Cellular Processes and Pathways That Protect Saccharomyces cerevisiae Cells against the Plasma Membrane-Perturbing Compound Chitosan

Global fitness analysis makes use of a genomic library of taggeddeletion strains. We used this approach to study the effectof chitosan, which causes plasma membrane stress. The data wereanalyzed using T-profiler, which was based on determining thesensitivities of groups of deletion strains to chitosan, asdefined by Gene Ontology (GO) and by genomic synthetic lethalityscreens, in combination with t statistics. The chitosan-hypersensitivegroups included a group of deletion strains characterized bya defective HOG (high-osmolarity glycerol) signaling pathway,indicating that the HOG pathway is required for counteractingchitosan-induced stress. Consistent with this, activation ofthis pathway in wild-type cells by hypertonic conditions offeredpartial protection against chitosan, whereas hypotonic conditionssensitized the cells to chitosan. Other chitosan-hypersensitivegroups were defective in RNA synthesis and processing, actincytoskeleton organization, protein N-glycosylation, ergosterolsynthesis, endocytosis, or cell wall formation, predicting thatthese cellular functions buffer the cell against the deleteriouseffect of chitosan. These predictions were supported by showingthat tunicamycin, miconazole, and staurosporine (which targetprotein N-glycosylation, ergosterol synthesis, and the cellwall integrity pathway, respectively) sensitized Saccharomycescerevisiae cells to chitosan. Intriguingly, the GO-defined groupof deletion strains belonging to the "cytosolic large ribosomalsubunit" was more resistant to chitosan. We propose that globalfitness analysis of yeast in combination with T-profiler isa powerful tool to identify specific cellular processes andpathways that are required for survival under stress conditions.

INTRODUCTION

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Introduction

Chitosan, a linear ß-1,4-D-glucosamine polymer, isa deacetylated derivative of chitin. When added exogenously,it is known to cause cell leakage and to inhibit the growthof fungi and bacteria; importantly, it seems to be less toxicto mammalian cells. These properties make it a valuable antimicrobialcompound with potential applications in medicine and in thefood industry (37, 45, 46, 48, 56, 63). Chitosan has a pK_a valueof around 6.5. Thus, at neutral and acidic pHs, chitosan moleculesbecome positively charged as a result of the protonation ofthe amino groups in the glucosamine residues, enabling themto interact with negatively charged components of the plasmamembrane, such as phospholipids (37). Chitosan also inducesthe formation of mass transfer channels in artificially createdlipid bilayers, thus providing additional evidence for its disturbingeffect on the plasma membrane (61). When Saccharomyces cerevisiaeis challenged with sublethal concentrations of chitosan, itinduces a specific transcriptional expression program comprisingthe environmental stress response and three more major transcriptionalresponses mediated by the transcription factors Cin5p, Crz1p,and Rlm1p, respectively (63). This is accompanied by structuralchanges in the cell wall, as reflected by the increased resistanceof living cells to ß-1,3-glucanase.

Here, we report a genome-wide screen of heterozygous essentialand homozygous nonessential deletion strains for altered sensitivityto chitosan. This type of approach is complementary to transcriptionalprofiling (4, 6, 21, 23, 38, 43, 44, 60, 64). Homozygous nonessentialdeletion strains have been extensively used to examine the responseof yeast deletion strains to various environmental conditions.Strains that are heterozygous for a deletion mutation of anessential gene are assumed to produce less of the essentialgene's product and therefore tend to be hypersensitive to drugsthat target that protein. For this reason, they may be usedto identify novel drug targets.

We have used T-profiler (5) to identify predefined groups ofgene deletion strains that are hypersensitive to chitosan stress.Chitosan-hypersensitive categories of deletion strains includedgroups with defects in RNA synthesis and processing, actin cytoskeletonorganization, protein N-glycosylation, ergosterol synthesis,endocytosis, and cell wall formation. The results of this analysiswere supported by using drugs that target specific cellularprocesses. For example, tunicamycin, which blocks the N-glycosylationof secretory proteins and thus causes the hypoglycosylationof yeast mannoproteins, strongly sensitized yeast cells to chitosan.Chitosan sensitivity was also elevated when it was combinedwith miconazole (an inhibitor of ergosterol biosynthesis) andwith staurosporine, which inhibits the Pkc1p complex, a proteinkinase complex that controls the cell wall integrity pathway.In addition, we demonstrate that various forms of plasma membranestress, such as that caused by hypotonic conditions, treatmentwith chlorpromazine (a drug that causes membrane stretch), ora high growth temperature, also dramatically increase the sensitivityof the cells to chitosan. We conclude that analyzing globalfitness experiments with T-profiler is a powerful approach toidentify cellular processes and pathways that are essentialfor survival under drug-induced or other stress conditions.

