synergy of the antibiotic colistin with echinocandin antifungals in

Synergy of the antibiotic colistin with echinocandin antifungalsin Candida species

Ute Zeidler1,2†, Marie-Elisabeth Bougnoux1–3, Alexandru Lupan4,5‡, Olivier Helynck4,5, Antonia Doyen6,7,Zacarias Garcia8, Natacha Sertour1,2, Cecile Clavaud9§, Helene Munier-Lehmann4,5}, Cosmin Saveanu6,7}

and Christophe d’Enfert1,2*

1Institut Pasteur, Unite Biologie et Pathogenicite Fongiques, Departement Genomes et Genetique, F-75015 Paris, France; 2INRA, USC2019, F-75015 Paris, France; 3Service de Microbiologie, Hopital Necker Enfants Malades, Universite Paris-Descartes, F-75015 Paris, France;

4Institut Pasteur, Unite de Chimie et Biocatalyse, Departement de Biologie Structurale et Chimie, F-75015 Paris, France; 5CNRS, UMR3523, F-75015 Paris, France; 6Institut Pasteur, Unite de Genetique des Interactions Macromoleculaires, Departement Genomes et

Genetique, F-75015 Paris, France; 7CNRS, UMR3525, F-75015 Paris, France; 8Institut Pasteur, Unite Dynamiques des Reponses Immunes,Departement d’Immunologie, F-75015 Paris, France; 9Institut Pasteur, Unite des Aspergillus, Departement Parasitologie et Mycologie,

F-75015 Paris, France

*Corresponding author. Unite Biologie et Pathogenicite Fongiques—INRA USC 2019, Institut Pasteur, 25-28 rue du Docteur Roux, 75724 Paris cedex 15,France. Tel: +33-140613257; Fax: +33-145688938; E-mail: [email protected]

†Present address: Sandoz, Austria.‡Present address: Chemistry Department, Faculty of Chemistry, Babes Bolyai University, Cluj Napoca, Romania.

§Present address: L’Oreal, France.}These authors contributed equally to this work.

Received 18 September 2012; returned 26 October 2012; revised 10 December 2012; accepted 19 December 2012

Objectives: Candida albicans is the most prevalent fungal pathogen of humans, causing a wide range of infec-tions from harmless superficial to severe systemic infections. Improvement of the antifungal arsenal is neededsince existing antifungals can be associated with limited efficacy, toxicity and antifungal resistance. Here weaimed to identify compounds that act synergistically with echinocandin antifungals and that could contributeto a faster reduction of the fungal burden.

Methods: A total of 38758 compounds were tested for their ability to act synergistically with aminocandin, ab-1,3-glucan synthase inhibitor of the echinocandin family of antifungals. The synergy between echinocandinsand an identified hit was studied with chemogenomic screens and testing of individual Saccharomyces cerevi-siae and C. albicans mutant strains.

Results: We found that colistin, an antibiotic that targets membranes in Gram-negative bacteria, is synergisticwith drugs of the echinocandin family against all Candida species tested. The combination of colistin and ami-nocandin led to faster and increased permeabilization of C. albicans cells than either colistin or aminocandinalone. Echinocandin susceptibility was a prerequisite to be able to observe the synergy. A large-scale screenfor genes involved in natural resistance of yeast cells to low doses of the drugs, alone or in combination, iden-tified efficient sphingolipid and chitin biosynthesis as necessary to protect S. cerevisiae and C. albicans cellsagainst the antifungal combination.

Conclusions: These results suggest that echinocandin-mediated weakening of the cell wall facilitates colistintargeting of fungal membranes, which in turn reinforces the antifungal activity of echinocandins.

Keywords: Candida albicans, polymyxin, combination therapy, caspofungin, sphingolipids

IntroductionInvasive fungal infections have emerged as a major cause of in-fectious complications in hospitalized patients with seriousunderlying diseases, especially those undergoing treatment ofhaematological malignancies or hospitalized in intensive care

units.1,2 Recent and comprehensive epidemiological surveyshave reported that invasive candidiasis accounts for �75% ofall invasive fungal infections,3,4 and Candida species are now re-currently found among the five leading causes of nosocomialbloodstream infections.5,6 Invasive candidiasis remains

# The Author 2013. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.For Permissions, please e-mail: [email protected]

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associated with a significant mortality, in the range of 40%,5,7

even in patients receiving antifungals of the echinocandinclass. However, recent studies suggest that early antifungaltreatment and the use of echinocandins might be optimal forpatient survival and therapeutic success.1,8 – 10 In this respect, itcan be postulated that compounds that enhance the efficacyof echinocandins could contribute to a faster reduction of thefungal burden and have the potential to provide a breakthroughin the management of invasive candidiasis.

