Genome-Wide Screening of Saccharomyces cerevisiae To Identify Genes Required for Antibiotic Insusceptibility of Eukaryotes

The adverse reactions provoked by many antibiotics in humansare well documented but are generally poorly understood at themolecular level. To elucidate potential genetic defects thatcould give rise to susceptibility to prokaryote-specific antibioticsin eukaryotes, we undertook genome-wide screens using the yeastSaccharomyces cerevisiae as a model of eukaryotes; our previouswork with a small number of yeast mutants revealed some specificgene functions required for oxytetracycline resistance. Here,the complete yeast deletion strain collection was tested forgrowth in the presence of a range of antibiotics. The sensitivitiesof mutants revealed by these screens were validated in independenttests. None of the $~$ 4,800 defined deletion strains tested werefound to be sensitive to amoxicillin, penicillin G, rifampin,or vancomycin. However, two of the yeast mutants were tetracyclinesensitive and four were oxytetracycline sensitive; encompassedamong the latter were mutants carrying deletions in the samegenes that we had characterized previously. Seventeen deletionstrains were found to exhibit growth defects in the presenceof gentamicin, with MICs for the strains being as low as 32µg ml^-1 (the wild type exhibited no growth defects atany gentamicin concentration tested up to 512 µg ml^-1).Strikingly, 11 of the strains that were most sensitive to gentamicincarried deletions in genes whose products are all involved invarious aspects of vacuolar and Golgi complex (or endoplasmicreticulum) function. Therefore, these and analogous organelles,which are also the principal sites of gentamicin localizationin human cells, appear to be essential for normal resistanceto gentamicin in eukaryotes. The approach and data describedhere offer a new route to gaining insight into the potentialgenetic bases of antibiotic insusceptibilities in eukaryotes.

INTRODUCTION

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Introduction

To be effective as chemotherapeutic agents, antibiotics notonly should inhibit target microorganisms but also should notexert adverse effects on host organisms. Thus, the well-documentedspread of antibiotic resistance among pathogenic microorganismsis one important obstacle to effective antibiotic treatment(12). At the same time, adverse reactions to antibiotics arisein about 5 to 10% of patients to whom they are prescribed (3),and this further erodes the perception that many antibioticsare "magic bullets." Adverse reactions range from mild effectssuch as hypersensitivity, rashes, and gastrointestinal intoleranceto more serious complications such as toxicity to various organsand in some cases death (3, 20). Despite this incidence of adverseresponses to antibiotics among humans, the underlying causesof these effects at the molecular level are in many cases unknown(unlike the causes of bacterial resistance). This is an importantgap in our knowledge, as a clearer understanding of adverseeffects is a prerequisite if these are to be averted in thefuture. For example, if it was possible to predict (e.g., genetically)which patients might be susceptible to the adverse effects ofparticular antibiotics, then it should be possible to tailorantibiotic prescriptions accordingly (or develop modified antibioticswith lower levels of toxicity), so improving the overall efficacyof antibiotic therapy.

In order to be able to use genetic tools to predict potentialdrug susceptibilities in humans, it is first necessary to haveestablished any genetic bases for such conditions. However,as for inheritable susceptibilities to diseases, this remainsa challenging task, despite the recent availability of the humangenome sequence and improved techniques for single-nucleotide-polymorphismanalysis (16). The yeast Saccharomyces cerevisiae provides arelatively simple model system for eukaryotes that is very wellunderstood genetically and has been at the forefront of recentadvances in functional genomics technologies (6, 9, 14, 21).Moreover, there is remarkable conservation of gene functionsbetween the yeast and humans. For example, greater than 40%of single-gene determinants of human heritable diseases haveyeast homologs (5). Therefore, with S. cerevisiae it is possibleto gain valuable insight into eukaryotic cell biology and geneticsthat would be very difficult to accomplish with higher eukaryoticcell systems.