MATERIALS AND METHODS

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Materials and Methods

Strains and growth conditions. S. cerevisiae strain BY4743 (MATa/MAT ${alpha}$ his3 ${Delta}$ 1/his3 ${Delta}$ 1 leu2 ${Delta}$ 0/leu2 ${Delta}$ 0MET15/met15 ${Delta}$ 0 LYS2/lys2 ${Delta}$ 0 ura3 ${Delta}$ 0/ura3 ${Delta}$ 0) and individual deletionstrains in this genetic background were used in this study.Synthetic complete (SC) medium (0.67% yeast nitrogen base withoutamino acids, 0.5% Casamino Acids, 2% glucose, with 25 mM phthalate-NaOHbuffer [pH 5.5]) was used to culture the wild-type (WT) strain,the homozygous deletion strains (in the case of nonessentialgenes), and the heterozygous deletion strains (in the case ofessential genes). Cells were precultured overnight in SC medium(in the case of the WT), for 12 h in yeast-peptone-dextrose(homozygous deletion pool), or for 5 h in SC medium (heterozygousessential pool). Batch fermentors were inoculated with cellsto an optical density at 600 nm (OD₆₀₀) of 0.07 ( $~$ 0.7 x 10⁶ cells/ml).The cultures were grown in 1 liter of SC medium at 30°C,with an aeration rate of 1.0 liter/min, fast stirring (200 rpm),and monitoring of pH. Chitosan (final concentration, 25 µg/ml)was added at an OD₆₀₀ of 0.1 ( $~$ 1 x 10⁶ cells/ml). Growth wasmeasured by monitoring the OD₆₀₀. Samples for genomic DNA isolationwere taken after 5 and 9 h of growth. Reference samples weretaken from a culture grown without chitosan at the same timepoints. An appropriate volume (20 to 25 ml) of the culture wascentrifuged at 4,000 rpm for 5 min, and the cell pellets werestored at –20°C.

Preparation and analysis of chitosan. Chitosan was obtained from Sigma (crab shells, minimum 85% deacetylated;average molecular mass of $≥$ 600 kDa) (41). It was fragmented usingnitrous deamination. Chitosan was dissolved in 10% acetic acidand incubated with sodium nitrite at a concentration of 20 mgper gram of chitosan at room temperature for 17 h. To stop thereaction, the pH of the preparation was adjusted to 5.5 withNaOH. The final concentration of chitosan was measured as glucosamineequivalents after hydrolysis in 6 M HCl at 100°C for 17h (31). The average size of the chitosan fragments was definedas the ratio of the number of glucosamine residues measuredafter acid hydrolysis of chitosan and the number of reducingends measured after fragmentation of chitosan by using a methoddescribed previously by Lever et al. (35) and was $~$ 50 glucosamineresidues. This was supported by gel filtration, which indicateda molecular mass of about 10 kDa ( $~$ 60 glucosamine residues).We used chitosan fragments because we reasoned that native chitosan(average molecular mass of $≥$ 600 kDa) might be too large to easilypass the cell wall (14) and because a previous study had shownthat fragmented chitosan had higher antifungal activity thannative chitosan (46).

SYTOX green uptake. Cell samples from exponential-phase cultures of S. cerevisiaewild-type strain BY4741 grown in either the absence or presenceof 25 µg/ml chitosan were spun down at 3,500 rpm, andthe cell pellet was washed twice with 50 mM MES (morpholineethanesulfonicacid)-NaOH buffer (pH 5.5). Ninety microliters of cell suspensionin buffer (OD₆₀₀ of 1) was transferred onto a 96-well platewith flat-bottom wells, and SYTOX green was added to a finalconcentration of 1 µM. The SYTOX green fluorescence wasread at 1-min intervals for 30 min in a GeminiXS microtiterplate reader using 488 nm as the excitation wavelength, 544nm as the emission wavelength, and 530 nm as the cutoff. Tocalculate relative fluorescence units, all measurements wereexpressed as a percentage of the fluorescence of a sample ofcells treated with 70% (vol/vol) ethanol for 5 min for completecell permeabilization, spun down at 3,500 rpm, resuspended inbuffer, and measured as described above.

PCR amplification, hybridization, and data acquisition. Yeast cells were lysed at 37°C with 0.5 mg/ml Zymolyase100T for 60 min in sorbitol buffer (1 M sorbitol, 100 mM sodiumEDTA, 14 mM ß-mercaptoethanol). Genomic DNA was isolatedwith the QIAGEN DNeasy kit. The PCR amplification of the DNAtags was performed on 200 ng of genomic DNA template. Both UPTAGsand DOWNTAGs were amplified in separate reactions using biotinylatedPCR primers complementary to common regions in the replacementcassette. PCR conditions and primers used were described previously(21). The final labeled UPTAGs and DOWNTAGs were purified ona YM10 Microcon column, combined, and hybridized to custom-madeoligonucleotide microarrays (DNA TAG3; Affymetrix, Santa Clara,CA) as described previously (21), with the exception that fourblocking primers were used in the hybridization mixture. After16 h of hybridization at 42°C, the arrays were stained withstreptavidin-phycoerythrin (Molecular Probes) and scanned withan Affymetrix GeneChip scanner. The hybridization intensitiesfor each probe were determined using Affymetrix GeneChip operatingsoftware.