The main objective of the present study was to identify com-pounds acting synergistically with echinocandins, i.e. compoundsthat when combined with echinocandins would have a greaterantifungal effect than the sum of the effects of the two individ-ual compounds.11 We have screened a collection of 38758 com-pounds in order to identify those that would increase theantifungal activity of aminocandin (IP960; HMR3270;NXL201),12 a non-marketed echinocandin to which we hadeasy access. We found that the antibiotic colistin showed syner-gism with aminocandin and another echinocandin, caspofun-gin,13 in all Candida species tested. Colistin (polymyxin E) is amember of the polymyxin family of antibiotics, which show effi-cacy against Gram-negative bacteria.14 Polymyxins are cationiccyclic heptapeptides with a hydrophobic tail that interacts withthe bacterial cytoplasmic membrane, therefore changing its per-meability and triggering cell death.14 Notably, weak antifungalactivity of colistin and polymyxin B has been previouslyreported.15,16 Moreover, polymyxin B was shown to synergizewith polyenes and azole antifungals and it has been proposedthat azole-induced changes in the ergosterol content of fungalmembranes might enhance the pore-forming activity of poly-myxins.17 – 19 In contrast to azoles and polyenes, which impactfungal membranes, echinocandins are lipopeptides, which targetcell wall biosynthesis through non-competitive inhibition of b-1,3-glucan synthase, the product of the FKS1 gene in Candida albi-cans.20 Moreover, echinocandins are proposed to act at theouter face of the plasma membrane,21 making it unlikely thattheir synergy with colistin has the same basis as that observedwith azoles. Hence, we have further investigated the synergybetween echinocandins and colistin with chemogenomic screensand by testing individual mutant strains. Our data suggest thatechinocandin-mediated alteration of the cell wall might facilitatecolistin’s access to and perturbation of fungal membranes, andthis in turn facilitates echinocandin activity.

Materials and methods

Strains and mediaClinical or reference isolates of C. albicans (SC531422), clinical isolatesof Candida lusitaniae (NEM_7104301), Candida parapsilosis (NEM_7103546),Candida glabrata (NEM_7118432), Candida tropicalis (NEM_7114236),Candida krusei (ATCC 6258; LGC Standards, Molsheim, France) and Yarro-wia lipolytica (NEM_7115511) and laboratory isolates of S. cerevisiae(BY4741, BY474323) were used in this study. Fluconazole- andcaspofungin-resistant strains used in this study are listed in Table 1 andwere obtained from D. Perlin (PHRI, New Jersey, USA) and D. Sanglard(CHUV, Lausanne, Switzerland). Strains were routinely grown at 308C inyeast/peptone/dextrose (YPD) medium (1% yeast extract, 2% peptoneand 2% glucose) or synthetic dextrose (SD) minimal medium [0.67%yeast nitrogen base without amino acids (Difco) and 2% glucose] and2% agar for solid media. RPMI 1640 GlutaMAX medium (Invitrogen)

buffered with 50 mM HEPES, pH 7, was used for screening compoundsthat synergize with aminocandin and for chequerboard assays. Alterna-tively, RPMI 1640 GlutaMAX medium buffered with 0.165 M MOPS wasused. Aminocandin (a kind gift from Dr Dominique Lebeller), caspofungin(MSD), colistin sulphate (Sigma-Aldrich, Saint-Quentin Fallavier, France),colistin sodium methanesulfonate (Sigma-Aldrich), hygromycin B(Sigma-Aldrich), nourseothricin (Werner BioAgents, Jena, Germany) and5-fluorocytosine (Sigma-Aldrich) were dissolved in H2O. Fluconazole(Sigma-Aldrich) and amphotericin B (Sigma-Aldrich) were dissolved inDMSO.

Screening of a chemical library for synergy withaminocandinA large-scale screen for antifungal synergy between a subinhibitory con-centration of aminocandin and a collection of 38758 chemicals obtainedfrom Prestwick, ChemDiv and the French ‘Chimiotheque Nationale’24 wasperformed in 96-well plates using a Tecan Freedom EVO 200 platform.C. albicans wild-type cells of strain SC5314 were grown in the presenceof 0.0156 mg/L aminocandin and 50 mM (on average) of each testedcompound (dissolved in DMSO) in 100 mL of RPMI medium, startingfrom an OD600 of 0.01 (where OD stands for optical density). Columns1 and 12 were used for controls: DMSO was used as a negative controlto define growth of the cells without compound; 10 mg/L of aminocandinwas used as a positive control to kill all cells. Cells were incubated for 24 hat 308C and cell viability was measured using a resazurin assay25 by theaddition of resazurin sodium salt to a final concentration of 100 mg/L.Plates were incubated for 2 h at room temperature. While resazurin isnaturally blue, its oxidation is associated with a change to red colour.To measure the colour change, a dual-wavelength measurement wasperformed (reference wavelength 604 nm, measurement wavelength570 nm) using a Tecan Safire2 microplate reader. For each plate the Z′

factor26 was calculated and was above 0.55 (Figure S1A, available asSupplementary data at JAC Online). The data were normalized usingthe following formula: % viability¼ 100× (sample value – average valueof positive controls)/(average of negative controls – average of positivecontrols). Active compounds were defined as compounds causing ,1%viability. Chemical families were defined using clustering methodsbased on the similarity concept, using the Tanimoto coefficient and theK-modes clustering algorithm.27 – 29 The best compounds were orderedfrom the different libraries and validated using MIC assays.

MIC and chequerboard microdilution assaysIn order to define the antifungal activity of a compound, MIC assays wereperformed in RPMI 1640 GlutaMAX medium buffered with 50 mM HEPES.Compounds were diluted 2-fold in the concentration range 0.1 mg/L–50 mg/L and 1.3×105 cells/mL of the C. albicans wild-type strainSC5314 were added. The MIC assays were incubated for 24 h at 308Cand viability of the cells was measured using the resazurin assay asdescribed above.