Using S. cerevisiae, we recently identified antioxidant functionsthat were essential for normal resistance to certain tetracyclineantibiotics (1, 2). Whereas the growth of wild-type S. cerevisiaewas unaffected at concentrations of tetracyclines close to theantibiotics' limits of solubility, deletion mutants deficientin Sod1p (Cu,Zn superoxide dismutase), Ctr1p (high-affinityCu transporter), and Mac1p (metalloregulatory transcriptionfactor) exhibited marked sensitivities to oxytetracycline anddoxycycline. These susceptibilities were shown to be due toa novel mode of oxytetracycline and doxycycline action thatwas dependent on oxidative damage and that is normally suppressedin cells by Sod1p and copper (1, 2). It was considered likelythat the insusceptibilities of humans to these antibiotics maywell also rely on these functions (1).

To build on the findings described above and broaden the workbeyond antioxidant gene functions alone, in this study we presentthe results of the first genome-wide screen for eukaryotic genefunctions that may be required to avert the adverse effectsof antibiotics. This is possible with the availability of thecomplete yeast deletion strain collection, which has been generatedthrough an international effort to delete systematically everyyeast open reading frame (21). We screened the collection witha range of antibiotics and report here several new gene functionsthat are required for normal antibiotic resistance in this yeastmodel of eukaryotes. In particular, the data reveal that normalvacuolar and Golgi complex functions are essential for insusceptibilityto the aminoglycoside antibiotic gentamicin in eukaryotes.

MATERIALS AND METHODS

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Materials and Methods

S. cerevisiae deletion strain collection. The S. cerevisiae deletion strain collection, constructed inthe BY4741 background (MATa his3 ${Delta}$ 1 leu2 ${Delta}$ 0 met15 ${Delta}$ 0 ura3 ${Delta}$ 0), was obtainedfrom Euroscarf (Frankfurt, Germany) in a 96-well format. Eachdeletion strain (of a total of $~$ 4,800 in the library) carriesa defined deletion of a characterized or putative open readingframe, in which the open reading frame has been replaced withthe kanMX4 marker by PCR (21). Strains were routinely storedin the 96-well format at -80°C in YEPD medium (8): 2% (wt/vol)bacteriological peptone (Oxoid), 1% yeast extract (Oxoid), 2%glucose supplemented with 15% (vol/vol) glycerol, and 150 µgof Geneticin (G418; Sigma) ml^-1 for selection.

Screening for antibiotic-sensitive deletion mutants. Deletion strains were inoculated from frozen stocks into Geneticin-supplementedYEPD medium in 96-well plates by using a 96-pin tool (1 to 2µl of inoculum per pin). The strains were cultured for2 days at 30°C and then replica inoculated onto YEPD agarsupplemented or not supplemented with the appropriate antibioticat 256 µg ml^-1. All antibiotics were purchased from Sigma.The plates were incubated at 30°C for 3 to 5 days beforethey were examined for growth. A positive result was scoredwhen the growth of a mutant in antibiotic-supplemented plateswas visibly diminished compared to its growth in control plates(e.g., see Fig. 1). The functions of genes that were deletedin mutants of interest were derived from databases on the WorldWide Web (http://genome-www.stanford.edu and http://mips.gsf.de/proj/yeast).

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FIG. 1. Screening for gentamicin sensitivity using the S. cerevisiae deletion strain collection. Strains were cultured in liquid YEPD medium in a 96-well format and replica inoculated onto YEPD agar supplemented with gentamicin (256 µg ml^-1). The results are for 1 strain set (strain set 4_3; Euroscarf) of a total of 76 strain sets examined with each antibiotic after incubation for 3 days at 30°C. Circles highlight strains that exhibited slight (position C12; gcs1 ${Delta}$ ) and strong (position G6; luv1 ${Delta}$ ) sensitivities to gentamicin relative to their growth on the control plate lacking gentamicin. The gentamicin sensitivities of these strains were subsequently validated (Fig. 2 and Table 1). Empty inocula on the control plate correspond to positions at which essential open reading frames were originally deleted, producing nonviable mutants.