Data processing. The majority of knockout strains are represented by four valuesof signal intensity (sense and antisense array elements foreach UPTAG and DOWNTAG). We have calculated the mean of thesignal intensities of tags representing each open reading frame(ORF) on the basis of two biological replicates of the experiment.The signal intensities of the UPTAGs and DOWNTAGs were calculatedseparately. Next, the ORFs whose averaged signal intensitieswere below the background level in the control experiment werediscarded from further analysis. We defined the background asthe average of all signal intensities of nonhybridizing spotsand found that, for all arrays, it was below 20. To quantifythe strain susceptibility to chitosan, we calculated the log₂ratio (log₂ R) of normalized signal intensities for each strainin the control condition to those with chitosan treatment. Thisresults in positive values for sensitive strains. To definesignificantly sensitive or resistant mutants in strains homozygousfor the deletion of nonessential genes or heterozygous for essentialgenes, we used a cutoff of $≥$ 1 for the log₂ R at 5 h (correspondingto a ratio of 2) and a cutoff of $≥$ 1.585 for the log₂ R at 9 h(corresponding to a ratio of 3).

Data analysis using T-profiler. We used an unpaired t test, which gives a measure of significanceto the difference between the mean of a specific group of deletionmutants and the mean of the remaining deletion mutants of thetotal data set (5). To increase the robustness of the t test,we discarded the highest and lowest mutant log₂ R of all deletionmutant groups. This method is comparable to the "jackknife"procedure and reduces the effect of outliers, which might causefalse-positive or false-negative results. Only groups consistingof at least five members were used for analysis. The P valuesobtained in this way were Bonferroni corrected for multipletesting by multiplying them by the number of mutant groups testedin parallel. Resulting E values that were $≤$ 0.05 were consideredto be significant.

The following two types of predefined groups of deletion strainswere used in our analysis. The first type of predefined groupsconsisted of Gene Ontology (GO) categories. These are definedaccording to function, biological process, or cellular localization(15). Deletion mutant groups containing more than 100 memberswere left out. In total, 961 categories were tested. The secondtype consisted of synthetic lethality (SL)-based deletion mutantgroups. These groups have been obtained by crossing a querydeletion strain into a set of $~$ 4,900 viable deletion mutantsand by screening the resulting double mutants for syntheticlethality or slow growth (54, 55). There have been $~$ 500 deletionstrains used as queries in such experiments, resulting in the462 deletion mutant groups used in our analysis. The completegene composition of each SL- and GO-based deletion mutant groupcan be found on the SGD website (27).

Growth experiments. Exponential-phase cultures of Saccharomyces cerevisiae wild-typestrain BY4741 were diluted to an OD₆₀₀ of 0.1, and culturesof 200 µl were grown further in 96-well plates at 30°C.Either no drug, 25 or 50 µg/ml chitosan, 1.25 µg/mltunicamycin, 250 µM chlorpromazine, 1.6 µM staurosporine,2 µg/ml miconazole, or a combination of chitosan withone of the drugs was added. To combine chitosan with hypo-osmoticstress, the cells were suspended in SC medium (control culture)or 75% SC medium, and chitosan was added at 50 µg/ml.To test growth at 37°C, cells were suspended in SC medium,with or without 50 µg/ml chitosan, and placed in a 96-wellmicrotiter plate, which was positioned in the reader that wasset at 37°C. Growth was monitored for 16 h by measuringthe OD₆₀₀ at 20-min intervals. The relative growth rates werecalculated by linear regression from logarithmic plots of theOD₆₀₀ data versus time.

Chitosan susceptibility assays were carried out as describedpreviously (63). In short, 10-fold serial dilutions of yeastcells were spotted onto solid SC medium prepared by using 2%agarose instead of agar, and the plates were incubated at 30°Cfor 3 days.

RESULTS

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Results

Overview of the chitosan susceptibilities of all deletion mutants. Treatment of the S. cerevisiae wild-type strain with 25 µg/mlor 50 µg/ml chitosan resulted in a mild reduction in itsrelative growth rate (Table 1). SYTOX green is a dye that fluoresceswhen bound to nucleic acids but that can pass the plasma membraneonly when that membrane's integrity is compromised. When exponentiallygrowing cells were treated with 25 µg/ml chitosan andstained with this dye, the fluorescence increased from about0.5% of the fluorescence of ethanol-treated cells to $~$ 6% at 15min and increased further with time (7% at 30 min, 11% at 60min, and 13% at 120 min), whereas the fluorescence of untreatedcells remained unchanged (Fig. 1). These results suggest thatchitosan at 25 µg/ml slightly affected the integrity ofthe plasma membrane. However, after a 4-h exposure to chitosan,the fluorescence decreased to 8% and to 2% at 6 h, suggestingthat the cells may partially recover from treatment with thismild chitosan concentration.