In order to assess the synergy between two compounds, chequer-board microdilution assays were performed in RPMI 1640 GlutaMAXmedium using 96-well plates.30 The tested concentrations were0.01–1 mg/L for aminocandin and caspofungin, 0.02–1.25 mg/L forfluconazole, 0.4–2.5 mg/L for amphotericin B, 0.008–0.5 mg/L for5-fluorocytosine, 8–500 mg/L for hygromycin B, 0.08–5 mg/L for nour-seothricin and 0.4–25 mg/L for colistin. The starting inoculum was2.5×103 cells/mL. Chequerboards were incubated for 24 h at 308C andviability of the cells was measured using the resazurin assay as describedabove. The fractional inhibitory concentration index (FICI) was calculatedby using the formula: MIC of drug A tested in combination/MIC of drug Aalone+MIC of drug B tested in combination/MIC of drug B alone. Com-pounds qualified as synergistic when the FICI was ≤0.5.11

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Table 1. Yeast strains used in this study

Species Strain Type of strain Genotype Reference

C. albicans DPL1000 wild-type, parent of 20S, 22S and 36S D. PerlinC. albicans 20S caspofungin-resistant, Fks1S645F FKS1C1934T/FKS1C1934T 67C. albicans 22S caspofungin-resistant, Fks1S645Y FKS1C1934A/FKS1C1934A 67C. albicans 36S caspofungin-resistant, Fks1S645P FKS1T1933C/FKS1T1933C 67C. albicans M70 wild-type, parent of C42C. albicans C42 caspofungin-resistant, Fks1F641S FKS1/FKS1C. albicans DSY294 fluconazole-susceptible clinical strain TAC1-3/TAC1-4 68

ERG11-3/ERG11-4C. albicans DSY296 fluconazole-resistant TAC1-5/TAC1-5 ERG11-5/ERG11-5 68C. albicans DSY3987 fluconazole-resistant ura3D::FRT/ura3D::FRT TAC1-5/TAC1-5 ERG11-5/ERG11-5 RP10::CIp10 D. SanglardC. albicans DSY3988 fluconazole-resistant ura3D::FRT/ura3D::FRT tac1-5D::hisG/tac1-5D::hisG ERG11-5/ERG11-5 RP10::CIp10 D. SanglardC. albicans CAI4 Dura3::limm434/Dura3::limm434 69C. albicans TPIPT1-4 Dura3::limm434/Dura3::limm434 Dipt1::hisG/Dipt1::hisG 53C. albicans TPIPT1-5 Dura3::limm434/Dura3::limm434 Dipt1::hisG/Dipt1::hisG::IPT1-URA3 53C. albicans CACB3B-5 Dura3::limm434/1Dura3::limm434 Dchs3-2::hisG/Dchs3-3::hisG_URA3_hisG_URA3_hisG 56C. albicans SN152 arg4D/arg4D leu2D/leu2D his1D/his1D URA3/ura3D::limm434 IRO1/iro1D::limm434 70C. albicans SN250_ARGplus his1D/his1D leu2D::CdHIS1/leu2D::CmLEU2 arg4D::CaARG4/arg4D URA3/ura3D::imm434

IRO1/iro1D::imm43454

C. albicans DCHS5_ARGplus SN152 chs5D::CmLEU2/chs5D::CdHIS1 arg4D::CaARG4 54C. albicans DCHS7_ARGplus SN152 chs7D::CmLEU2/chs7D::CdHIS1 arg4D::CaARG4 54C. albicans DCSG2_ARGplus SN152 csg2D::CmLEU2/csg2D::CdHIS1 arg4D::CaARG4 54C. albicans S11.A14 mit1-D::hisG/mit1-D::hisG-URA3-hisG 52S. cerevisiae BY4741 MATa his3D1 leu2D0 met15D0 ura3D0 23S. cerevisiae BY4743 MATa his3D1 leu2D0 met15D0 ura3D0/MATa his3D1 leu2D0 met15D0 ura3D0 23S. cerevisiae surD BY4741 sur1DKanMX4 31S. cerevisiae sur1D het BY4743 sur1DKanMX4/SUR1 InvitrogenS. cerevisiae csg2D BY4741 csg2DKanMX4 31S. cerevisiae gef1D BY4741 gef1DKanMX4 31S. cerevisiae erg6D BY4741 erg6DKanMX4 31S. cerevisiae vps74D BY4741 vps74DKanMX4 31S. cerevisiae trp1D BY4741 trp1DKanMX4 31S. cerevisiae chs3D BY4741 chs3DKanMX4 31S. cerevisiae chs5D BY4741 chs5DKanMX4 31S. cerevisiae chs6D BY4741 chs6DKanMX4 31S. cerevisiae chs7D BY4741 chs7DKanMX4 31S. cerevisiae skt5D BY4741 skt5DKanMX4 31S. cerevisiae ipt1Dhet BY4743 ipt1DKanMX4/IPT1 InvitrogenS. cerevisiae sur4Dhet BY4743 sur4DKanMX4/SUR4 Invitrogen

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Fitness assay with barcoded S. cerevisiaeknock-out collectionsFitness assays were performed with a homozygous and a heterozygousS. cerevisiae knock-out collection: 4885 S. cerevisiae mutants from thesystematic deletion collection of Giaever et al. (strain BY474131) weregrown in individual wells in 96 deep-well plates at 308C for 2 days inYPD medium and then pooled, and aliquots were stored at 2808C. Adiploid heterozygous mutant collection (Invitrogen 95401.H4Pool; LifeTechnologies, Saint Aubin, France) consisting of 5936 mutants wasgrown for 15 generations in YPD medium and then pooled, and aliquotswere stored at 2808C.