Validation of antibiotic sensitivity. The antibiotic sensitivities of mutant strains of interest,identified during screening of the deletion strain collection(see above), were validated by spotting tests. Strains of interestwere cultured in 96-well plates under the same conditions describedabove and then adjusted to an optical density at 600 nm (OD₆₀₀)of $~$ 0.01 with sterile water. Aliquots (4 µl) were spottedonto plates supplemented with the appropriate antibiotic, suppliedat the same concentration used for initial screening (256 µgml^-1). Growth was examined after incubation at 30°C for3 to 5 days. All plates were prepared and inoculated at leastin duplicate.

Determination of MICs. Strains of interest were cultured in 96-well plates under thesame conditions described above and then adjusted to an OD₆₀₀of $~$ 0.03 with sterile water. These cell suspensions were replicainoculated by using a 96-pin tool ( $~$ 400 to 500 cells per inoculum)to YEPD agar supplemented or not supplemented with antibiotics;antibiotics were supplied in twofold dilution series at finalconcentrations ranging between 1 and 512 µg ml^-1. Theplates were examined after incubation for 3 to 5 days at 30°C.The MICs for each sensitive mutant strain were determined asthe lowest antibiotic concentrations that resulted in full inhibitionof visible growth in replicate incubations.

RESULTS

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Results

Screening the S. cerevisiae deletion strain collection for antibiotic-sensitive mutants. To elucidate the gene functions that may be required for normalantibiotic resistance in eukaryotes, the full collection ofhaploid S. cerevisiae deletion strains was screened for growthin the presence of a range of test antibiotics. The antibioticsselected for this study (Table 1) are well characterized andare in use for human therapy, and most are also commonly associatedwith adverse effects in humans (3, 10). It was considered worthwhileto include two tetracycline antibiotics in the study since theoxytetracycline-sensitive mutants that we identified previouslywere tetracycline resistant (1, 2), indicating that these similarantibiotics can exert different effects on eukaryotic cells.

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TABLE 1. Numerical breakdown of strains of interest identified during screening of the deletion strain collection and subsequent validation

It was confirmed in preliminary experiments that the growthof wild-type S. cerevisiae was unaffected by each of the testantibiotics at concentrations up to 512 µg ml^-1 (the highestconcentration tested). To screen for antibiotic-sensitive mutants,256 µg ml^-1 was used as the test antibiotic concentration.Putative antibiotic-sensitive yeast mutants were identifiedin the screens outlined in the Materials and Methods. Thesemutants exhibited various degrees of diminished growth in thepresence of antibiotic compared with the growth in the controlincubations lacking antibiotic. In this way, screens with oxytetracycline(12 strains) and gentamicin (19 strains) yielded the greatestnumber of putative sensitive mutants (Table 1). Furthermore,many of the putative gentamicin-sensitive strains exhibitedcomplete inhibition of growth in the presence of gentamicin(for example, see Fig. 1). In contrast, only two putative rifampin-sensitivemutants and two putative vancomycin-resistant mutants were identified,and these strains still exhibited some (albeit apparently diminished)growth in the presence of the antibiotics (data not shown).A total of 61 putative antibiotic-sensitive mutants were identifiedin the screens with the seven test antibiotics (Table 1).