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TABLE 1. Conditions and drugs that sensitize yeast cells to chitosan^a

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FIG. 1. SYTOX green uptake of yeast cells grown in the presence of chitosan. Yeast cells were grown exponentially in the absence or presence of 25 µg/ml chitosan. Samples for fluorescence measurements were taken at the indicated time points. RFU, relative fluorescence units expressed as a percentage of the fluorescence obtained by treating the same number of cells with 70% ethanol. After 4 and 6 h of incubation, the relative fluorescence unit values were 8% and 2%, respectively. Closed triangles, control; closed squares, 25 µg/ml chitosan. Means and standard deviations are shown (n = 3).

To identify the genes and the cellular processes involved inchitosan sensitivity, a global fitness experiment was carriedout in the presence of 25 µg/ml of chitosan. The abundanceof the deletion strains in the control culture and in the chitosan-treatedculture was determined after 5 and 9 h of growth, which correspondsto 3 to 4 and 6 to 7 generation times of the wild-type strain,respectively. These late time points were chosen to be ableto detect even relatively small differences in growth. Amongthe $~$ 4,900 homozygous deletion mutants of nonessential genes,we found 184 strains with a log₂ R of $≥$ 1 (this corresponds toa twofold reduction in strain abundance in the presence of chitosan)at 5 h and 153 strains with a log₂ R of $≥$ 1.585 (this correspondsto a threefold reduction) at 9 h (see Fig. 2 for Venn diagrams)(for the whole list of mutants, see Table SD1 in the supplementalmaterial). A total of 101 mutants were chitosan hypersensitiveat both time points. To establish how reliable the results obtainedby the genomic approach were, we retested a number of individualstrains that varied widely in their sensitivities to chitosan.Both approaches generally gave the same results for both thesensitive and the resistant strains (see Table SD2 in the supplementalmaterial). As shown previously (63), rlm1 ${Delta}$ cells were less sensitiveto chitosan than wild-type cells. Interestingly, mnn4 ${Delta}$ and mnn6 ${Delta}$ cells, which are responsible for the introduction of (negativelycharged) phosphodiester groups in N- and O-linked protein sidechains, were also less sensitive to chitosan, indicating thatthe binding of chitosan to phosphodiester linkages enhancesits effect. Conceivably, the binding of chitosan to such groupsin plasma membrane proteins, and the resulting local increasein the concentration of chitosan, is largely responsible forthis stimulatory effect.

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FIG. 2. Overview of chitosan-hypersensitive deletion mutants at 5 and 9 h. (A) Venn diagram summarizing the results for strains heterozygous for the deletion of an essential gene. (B) Venn diagram summarizing the results for strains homozygous for the deletion of a nonessential gene. The cutoffs for the log₂ R were 1 at 5 h (ratio of 2), and 1.585 at 9 h (ratio of 3). The numbers in parentheses refer to the total numbers of sensitive strains identified at the indicated time points.

Among the $~$ 1,100 strains heterozygous for the deletion of anessential gene, we detected only 11 and 3 chitosan-hypersensitivemutants at 5 and 9 h, respectively. In addition, the highestobserved sensitivity of the mutants, expressed as log₂ R, wasmuch lower than in the homozygous deletion strains both at 5h (1.46 and 9.57, respectively) and at 9 h (1.75 and 9.39, respectively).These observations indicate that in most heterozygotes, theactivity of the remaining copy of the gene is sufficient towithstand chitosan-induced stress to a large extent. Since previousstudies have shown that heterozygous deletants are often highlysensitive to drugs that specifically target the product of thedeleted gene (20, 22, 23, 38), our data also indicate that chitosandoes not have a unique protein target.

Identification of GO-based groups of deletion strains with altered fitness in the presence of chitosan. To acquire easily interpretable biological information aboutthe response of deletion strains to chitosan treatment, we usedT-profiler (which is based on t statistics) to identify changesin susceptibilities of predefined groups of mutants (see Materialsand Methods). We first tested groups of strains with a deletionof a gene with a common regulatory motif in their upstream promoterregion and groups with a deletion of a gene bound by a commontranscription factor as determined by transcription factor occupancyanalysis (25, 34). None of those groups of deletants had significantt values, indicating that there is no simple relationship betweenthe transcriptional response of coregulated genes in responseto stress and the fitness of the corresponding deletion mutantsunder the same conditions.

Next, we investigated the response of GO- and SL-based deletionmutant groups to chitosan. GO categories were included in ourstudy in order to investigate whether the genes that were deletedin chitosan-hypersensitive GO-based groups of mutants shareany functional features or participate in the same biologicalprocesses. The advantage of this approach is that all yeastgenes are annotated in these categories, thus giving a comprehensiveoverview of the whole yeast genome. On the other hand, due tothe hierarchical structure of the categories, there is considerableoverlap between them. We further used 462 SL-based gene groupsin our analysis, representing a wide spectrum of biologicalprocesses and cellular functions (for a detailed discussionof these results, see below). The major advantage of this approachis that it represents experimentally validated relationshipsbetween genes rather than the researchers' view of those relationships.However, it is incomplete, as only around 10% of the genes havebeen tested as query genes (54, 55).