For each chemogenomics experiment, aliquots were thawed and cellswere allowed to recover in YPD medium for 8 h at 308C with shaking.Pools of 3×106 cells of the homozygous mutants were transferred to100 mL flasks in YPD medium and cells were grown for about 11 genera-tions at 308C with (i) no compound, (ii) 0.00125 mg/L aminocandin, (iii)20 mg/L colistin or (iv) the combination of 0.00125 mg/L aminocandinand 5 mg/L colistin. The pool of heterozygous mutants was grown with(i) no compound, (ii) 0.002 mg/L aminocandin, (iii) 30 mg/L colistin or(iv) the combination of 0.002 mg/L aminocandin or 10 mg/L colistin.

Subsequently, 7×107 cells were recovered by centrifugation, washedonce with cold sterile water and stored at 2808C until processing. Thegrowth of individual strains in the different cultures was determined byamplifying, labelling and hybridizing the barcodes on custom barcodemicroarrays (Agilent G2509F—AMADID N8026035; Agilent, Massy,France). Briefly, genomic DNA from the collected cells was extractedwith phenol–chloroform by extensive vortexing in the presence of glassbeads (size 425–600 nm). Primers U1 and KU (for primer sequencessee Table S1, available as Supplementary data at JAC Online) wereused to amplify the upstream barcodes and primers KD and D1 toamplify the downstream barcodes. Twenty-five cycles of PCR with anannealing temperature of 508C were used. The resulting PCR productswere verified by electrophoresis on an agarose gel and used in a labellingPCR with the Cy3 or Cy5 5′-labelled oligonucleotides U2comp for the up-stream tags and D2comp for the downstream tags and unlabelled U1and D1 as a control. Only 15 cycles of amplification were used in the la-belling step. The labelled PCR products were mixed and precipitated in thepresence of linear acrylamide and a mixture of complementary oligonu-cleotides (U1, D1, U2block, D2block) in 4-fold molar excess to avoidbinding of the fluorescently labelled oligonucleotides to the microarrayprobes. Hybridization was performed using the DIG Easy Hyb buffer(Roche Applied Science, Meylan, France), at 248C, overnight, in a rotatingAgilent hybridization chamber. The slides were washed in decreasingconcentrations of SSPE buffer [10 mM potassium phosphate (pH 7.4),150 mM NaCl, 0.5 mM EDTA and 0.05% (w/v) Triton X100] down to0.2× SSPE, dried and treated immediately with the Agilent Stabilizationand Drying Solution to avoid ozone-induced degradation of the Cy5 fluor-ophore. Scanning was performed on a Genepix 4200AL scanner (Molecu-lar Devices, Saint Gregoire, France) and the images were analysed usingAxon Genepix Pro 7 (Molecular Devices). We filtered the data accordingto our previous estimates of the reliability of the microarray signal.32 Fil-tered data were normalized using the Loess algorithm (R package marrayincluded in Bioconductor33) separately for signals coming from upstreamor downstream barcodes. The average of the values for the upstreambarcode and the downstream barcode was calculated. The log2 of theratio between the signal obtained for a given mutant growing with andwithout drug was used as an estimate of the drug’s effect on thegrowth rate of the mutant.

Confirmation of selected S. cerevisiaeand C. albicans mutantsIn order to confirm the data of the fitness screen, we selected severalstrains of the homozygous and heterozygous deletion mutant collections

(Table 1) and retested them singly. The respective strains were streakedon YPD plates and single colonies were used to isolate genomic DNA ofthe strains using the Epicentre MasterPure Yeast DNA purification kit(Tebu-bio, Le Perray en Yvelines, France). PCRs were performed using oli-gonucleotides binding to the upstream region of the respective gene andon the KANMX4 cassette, which was used to delete the genes. Confirmedstrains, i.e. those harbouring a correctly integrated marker cassette, wereinoculated from an overnight culture to OD600 0.002 in YPD medium andgrown with (i) no compound, (ii) 0.00125 mg/L aminocandin, (iii) 20 mg/Lcolistin or (iv) the combination of 0.00125 mg/L aminocandin and 5 mg/Lcolistin overnight at 308C (16 h). Subsequently, the OD600 of the strainswas measured to determine the susceptibility of the respective strains.C. albicans mutant strains (Table 1) were tested using the same proced-ure except that cells were grown with (i) no compound, (ii) 0.00125 mg/Laminocandin, (iii) 30 mg/L colistin or (iv) the combination of 0.00125 mg/L aminocandin and 20 mg/L colistin.