Validation of antibiotic sensitivity. Since $~$ 4,800 different strains were involved in the screens describedabove, it was not feasible to standardize conditions (e.g.,cell densities) rigorously during screening. Therefore, to validatethe antibiotic sensitivities of the strains of interest identifiedabove, organisms were applied to agar supplemented with theappropriate antibiotic (at 256 µg ml^-1) as spots of standardizedcell density. These 4-µl spots also diffused further inthe agar than the smaller inocula that were necessary for thescreening tests (described above), thereby giving a better resolutionof individual cell colonies in this case and a greater sensitivityof detection (Fig. 2B). Of the 61 putative sensitive mutantsidentified in the screens described above, 23 strains were confirmedto be sensitive when retested with the relevant antibiotic underthese more uniform conditions. None of the putative amoxicillin-,penicillin G-, rifampin-, or vancomycin-sensitive mutants identifiedin the screens were found to exhibit genuine sensitivities tothese antibiotics. In contrast, 17 of the 19 putative gentamicin-sensitivestrains were confirmed to be gentamicin sensitive in spottingtests (Fig. 2A and B). The growth of each of these 17 strainswas completely or almost completely inhibited by gentamicinat 256 µg ml^-1, with the exception of S. cerevisiae chs1 ${Delta}$ ,which did grow in the presence of gentamicin at this concentration,but with a diminished colony density (Fig. 2B). Strikingly,11 of the 17 gentamicin-sensitive mutants were defective ingene functions that are involved directly with organellar proteinsorting or processing (Table 2). These included genes importantfor Golgi complex or endoplasmic reticulum (ER) functions (e.g.,CAX4, GCS1, MNN9, and SAC1) as well as several VPS (PEP) genesthat are involved specifically with vacuolar protein sortingor biogenesis (PEP3, PEP5, VPS15, VPS16, VPS33, VPS34). Othergentamicin-sensitive mutants were defective in various othertypes of function or had no characterized function.

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FIG. 2. Validation and quantification of antibiotic (gentamicin) sensitivity. All 19 putative gentamicin-sensitive strains identified during initial screening of the deletion strain collection were tested quantitatively for antibiotic sensitivity. (A) Grid of putative gentamicin-sensitive mutants identified from screening (WT, wild type). (B) Mutants of interest were cultured in liquid YEPD medium and adjusted to an OD₆₀₀ of $~$ 0.01 before they were spotted (4 µl) onto unsupplemented and gentamicin-supplemented YEPD agar (the strains in the grid correspond to those shown in panel A). (C) MIC determination. Mutants were cultured as described above for panel B and adjusted to an OD₆₀₀ of $~$ 0.03 before replica inoculation with a 1- to 2-µl pin tool onto YEPD agar supplemented with a range of gentamicin concentrations (1 to 512 µg ml^-1); the results obtained with 0, 64, and 512 µg of gentamicin ml^-1 are shown. All plates were incubated for 3 days at 30°C before examination. Typical results from one of several replicates are shown.

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TABLE 2. Gentamicin-sensitive S. cerevisiae mutants

Mutants that appeared to be sensitive to the tetracycline antibioticsduring screening were also tested by spotting. From a totalof 21 putative sensitive strains, 4 were confirmed to be oxytetracyclinesensitive and 2 were confirmed to be tetracycline sensitive;the last 2 strains (erg28 ${Delta}$ , adh1 ${Delta}$ ) were also among the oxytetracycline-sensitivestrains (Table 3). The two mutants that were confirmed to beoxytetracycline sensitive but not tetracycline sensitive carrieddeletions in the SOD1 and MAC1 genes, in keeping with our previousfindings (1, 2). However, the ctr1 ${Delta}$ mutant described above wasnot among those identified by screening for oxytetracyclinesensitivity in this study.

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TABLE 3. Tetracycline- and oxytetracycline-sensitive S. cerevisiae mutants

MICs for antibiotic-sensitive yeast mutants. To provide a more quantitative analysis of antibiotic sensitivity,the MICs were determined as outlined in Materials and Methodsfor each of the mutants that were confirmed to be antibioticsensitive in the spotting tests (described above). GentamicinMICs were between 32 and 64 µg ml^-1 for the majority ofthe gentamicin-sensitive mutants, and these included all ofthe VPS mutants (Fig. 2C; Table 2). Gentamicin MICs were greaterthan 512 µg ml^-1 for two mutants (gcs1 ${Delta}$ and chs1 ${Delta}$ ): theywere not inhibited fully at this concentration, the highestconcentration tested. Note that the growth of these mutantsin the MIC tests could be attributable to the outgrowth of onlyone or two cells, as suggested by the spotting test for thegcs1 ${Delta}$ mutant, in which just one colony was apparent in the presenceof gentamicin at 256 µg ml^-1 (Fig. 2B). Overall, the gentamicinsensitivities of the test strains (Table 2) were more markedthan the oxytetracycline or tetracycline sensitivities, withthe MICs of the last two antibiotics ranging between 128 and512 µg ml^-1 (Table 3). It should be noted that for mostof the antibiotic-sensitive mutants, some degree of growth inhibitionwas evident at concentrations considerably lower than the MICsfor full inhibition (data not shown).