We detected 13 significant GO groups, 12 of which were hypersensitiveto chitosan, whereas 1 of them contained resistant mutants (Table2). These 13 groups can be further ordered into several biologicallyrelevant categories.

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TABLE 2. Identification of GO-based gene groups with altered fitness in the presence of chitosan^a

Four groups of strains that carried deletions of genes involvedin RNA polymerase II transcription or in RNA splicing were foundto be hypersensitive to chitosan. The five most sensitive mutantsare paf1 ${Delta}$ , snf5 ${Delta}$ , rtf1 ${Delta}$ , srb5 ${Delta}$ , and med1 ${Delta}$ . Interestingly, Paf1p andRtf1p belong to the Paf1p complex, which is required for thefull transcription of several cell wall biosynthetic genes (9).Ten mutants with a deletion of genes involved in processes occurringat the endosome membrane (snf8 ${Delta}$ , snf7 ${Delta}$ , vps20 ${Delta}$ , vps28 ${Delta}$ , vps22 ${Delta}$ , vps36 ${Delta}$ ,vps25 ${Delta}$ , vps24 ${Delta}$ , did4 ${Delta}$ , and srn2 ${Delta}$ ) are highly sensitive to chitosan.Deletion mutants of five genes that encode proteins participatingin retrograde transport from the Golgi apparatus to the endoplasmicreticulum (ER) (get1 ${Delta}$ , get2 ${Delta}$ , get3 ${Delta}$ , glo3 ${Delta}$ , and sec22 ${Delta}$ ) also showedincreased sensitivity to chitosan. In addition, the mutantsin the "nuclear envelope-ER network" group are hypersensitiveto chitosan. These observations indicate that the proper functioningof the intracellular transport machinery is crucial for counteractingchitosan stress. We also found that mutants from the "glycoproteinmetabolism" group, which includes N-glycosylation, are highlysensitive to chitosan. In addition, we identified mutants inthe "response to osmotic stress" category to be hypersensitiveto chitosan. These findings will be the subject of a more extendeddiscussion later in this paper.

We also detected one GO functional category that contained geneswhose deletants exhibited increased resistance to chitosan.This category, designated "cytosolic large ribosomal subunit,"comprises genes coding for structural proteins of the largeribosomal subunit. Interestingly, a similar behavior of deletionmutants in ribosomal proteins was observed in other genome-widefitness studies of yeast (58, 60).

Deletion mutants of genes that synthesize a truncated lipid-linked oligosaccharide for protein N-glycosylation are highly susceptible to chitosan. The pathway of protein N-glycosylation in yeast begins withthe assembly of a lipid-linked oligosaccharide, which is latertransferred in the ER to suitable protein N-glycosylation sites.The first part of the lipid-linked oligosaccharide assemblyin yeast takes place on the cytoplasmic side of the ER membrane.The second part, which involves the addition of the final fourmannose residues and three glucose residues, occurs in the ERlumen (7). None of the heterozygote deletion strains of essentialgenes showed increased sensitivity to chitosan (log₂ R of $~$ 0),consistent with our previously noted observation that most heterozygousdeletants were not strongly affected by chitosan. We found thatthree of six homozygous deletion mutants of genes involved inthe second part of lipid-linked oligosaccharide biosynthesis(alg9 ${Delta}$ , alg6 ${Delta}$ , and alg8 ${Delta}$ ) are hypersensitive to chitosan (Table3). All three mutants exhibit hypoglycosylation of secretoryproteins. We found that the alg5 ${Delta}$ deletion mutant, which lacksthe gene coding for Dol-P-Glc synthase, is also highly sensitiveto chitosan. Dol-P-Glc is the sugar donor of the three glucoseresidues in the N-chain precursor.

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TABLE 3. Chitosan sensitivities of deletion strains of genes that synthesize a truncated lipid-linked oligosaccharide for protein N-glycosylation^a

The sensitizing effect of incomplete N-glycosylation was alsoobserved when yeast cells were treated with a combination ofchitosan and tunicamycin, which blocks N-glycosylation (Table1). Hypoglycosylation probably leads to a lower efficiency offolding of secretory proteins in the ER and thus to lower levelsof the mature proteins (47). As many known cell wall polysaccharidesynthases and cell wall construction enzymes are N-glycosylated,tunicamycin-induced hypoglycosylation may result in a weakenedcell wall and, indirectly, in increased membrane stress, potentiallyrendering the cells more prone to the membrane-perturbing effectof chitosan.