Analysis of cell permeability using propidiumiodide (PI) stainingThe wild-type strain SC5314 (2×105 cfu/mL) was inoculated from anovernight culture in RPMI and grown for 1 h at 308C. Subsequently,cells were grown in RPMI with (i) no compound, (ii) 0.00125 mg/L amino-candin, (iii) 20 mg/L colistin and (iv) the combination of 0.00125 mg/Laminocandin and 20 mg/L colistin. After 10, 30, 50 and 70 min growthat 308C, 4×106 cells were harvested, washed with 1× PBS, resuspendedin 1× PBS and incubated for 3 min with 5 mg of PI (Sigma-Aldrich) in thedark. Subsequently, the cells were washed with 1× PBS, resuspended in1× PBS and analysed using a Miltenyi Biotec MACSQuant FACS machine(Miltenyi, Paris, France). Cells treated with 70% ethanol for 1 h wereused as positive control while untreated cells were used as negativecontrol. In order to analyse the data and to calculate the percentageof PI-stained cells, the software FlowJo version 7.6.5 (TreeStar Inc.,Oregon, USA) was used. The raw data were analysed using the Mantel–Haenszel x2 test.

Mouse model of systemic candidiasisAll animal experiments adhered to the EU Directive 86/609 on the ap-proximation of laws, regulations and administrative provisions ofMember States regarding the protection of animals used for experimentaland other scientific purposes, and to related national regulations. Allexperiments were performed according to the guidelines of the EuropeanConvention for the Protection of Vertebrate Animals used for Experimen-tal and Other Scientific Purposes (ETS No. 123). The protocol wasapproved by Institut Pasteur Health Center Animal Care Committee(protocol number 10.455). Recovery of organs was performed followingeuthanasia of animals, and all efforts were made to minimize suffering.

In vivo interaction between colistin and caspofungin was evaluatedusing a mouse model of systemic infection. Ten-week-old BALB/c mice(Charles River, L’Arbresle, France) were immunosuppressed with200 mg/kg endoxan (cyclophosphamide; Baxter, Maurepas, France)4 days before infection and were infected intravenously with 104 cfuper mouse of C. albicans strain SC5314. Treatment with antifungalswas performed intraperitoneally in a volume of 200 mL and started24 h after infection. For in vivo studies the less toxic prodrug colistinsodium methanesulfonate was used. Colistin sodium methanesulfonate(Sigma-Aldrich) was injected twice per day at a concentration of40 mg/kg34 while CancidasTM (caspofungin; MSD) was used at a concen-tration of 0.3 mg/kg and injected once per day.35 Mice were kept ingroups of 10 for each treatment. One group of mice was treated with co-listin alone, one with caspofungin alone, and one with caspofungin andcolistin, at the given concentrations. The group of untreated micereceived 200 mL of sterile saline intraperitoneally. At 3 days post-

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infection, mice were euthanized by CO2 asphyxiation. Kidneys of eachanimal were removed, weighed and homogenized in 1 mL of sterile0.15 M NaCl. Serial 10-fold dilutions of the homogenates were platedonto SD agar containing 50 mg/L ticarcillin and 10 mg/L gentamycinand incubated for 2 days at 308C. The fungal burden was determinedby counting the cfu and analysed using the software GraphPad Prism5.

Results

Colistin is synergistic with echinocandins

In order to find compounds acting synergistically with the echi-nocandin aminocandin,12 a high-throughput screen was per-formed on a total of 38758 molecules with a large diversity ofchemical backbones. The compound collection comprised16224 commercial compounds [1120 obtained from PrestwickChemical (http://www.prestwickchemical.com/index.php?pa=26)and 15104 obtained from ChemDiv (http://chemistryondemand.com/compound-library)] and 22534 compounds from the Frenchacademic chemical library (Chimiotheque Nationale,24 http://chimiotheque-nationale.enscm.fr/). Each compound at a finalconcentration around 50 mM was combined with a subinhibitorydose of aminocandin (0.0156 mg/L) and growth of C. albicansSC5314 was recorded after 24 h at 308C using a resazurinassay for measuring cell viability. Data were normalized as per-centage of viability relative to positive and negative controls.The Z′ factor,26 which is a statistical test to define the qualityof a large-scale screen, was .0.55 for all plates, indicating therobustness and reliability of the assay (Figure S1A, available asSupplementary data at JAC Online). Five-hundred-and-fifty-fivecompounds were identified whose combination with aminocan-din resulted in 100% growth inhibition (Figure S1B, availableas Supplementary data at JAC Online) and were clustered intochemical families. One-hundred-and-fifty-six compounds selectedwithin the different families were re-evaluated using MIC tests andchequerboard assays. Among these, 98 showed antifungal activityindependently of the addition of aminocandin while 58 had no sig-nificant antifungal activity on their own but acted synergisticallywith aminocandin (data not shown).

Colistin sulphate (referred to below as colistin) was chosen forfurther study since it exhibited the highest synergy with amino-candin in a chequerboard microdilution assay (FICI was 0.14 inRPMI–HEPES and 0.25 in RPMI-MOPS; Table 2). Colistin showedantifungal activity against C. albicans strain SC5314 at concen-trations ≥60 mg/L (data not shown). Colistin was also tested incombination with different classes of antifungals using chequer-board assays and the FICI values were calculated for each com-bination (Table 2). We found synergy with the other b-1,3-glucansynthase inhibitor, caspofungin (FICI 0.26). With both caspofun-gin and aminocandin, synergy was observed from a concentra-tion of 0.4 mg/L colistin and C. albicans growth was preventedat all echinocandin concentrations tested in combination witha concentration of ≥3.1 mg/L colistin (data not shown). Ourdata also indicated synergy of colistin with amphotericin B(FICI 0.27) and fluconazole (FICI 0.47), consistent with previouslypublished data that showed synergy between these antifungalsand polymyxin B.17,18 In contrast, colistin did not show synergismwith nourseothricin and hygromycin B, which act on protein syn-thesis (FICI 0.55 and data not shown), and 5-fluorocytosine,which acts on DNA and RNA synthesis (FICI 1).