DISCUSSION

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Discussion

This is the first study in which a eukaryotic genome has beenscreened to identify genes that are required for normal resistanceto antibiotics in eukaryotes. This was possible with the yeastmodel thanks to the strides in functional genomics technologiesfor this organism that have arisen since the completion of itsgenome sequence in 1996. In particular, we exploited the yeastdeletion strain collection, which provides an outstanding resourcefor addressing biological questions such as this (7, 15, 21).

The molecular mechanisms underlying the broad range of adverseeffects that can arise during antibiotic administration areunknown in many cases. Our previous demonstration that threenonessential antioxidant genes in yeast (which have human homologs)are essential for resistance to certain tetracyclines was consistentwith the argument that susceptibility to adverse effects ofantibiotics, like susceptibility to many diseases, can be determinedgenetically (1, 2). There are already certain known examplesof genetically determined antibiotic susceptibility in humans.Thus, patients deficient in the enzyme glucose-6-phosphate dehydrogenasecan develop acute hemolysis when they are prescribed sulfonamidesor certain other antibiotics (3). In this study, we have identified21 new gene functions that are required for normal resistanceof the yeast model of eukaryotes to prokaryote-specific antibiotics,in particular, gentamicin. The same or similar functions inhumans may well also be required for insusceptibility to thesame antibiotics.

Since none of the $~$ 4,800 yeast mutants exhibited susceptibilityto amoxicillin, penicillin G, rifampin, or vancomycin, it seemsless likely that the common adverse reactions that these antibioticsmay elicit (3) are dependent on the defective activities ofspecific gene products. However, our results do not fully ruleout that possibility since susceptibility to these antibioticscould be (i) a result of polygenic traits, which are much moredifficult to elucidate; (ii) dependent on human gene functionsor processes that do not occur in yeast; (iii) dependent onpartial loss (e.g., due to heterozygosity) of essential genefunctions which are not encompassed in the haploid yeast deletionstrain collection (due to lethality); (iv) manifested in subtlerways that are not detectable as inhibition of growth; or (v)influenced by any differences in antibiotic uptake between mammalianand yeast cells. In addition, while our screens erred on theside of saturation—more than half of the mutants thatwere scored as potentially sensitive from the initial screensproved to have normal resistance when they were examined further—somemoderately sensitive mutants may have been missed. For example,only two (sod1 ${Delta}$ and mac1 ${Delta}$ ) of the three previously identifiedoxytetracycline-sensitive mutants were detected here (the screenswere performed blind). However, the oxytetracycline MICs forthese mutants were high at 512 µg ml^-1, and the thirdmutant, ctr1 ${Delta}$ , appeared to be slightly less sensitive than thesod1 ${Delta}$ and mac1 ${Delta}$ mutants in the previous study (1). Thus, it canbe estimated that an MIC of $~$ 512 µg ml^-1 is the approximatelimit above which any slight sensitivity may, in many cases,not have been detected by our screening methodology, and thisis borne out by the data in Tables 2 and 3. Of course, the antibioticconcentration used here for screening (256 µg ml^-1) couldbe raised or lowered to adjust the sensitivity of the screens,although the potential relevance to adverse reactions of genedefects that yield antibiotic MICs greater than 512 µgml^-1 is questionable: the peak concentrations of most antibioticsin the plasma or serum of treated patients are typically lessthan about 10 to 20 µg ml^-1 (18). Nonetheless, it shouldbe noted that even though the oxytetracycline MIC for the sod1 ${Delta}$ mutant, for example, was high ( $~$ 512 µg ml^-1), some inhibitionof sod1 ${Delta}$ mutant growth is still readily evident at 100 µgml^-1 and can be detectable in the presence of oxytetracyclineat a concentration as low as 10 µg ml^-1 (2). Inhibitoryeffects commencing at antibiotic concentrations lower than theMICs presented for full inhibition were also detected againstmost other mutants of interest in this study.