HOG pathway mutants are highly susceptible to chitosan. The HOG (high-osmolarity glycerol) pathway is crucial for theregulation of the intracellular osmotic balance of the yeastcell in response to osmotic changes in its surrounding environmentand also for cell wall maintenance (1, 18, 32). The HOG pathwaycomprises two upstream signaling branches mediated by the osmosensingtransmembrane proteins Sln1p and Sho1p and a downstream mitogen-activatedprotein (MAP) kinase cascade. In the Sln1p branch, the signalis transferred from Ssk1p to two redundant MAP kinase kinasekinases, Ssk2p and Ssk22p, which activate the MAP kinase kinasePbs2p, which in turn activates Hog1p. We found that the homozygousdeletion mutants of genes in this branch (ssk1 ${Delta}$ ) and in the MAPkinase cascade (ssk2 ${Delta}$ , pbs2 ${Delta}$ , and hog1 ${Delta}$ but not the redundant genessk22 ${Delta}$ ) are extremely sensitive to chitosan (Table 4). Consistentwith these observations, the PBS2 gene was initially identifiedby the sensitivity of the deletion mutant to polymyxin B, adecapeptide that (like chitosan) is a cationic molecule andcauses plasma membrane disintegration and leakage of cellularcomponents (50). The hypersensitivity of mutants of the HOGpathway to chitosan indicates that the proper functioning ofthis pathway is required for counteracting chitosan stress.This was experimentally validated by incubating yeast cellsbefore and during chitosan treatment in the presence of 1 Msorbitol, which partially protected the cells against the deleteriouseffect of chitosan on growth (Table 5). Conversely, hypotonicgrowth conditions made the cells more vulnerable to chitosan(Table 1).

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TABLE 4. Chitosan sensitivities of strains homozygous for the deletion of a nonessential gene from the HOG pathway^a

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TABLE 5. Pretreatment with 1 M sorbitol protects yeast against chitosan-induced stress^a

Identification of chitosan-hypersensitive SL-based groups of deletants. In synthetic genetic analysis, a strain bearing a mutation ina query gene is crossed with a set of $~$ 4,700 deletion mutants,and the resulting double mutants are screened for syntheticlethal (SL) or slow-growth ("sick") (SS) phenotypes (54). Thus,an SL group is composed of the strain with a deletion of thequery gene and all mutants that have an SL or SS interactionwith this deletion mutant. Twenty-five SL-based deletion mutantgroups were hypersensitive to chitosan (Table 6), comprising61 chitosan-hypersensitive mutants. The membership of theseSL groups only partially overlaps with that of the GO-basedmutant groups. For example, two SL groups are involved in proteinN-glycosylation (the HOC1 and the CWH41 groups), in agreementwith the results obtained with the GO group analysis (Table2). In addition, both GO and SL groups identify intercompartmentaltransport in the secretory pathway as being important for counteractingchitosan-induced stress. On the other hand, both approachespoint to several unique cellular functions that are needed tocounteract chitosan-induced stress, indicating that the twoapproaches complement each other.

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TABLE 6. Identification of chitosan-hypersensitive SL deletion mutant groups^a

The first four chitosan-hypersensitive groups are based on querygenes that are involved in the formation of the cell wall andits localization and regulation (Table 6). CHS3 and CHS4 aredirectly involved in chitin synthesis, whereas BNI4 is involvedin the polymer's localization (13). The SMI1 gene encodes asignaling protein that is involved in regulating cell wall synthesis(3). These observations indicate that cell wall integrity isrequired to withstand chitosan-induced stress. Consistent withthis, staurosporine (a potent inhibitor of the protein kinaseC complex, which controls the cell wall integrity signalingpathway) (36, 59, 62) showed a dramatic growth-inhibitory effectwhen combined with chitosan (Table 1).

Plasma membrane integrity also seems to be a major contributingfactor to the yeast cell's ability to cope with chitosan-inducedstress. Members of both the ERG11-based SL group and the CNB1-basedSL group are chitosan hypersensitive. Ergosterol is the maincomponent responsible for the rigidity of the plasma membrane,and ERG11 encodes an essential enzyme in ergosterol biosynthesis,the cytochrome P-450-dependent C14 lanosterol demethylase. Thehypersensitivity of the ERG11-based SL group to chitosan wasexperimentally confirmed by incubating yeast cells in the presenceof a combination of chitosan and miconazole (a specific inhibitorof Erg11p), resulting in a synergistic effect on growth (Table1). CNB1 encodes the regulatory subunit of calcineurin, whichis involved in coping with plasma membrane stress (11). Theidentification of the CNB1-based SL group is in agreement withour previous study, where cells exposed to the calcineurin inhibitorFK506 showed increased chitosan sensitivity (63). Additionalexperimental validation for a key role of plasma membrane integrityin coping with chitosan-induced stress was obtained by subjectingthe cells to hypotonic stress and to chlorpromazine, a cationicamphipathic drug that inserts itself into the plasma membranelipid bilayer, thus leading to plasma membrane stretching (30).Table 1 shows that both treatments acted synergistically incombination with chitosan. Consistent with this, cells grownat 37°C, a temperature that is believed to increase membranefluidity, were also more sensitive to chitosan.