We also tested whether colistin exhibited synergistic activitywith aminocandin in other yeast species. We found synergismin all species tested, with FICI ranging from 0.07 to 0.28(Table 2). Colistin also showed weak antifungal activity in C. lusi-taniae, C. krusei, C. tropicalis and Y. lipolytica whereas MICs were≥ 50 mg/L for the other species (Table 2).

Taken together, these results showed that colistin, an anti-biotic in clinical use, slightly increases the antifungal activity ofamphotericin B and fluconazole, two drugs that target fungalmembranes, and strongly potentiates the antifungal activity ofcell wall b-1,3-glucan synthase inhibitors of the echinocandinfamily in yeasts.

Synergism of colistin and caspofungin in caspofungin-and fluconazole-resistant strains

The rising number of caspofungin- and fluconazole-resistantstrains occurring in hospitals is of concern.5 Therefore, wetested whether colistin acted synergistically with caspofunginin caspofungin- and fluconazole-resistant strains of C. albicans(Table 1). While caspofungin-resistant strains harboured muta-tions in the FKS1 gene, which encodes b-1,3-glucan synthase,targeted by echinocandins,36 fluconazole-resistant strains hadmutations in ERG11, which encodes cytochrome P450 lanosterol14-a-demethylase, targeted by azoles,37 and/or TAC1, whichencodes a transcription factor regulating the expression of thedrug efflux pump-encoding genes CDR1 and CDR2.38 Consistentwith results obtained using C. albicans strain SC5314, we foundthat colistin acted synergistically with caspofungin or fluconazolein all C. albicans wild-type isolates tested (Table 2 and data notshown). Colistin acted synergistically with caspofungin influconazole-resistant strains but not in caspofungin-resistantstrains (Table 2 and data not shown). This suggested that the ef-ficacy of caspofungin against its target was a prerequisite for ob-serving synergy with colistin. Moreover, it indicated that colistintoxicity was not affected by increased expression of drug effluxproteins, including Cdr1 and Cdr2.

Genome-wide fitness profiling of S. cerevisiae knock-outmutants reveals a role of sphingolipid and chitinbiosynthesis in tolerance to the aminocandin/colistincombination

Collections of molecularly barcoded heterozygous and homozy-gous knock-out S. cerevisiae mutants are available.31 Exposureof pools of both types of mutants to a given compound allowsthe identification of strains that show reduced or increased sus-ceptibility to this compound. This strategy has been used suc-cessfully to obtain insights into the mode of action ofantifungals.39 Hence, we screened a collection of 4885 barcodedS. cerevisiae haploid knock-out mutants (excluding genes essen-tial for S. cerevisiae growth) and a collection of 5936 barcodedS. cerevisiae heterozygous diploid knock-out mutants (includingessential genes) for susceptibility to colistin, aminocandin andthe combination of aminocandin and colistin. Pools of deletionmutants were grown in YPD medium without compound, in thepresence of 20 mg/L colistin (which led to a decrease in growthrate of �10%, allowing us to test the susceptibility of S. cerevisiaeknock-out mutants to colistin alone), in the presence of0.00125 mg/L aminocandin and in the presence of the

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combination of 0.00125 mg/L aminocandin and 5 mg/L colistin. Sub-sequently, cells treated with the different compounds or combin-ation of compounds were compared with non-treated cells inorder to identify those mutants that showed increased ordecreased fitness in one of the three conditions tested (Tables S2and S3, available as Supplementary data at JAC Online). Selectedmutants identified in this manner were then tested individuallyin order to confirm the data of the high-throughput experiments(Figure S2, available as Supplementary data at JAC Online).

Table 3 lists S. cerevisiae genes whose corresponding haploidgene deletion strains were most affected by treatment with colis-tin [log2(treated/untreated),20.7], aminocandin [log2(treated/untreated),20.7] or the combination of the drugs [log2(treated/untreated),21.0]. Enriched functional annotations for haploidmutants showing hypersusceptibility to drugs were evaluatedusing the generic GO Term Finder (http://go.princeton.edu/cgi-bin/GOTermFinder). ‘Glycosphingolipid biosynthetic process’was the only term significantly enriched (corrected P value0.00119) among the 14 strains most affected by colistin treat-ment. The genes annotated to this term code for Sur1/Csg1and Csg2, two proteins that act in a complex to catalyse the for-mation of mannose-inositol-P-ceramide, one of the sphingolipidcomponents that concentrate with ergosterol in lipid rafts of theplasma membrane.40 – 42 Similarly, the term ‘glycosphingolipidbiosynthetic process’ was enriched when functional annotationsfor the 20 heterozygous diploid mutants most susceptible to colistinwere analysed (P¼0.00598). Deletion of one copy of SUR1/CSG1led to hypersusceptibility of the heterozygous diploid strain tocolistin, as did the deletion of one copy of IPT1, coding for theenzyme that catalyses the next step in this pathway, i.e. synthe-sis of mannose-(inositol-P)2-ceramide from mannose-inositol-P-ceramide,43 and the deletion of SUR4/ELO3, which encodes

an elongase involved in sphingolipid biosynthesis44 (Table S3, avail-able as Supplementary data at JAC Online). As shown in Figure 1and Table 3, growth of haploid strains with deletions in sphingolipidbiosynthesis genes was not altered in the presence of aminocandin.Therefore, the combination of colistin and aminocandin did not ag-gravate the fitness defect of these strains observed upon treat-ment with colistin alone, even though ‘glycosphingolipidbiosynthetic process’ was among the enriched GO terms(P¼0.0085) found upon analysis of the 25 genes whose deletionconferred highest susceptibility to the aminocandin/colistin com-bination treatment.

