact with DNA. This was supported by in vitro gel retardation, DNase protection, and strand break assays that indicated that both peptides bound DNA, that Mag 2 appeared to bind to DNA with greater affinity than DsS3(1-16), and, finally, that Mag 2, but not DsS3(1-16), could induce strand breaks in vitro. Further differences in the nature of the peptides' interactions with DNA were highlighted by CD analysis. These experiments showed that DsS3(1-16) was largely disordered in solution, which was supported by previous observations (15), and this did not change in the presence of DNA. Mag 2, however, was disordered in solution but in the presence of DNA adopted an {alpha}-helical conformation similar to that observed in a hydrophobic environment. Thus, despite interaction with DNA being a possible common mechanism of inhibition of both peptides, each peptide appears to interact with DNA differently in vitro. Crucially, this could account for some of the differences observed with the deletion mutant screen and gene expression profiles obtained from cells exposed to each of the inhibitory peptides. For example, the specific up-regulation of genes involved in DNA-dependent DNA replication and DNA repair that occurred upon exposure to Mag 2 further supported our in vitro observations that this peptide had a more significant detrimental effect on the integrity of DNA than DsS3(1-16) did.

Collectively, our data imply that both peptides must be able to pass though the cytoplasmic membrane of yeast cells to mediate inhibitory effects inside the cell. Supporting this conclusion, DsS3 has been shown to affect the viability of intraerythrocytic parasites at concentrations that did not permeate the host cell, indicating that the peptide can cross lipid bilayers and enter cells via some unknown mechanism (15). Similarly, Mag 2 has been shown to enter the cytosol of microbial cells (17). Therefore, it is clear that the two peptides have inhibitory actions not necessarily linked to membrane disruption. Indeed, we have shown recently that a principal antifungal action of DsS3(1-16), but not of Mag 2, is the induction of programmed cell death (34).

In summary, we have shown that DsS3(1-16) and Mag 2 have both common and unique inhibitory actions on yeast cells that are not simply due to membrane disruption. In fact, both DsS3(1-16) and Mag 2 interact with DNA in vitro in subtly different ways, but more importantly, functional genomics experiments have provided evidence that both peptides also interact and interfere with DNA integrity differently in vivo. This implies that both peptides are able to pass through the cytoplasmic membrane and interfere with the function of DNA in vivo, an inhibitory action that has not previously been observed for either of these peptides.


   ACKNOWLEDGMENTS
 
We thank J. Potter for expert assistance with CD.

The microarray work was supported by the Consortium for the Functional Genomics of Microbial Eukaryotes, which is funded by the UK Biotechnology and Biological Sciences Research Council (34/IGF13036). P.C. and C.O.M. also thank the BBSRC for supporting this research (grant no. P20473).


   FOOTNOTES
 
* Corresponding author. Mailing address: Centre for Biomolecular Sciences, School of Biology, University of St. Andrews, The North Haugh, St. Andrews KY16 9ST, United Kingdom. Phone: (44) (0)1334 463406. Fax: (44) (0)1334 462595. E-mail: pjc5{at}st-andrews.ac.uk Back

{triangledown} Published ahead of print on 10 September 2007. Back

{dagger} Supplemental material for this article may be found at http://aac.asm.org/. Back

{ddagger} Present address: Centre for Plant Integrative Biology, School of Biosciences, University of Nottingham, Sutton Bonington LE12 5RD, United Kingdom. Back


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Antimicrobial Agents and Chemotherapy, November 2007, p. 3948-3959, Vol. 51, No. 11
0066-4804/07/$08.00+0     doi:10.1128/AAC.01007-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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