Finally, eight groups consist of mutants that show SL with mutantsof genes encoding various cytoskeleton-related proteins. Theseinclude the four members of cytoplasmic prefoldin protein complex(GIM1, GIM2, GIM3, and GIM5), which participates in the foldingof tubulin and actin; RVS161 and RVS167, both subunits of thesame complex that regulates cell polarity, actin cytoskeletonpolarization, and endocytosis; and, finally, ARC40 and ARP2,the components of the actin nucleation center, involved in endocytosis,membrane growth, and polarity. In addition, we detected fourgroups of chitosan-hypersensitive mutants that have SL interactionswith CFT4, SET2, ARP6, and CDC7, genes participating in DNAbinding and histone methylation.

DISCUSSION

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Discussion

Like many natural antimicrobial compounds found in vertebrates,invertebrates, and plants, chitosan is positively charged (atleast at neutral and acidic pHs) and perturbs the plasma membraneof bacteria and fungi (51-53). Cationic peptides and other cationiccompounds, including chitosan, have been shown to facilitatethe entry of molecules into mammalian cells (17). Chitosan maythus function as an antimicrobial compound itself and as a modelcompound to understand the deleterious effects of other cationicantimicrobial compounds on microorganisms. In a previous study,we analyzed the transcriptional response of yeast cells to chitosan(63). Here, we have complemented the transcriptional analysisof chitosan-treated cells with a global fitness study. Globalfitness studies make use of a genomic library of deletion strains,allowing the parallel measurement of their growth response toa chosen stress condition (43, 44, 54). For the analysis andinterpretation of our data, we have applied and extended theapproach developed for global transcriptional analysis, calledT-profiler, which is based on the study of the transcriptionalbehavior of related groups of genes using t statistics (5, 63).In this study, we have determined the response to chitosan ofpredefined groups of mutant strains carrying a deletion in relatedgenes. Two types of deletion mutant groups were successfullytested: (i) groups based on GO categories (2), and (ii) groupsbased on global SL screens (54, 55). GO groups have the advantagethat they cover all deletions, in contrast to the incompletecoverage by SL groups. However, the SL groups have the intrinsicadvantage that they are defined by the yeast cell rather thanthe investigator.

Both approaches led to testable predictions. For example, theanalysis based on GO groups identified a role for the Sln1 signalingbranch and the Hog1 MAP kinase module of the HOG pathway inprotecting the cell against chitosan-induced stress. In contrast,the Sho1 signaling branch is not involved. We hypothesize thatchitosan may make the plasma membrane leaky, for example, byinducing the formation of mass transfer channels as has beenobserved in artificial lipid bilayers (61). This would resultin the uptake of (acidic) medium and, consequently, cytosolicacidification. The HOG pathway genes SSK1, SSK2, PBS2, and HOG1,which function in the Sln1 branch and the Hog MAP kinase module,respectively, might then be expected to offer protection notonly against chitosan (this paper) but also against cytosolicacidification resulting from other stresses. Evidence for thiscomes from the work of Mollapour and Piper (40), who found thatmutants with a deletion of either SSK1, PBS2, or HOG1 are highlysensitive to acetic acid/acetate at pH 4.5. At this pH, whichis below the pK_a value of acetic acid, undissociated, and thusuncharged, acetic acid molecules form the majority. The unchargedacetic acid molecules can easily pass the plasma membrane. Theywill immediately dissociate upon arrival in the cytosol, resultingin acid stress. Mollapour and Piper (40) also found that thesho1 ${Delta}$ strain does not show increased sensitivity to acetic acid/acetate,demonstrating that the Sho1 signaling branch is not involvedin coping with acid stress. Similar results have been describedpreviously by Lawrence et al. (33), who found that ssk1 ${Delta}$ , pbs2 ${Delta}$ ,and hog1 ${Delta}$ cells, but not sho1 ${Delta}$ cells, were more sensitive to citricacid/citrate at pH 3.5, and by Kapteyn et al. (32), who showedthat Hog1p is required for a cell wall-strengthening responseinduced by culturing cells at pH 3.5. Using global transcriptanalysis, we have previously shown that in the presence of chitosan,yeast cells strongly and continuously activate the cell wallintegrity pathway (63). This raises the question of how cellschallenged with chitosan can have the cell wall integrity pathwayturned on, whereas simultaneously, the HOG pathway is functionalin protecting the cells against chitosan. Interestingly, Mollapourand Piper (40) showed that in yeast cells challenged with aceticacid/acetate at pH 4.5, the doubly phosphorylated forms of Slt2pand Hog1p can be present in the cell simultaneously, indicatingthat at least under some stress conditions, the simultaneousactivation of both MAP kinases is possible. This further suggeststhat the HOG pathway might have functions other than organizinga response to hypertonic stress, as also indicated by the observationsthat the HOG pathway is induced by cold stress and dimethylsulfoxide (26, 42).