We could not find any significant enrichment for a commonannotation for the most affected haploid or heterozygousdiploid strains from the screen using aminocandin alone. In con-trast, two GO terms were highly enriched among the 25 haploidstrains most susceptible to the combination of aminocandin andcolistin: ‘fungal-type cell wall chitin biosynthetic process’(P¼1.35×1025, four genes—CHS3, CHS7, CHS6 and SKT5) andthe broader category ‘cellular cell wall organization or biogenesis’(P¼1.51×1025, nine genes, including chitin biosynthesis genesand SLT2, YPK1, FKS1, ROM2 and TUS1). As shown in Figure 1, in-activation of these genes also resulted in increased susceptibilityto aminocandin alone but the increase was smaller than thatobserved on treatment with the combination. The observationthat chitin synthesis mutants with a decreased amount ofchitin in their cell wall45 – 49 have increased susceptibility to ami-nocandin is consistent with previously published data39 and therole of chitin synthesis in protecting yeast cells against the cellwall alterations resulting from b-1,3-glucan synthase inhib-ition.50 However, the enhanced reduction in fitness observedwith the combination of aminocandin and colistin suggestedthat combined defects in b-1,3-glucan and chitin synthesis

Table 2. Synergistic activity of colistin with echinocandins against different yeast species

Strain (phenotype) Test compound

MIC of individual compoundOptimal compound concentration for

observing synergy

FICItest compound (mg/L) colistin (mg/L) test compound (mg/L) colistin (mg/L)

C. albicans SC5314 aminocandin 0.5 60 0.06 0.39 0.14C. albicans SC5314 caspofungin 0.5 60 0.125 0.39 0.26C. albicans SC5314 fluconazole 0.31 60 0.08 12.5 0.47C albicans SC5314 amphotericin B 0.63 60 0.16 0.8 0.27C. albicans SC5314 nourseothricin 2.5 60 1.25 3.1 0.55C. albicans SC5314 5-fluorocytosine 0.25 60 0.25 0.39 1

C. albicans DPL1000 (CasS FluS) caspofungin 2.0 250 0.25 0.78 0.12C. albicans 36S (CasR FluS) caspofungin 20 250 0.5 25 1.1C. albicans DSY294 (CasS FluS) caspofungin 1.0 50 0.25 0.78 0.26C. albicans DSY3987 (CasS FluR) caspofungin 0.63 125 0.16 0.39 0.26C. albicans DSY3988 (CasS FluR) caspofungin 0.63 125 0.16 0.39 0.26

C. glabrata NEM_7118432 aminocandin 0.5 50 0.06 0.39 0.14C. lusitaniae NEM_7104301 aminocandin 1.0 25 0.03 0.39 0.07C. krusei ATCC 6258 aminocandin 5.0 25 0.06 0.78 0.09C. tropicalis NEM_7114236 aminocandin 2.5 12.5 0.63 0.39 0.14C. parapsilosis NEM_7103546 aminocandin 5.0 100 0.08 1.56 0.15Y. lipolytica NEM_7115511 aminocandin 12.5 12.5 1.25 1.56 0.25S. cerevisiae BY4741 aminocandin 0.13 25 0.13 0.39 0.28

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http://go.princeton.edu/cgi-bin/GOTermFinder







Table 3. S. cerevisiae haploid gene deletion strains most affected by treatment with colistin, aminocandin or combination of the drugs; selectionwas done using a threshold value of 20.7 for single-drug treatment and 21.0 for the combination

ORF Name Colistina Aminocandina Combineda Descriptionb

Glucan synthesisYKL126W YPK1 21.3 21.0 21.3 protein kinase that affects FKS1, sphingolipid-mediated cell integrity signallingYLR425W TUS1 0.1 20.3 21.7 GEF that modulates Rho1 GTPaseYLR371W ROM2 1.1 20.3 21.6 GDP/GTP exchange protein (GEP) for Rho1p and Rho2pYLR342W FKS1 1.3 21.3 21.6 catalytic subunit of 1,3-b-D-glucan synthase, binds regulatory subunit Rho1

Chitin synthesisYBL062W overlaps

SKT520.1 20.6 21.4 overlaps MRM1, chitin synthesis

YBR023C CHS3 0.1 20.6 22.2 chitin synthaseYBL061C SKT5 0.2 20.6 22.6 activator of Chs3, chitin biosynthesisYHR142W CHS7 0.3 20.4 22.1 unknown function, chitin biosynthesisYJL099W CHS6 0.3 20.3 21.4 mediates export of cargo proteins, including Chs3, from Golgi to

plasma membrane

PKC pathwayYJL095W BCK1 20.1 20.4 22.5 MAP kinase kinase kinase in the PKC pathwayYHR030C SLT2 0.1 20.3 21.9 MAP kinase in the PKC pathway