The screen with the aminoglycoside antibiotic gentamicin yieldedthe greatest number of sensitive yeast mutants. Gentamicin isan inhibitor of bacterial protein synthesis, but it also haswell-documented nephrotoxic and ototoxic side effects in humans.The molecular bases for these adverse effects are not yet fullyunderstood (4, 11), although a mutation in a mitochondrial rRNAgene has been linked to familial aminoglycoside ototoxicity(13). It is known that gentamicin is internalized through endocytosisin mammalian cells and it becomes localized principally to endosomaland lysosomal vacuoles as well as to the Golgi complex (17,19). Therefore, it is particularly interesting that most ofthe gentamicin-sensitive strains identified in this study weredefective in genes associated with various aspects of vacuolarand Golgi complex (or ER) function. Thus, normal operation ofthese organelles is required for the insusceptibility of yeastto gentamicin. This evidence supports a previously suggestedhypothesis that the normal localization of gentamicin in eukaryoticsubcellular compartments such as lysosomes may serve to divertthe antibiotic from more critical cellular targets, so helpingto avert gentamicin toxicity (11). Presumably, patients withpotential defects in functions analogous to those identifiedhere (i.e., the vacuolar and Golgi complex functions as wellas certain others listed in Table 2) could be at a high riskof suffering gentamicin toxicity, and our approach has now pavedthe way for this novel hypothesis to be tested in a mammaliansystem. It is also of interest that one of the gentamicin-sensitiveyeast mutants identified here carried a deletion in a putativeopen reading frame (YDR455c) with no previously characterizedfunction. Assigning functions to such open reading frames isone of the major challenges in the postgenomics era. By association,there seems a good chance from our results that YDR455c mayencode a product that is involved in vacuolar or Golgi complexfunction.

As well as the antioxidant functions that we previously showedare required for oxytetracycline insusceptibility, two furthergenes required for both oxytetracycline and tetracycline insusceptibility,ADH1 and ERG28, were identified here. These two genes apparentlyplayed a more important role in antibiotic insusceptibilitysince the oxytetracycline or tetracycline MIC for the relevantdeletion mutant was lower (128 µg ml^-1). Erg28p is involvedin ergosterol biosynthesis in yeast, although its precise roleis unknown (9). We hypothesized that a possible defective membranefunction in an erg28 ${Delta}$ mutant could allow more tetracycline toenter cells. However, in preliminary experiments we found noevidence for elevated levels of tetracycline uptake in thismutant compared to those in wild-type yeast (data not shown).It is interesting that only ERG28 and none of the other yeastERG genes appeared to be required for tetracycline resistance,and this difference could help pinpoint the role of ERG28 inconferring tetracycline resistance as the molecular functionof Erg28p becomes unraveled in the future. Moreover, such knowledgeshould also provide the opportunity to determine whether anyfunctions equivalent to that of Erg28 involved in human cholesterolbiosynthesis could be important for human responses to tetracyclineantibiotics.

In conclusion, by exploiting the yeast model we have establishedthe first data sets from genome-wide screens to catalogue eukaryoticgenes that are required for antibiotic insusceptibility. Thedata obtained for the tetracyclines and gentamicin, in particular,are consistent with models in which the susceptibilities ofcertain individuals to the well-documented adverse effects ofthese antibiotics could have a genetic basis. Our data providethe necessary information with which such hypotheses can nowbe tested in higher systems. They also give new insight intothe mechanisms by which these prokaryote-specific antibioticsmay be processed in eukaryotic cells.

ACKNOWLEDGMENTS

The support of the National Institutes of Health (grant R01GM57945) and the University of Nottingham Research Committeeis gratefully acknowledged.

FOOTNOTES

* Corresponding author. Mailing address: School of Life and Environmental Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom. Phone: 44-115-9513315. Fax: 44-115-9513251. E-mail: Simon.Avery{at}nottingham.ac.uk.

REFERENCES

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