Analysis of both types of deletant groups identified defectiveprotein N-glycosylation as an important factor contributingto chitosan hypersensitivity, and this was supported experimentallyby the observation that tunicamycin sensitized yeast cells tochitosan (Table 1). As defective protein N-glycosylation affectsprotein folding in the ER, it seems likely that many secretoryproteins involved in cell wall construction will be producedin insufficient amounts, affecting cell wall integrity and indirectlyresulting in increased membrane stress.

Several SL groups are related to actin cytoskeleton organization.Previous studies have shown that actin cytoskeleton depolarizationoccurs upon heat and cell wall stress (12) as well as underhypo- and hyperosmotic conditions (10, 24). Interestingly, actincytoskeleton depolarization was also observed upon treatmentwith two plasma membrane perturbants, sodium dodecyl sulfateand chlorpromazine (12). The actin cytoskeleton is also requiredfor stress-dependent cellular redistribution of the cell wallbiosynthetic protein Fks1p (12). The transient depolarizationof the actin cytoskeleton may therefore act as a homeostaticmechanism to repair cell wall and membrane damage, which canexplain why mutants defective in actin cytoskeleton organizationare hypersensitive to membrane stress caused by chitosan. Interestingly,actin patches colocalize with sites of endocytosis and associatewith endosomes, and their movement is mediated by polarizedactin cables (28). Endocytosis in S. cerevisiae is essentialfor plasma membrane-associated functions, because it is requiredfor the recycling of plasma membrane components, uptake of nutrients,and regulation of cell surface signaling receptors (16, 57).The observation that mutants that are defective in endocytosisare hypersensitive to chitosan-induced plasma membrane perturbationsupports the idea that endocytosis is crucial for the maintenanceof plasma membrane integrity and functioning.

Recent studies of S. cerevisiae have demonstrated that on thelevel of individual genes, there is often no correlation betweenthe transcriptional activation of a gene upon stress and thesensitivity of the corresponding deletion strain to the samestress (4, 21). Similarly, we have found that there is no clearrelationship between the transcriptional responses of coregulatedgenes in case of stress and the stress sensitivities of thecorresponding groups of deletion strains. The transcriptionalresponse to stress is unavoidably confounded by a slow-growthresponse (8). We have separated these two responses by focusingour analyses at the level of cellular functions and pathways,thus gaining results in which transcriptional and fitness profilingsshow a larger degree of similarity.

We (63) showed previously that the main transcriptional responsesof yeast cells during the first 180 min of incubation in thepresence of chitosan are mediated by the transcription factorMsn2p/Msn4p, which mediates a general stress response (19);Crz1p, which probably mediates the transcriptional responseto plasma membrane stress (49); Rlm1p, which activates a groupof mostly cell wall-related genes in case of cell wall stress(29); and Cin5p, which mediates salt tolerance (39). Althoughthe global fitness experiment described in this paper coversa period of 9 h and the data are thus not directly comparableto the transcriptional profiling data obtained during a 180-minchitosan treatment, our current results clearly point to a majorrole for cell wall and plasma membrane integrity in counteractingand recovering from chitosan-induced stress. They also showthe benefit of analyzing the data on the level of functionalcategories of genes using T-profiler.

In summary, our genome-wide analysis of fitness of all yeastdeletion mutants in response to chitosan treatment indicatesthat the loss of genes that are involved (directly or indirectly)in maintaining plasma membrane integrity is the primary causeof chitosan hypersensitivity (summarized in Fig. 3). In addition,we have shown that T-profiler analysis of yeast fitness data,based on GO- and SL-based gene groups, is highly effective inidentifying cellular processes and pathways that are crucialfor defending the cell against chitosan-induced stress. Thisapproach is equally effective for the analysis of other fitnessprofiles (our own unpublished data) and may eventually providea useful platform for the comparison and integration of otherlarge-scale genomic studies. Finally, it will also allow thesystematic identification of combinations of drugs to preventor minimize fungal infections in food and hosts.

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FIG. 3. Factors contributing to chitosan hypersensitivity in S. cerevisiae. ${Delta}$ mutants refers to deletion mutants.

ACKNOWLEDGMENTS

This research was financially supported by grant APB.5504 fromThe Netherlands Technology Foundation (to F.M.K.) and the UKNatural Environment Research Council (to S.G.O.). We are gratefulfor the use of the resources of the COGEME facility at Manchester(established by a BBSRC Investigating Gene Function grant toS.G.O.).

We thank Andy Hayes and Bharat Rash for their advice and assistancewith the bar code array analyses. We thank Gertien Smits forstimulating discussions.

FOOTNOTES

* Corresponding author. Mailing address: Swammerdam Institute for Life Sciences, Nieuwe Achtergracht 166, 1018WV Amsterdam, The Netherlands. Phone: 31205256424. Fax: 31205257056. E-mail: A.M.Zakrzewska{at}uva.nl

Published ahead of print on 26 January 2007.

Supplemental material for this article may be found at http://ec.asm.org/.

REFERENCES

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