Sphingolipid synthesisYBR036C CSG2 21.4 0.0 21.2 ER membrane protein required for mannosylation of inositolphosphorylceramideYPL057C SUR1 21.2 0.0 21.0 mannosylinositol phosphorylceramide (MIPC) synthase in complex with Csg2YLR372W SUR4 20.6 20.4 21.1 fatty acid and sphingolipid biosynthesis

Golgi functionYER151C UBP3 21.1 20.4 20.9 ubiquitin-specific protease that acts in complex with Bre5 to co-regulate

transport between the ER and GolgiYKR001C VPS1 20.9 0.0 20.5 dynamin-like GTPase required for vacuolar sortingYDR372C VPS74 20.8 0.0 20.7 protein required for Golgi localization of glycosyltransferasesYFL025C BST1 20.8 20.5 21.4 GPI inositol deacylase in the ERYAL026C DRS2 20.6 0.0 21.3 aminophospholipid translocase (flippase)YJL183W MNN11 20.2 20.6 21.9 Golgi mannosyltransferase complex componentYOR327C SNC2 0.6 20.8 0.0 vesicle membrane receptor protein (v-SNARE)

Aromatic amino acid metabolismYBR069C TAT1 21.2 20.2 21.0 amino acid transporterYER090W TRP2 20.8 20.1 20.5 anthranilate synthase, tryptophan biosynthesisYGL148W ARO2 20.8 20.2 20.7 chorismate synthase, aromatic acid biosynthesis

Other rolesYOR200W 21.0 21.2 20.6 overlaps MRM1, mitochondrial enzymeYIR023W DAL81 20.8 0.8 0.0 positive regulator of genes in multiple nitrogen degradation pathwaysYKL048C ELM1 20.8 20.3 20.6 protein kinase involved in cellular morphogenesisYCR044C PER1 20.5 20.1 21.4 ER protein required for GPI-phospholipase A2 activityYOR080W DIA2 20.3 21.0 0.0 origin-binding F-box protein that forms an SCF ubiquitin ligase complex with

Skp1p and Cdc53YDR146C SWI5 20.2 20.6 21.5 transcription factorYER111C SWI4 20.1 0.0 21.8 transcription factorYPL066W 0.0 20.7 21.8 unknown functionYPL086C ELP3 0.1 20.7 20.4 subunit of Elongator complex, modifies tRNAsYPL158C AIM44 0.1 20.6 21.4 unknown functionYGR228W 0.2 20.2 21.1 overlaps SMI1, involved in the regulation of cell wall synthesisYFL001W DEG1 0.3 20.8 0.1 tRNA:pseudouridine synthaseYLR140W 0.3 21.2 0.0 overlaps RRN5, involved in pol I transcription of rDNAYBR200W BEM1 0.7 20.2 21.7 involved in establishing cell polarity and morphogenesis

aNumbers represent log2 of the ratio treated over control for each strain.bDescriptions were retrieved from the Saccharomyces Genome Database71 and simplified.


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might facilitate colistin antifungal activity. Intriguingly, strainsdeleted for the FKS1 and ROM2 genes, the former encodingone of the two catalytic subunits of the S. cerevisiaeb-1,3-glucan synthase and the latter encoding a GDP/GTP ex-change protein for the Rho1 regulatory subunit of b-1,3-glucansynthase,51 were less affected than the average population ofmutants by colistin treatment while they were hypersusceptibleto aminocandin and aminocandin/colistin combination treat-ments (Figure 1).

Mutations in sphingolipid and chitin biosynthesis resultin increased susceptibility to colistin in C. albicans

The results presented above indicated that defects in sphingo-lipid biosynthesis increased the susceptibility of S. cerevisiae tocolistin and the colistin/aminocandin combination whiledefects in chitin biosynthesis increased susceptibility to this com-bination and aminocandin alone. In order to test whether thisheld true in C. albicans, we took advantage of existing C. albicansknock-out mutants in the MIT1, CSG2, IPT1, CHS3, CHS5 and CHS7genes, which encode orthologues of S. cerevisiae SUR1/CSG1,CSG2, IPT1, CHS3, CHS5 and CHS7, respectively.52 – 56 Inactivationof MIT1 and CSG2 did not increase the susceptibility of C. albicansto colistin, aminocandin or their combination (data not shown).In contrast, inactivation of IPT1 resulted in enhanced susceptibil-ity of C. albicans to colistin alone or in combination with amino-candin (Figure 2a). Furthermore, this increased susceptibility wasabolished upon reintroduction of a wild-type allele of IPT1 in theipt1D mutant (Figure 2a). Data presented in Figure 2(b) showthat inactivation of CHS3 increased the susceptibility of C. albi-cans to the aminocandin/colistin combination while having nosignificant effect on susceptibility to the individual drugs. Inacti-vation of CHS5 enhanced the susceptibility of C. albicans to colis-tin alone or in combination with aminocandin while inactivationof CHS7 had no effect. Taken together, these results indicatedthat, as observed in S. cerevisiae, efficient sphingolipid andchitin biosynthesis protected C. albicans against the antifungalactivity of colistin and the aminocandin/colistin combination, al-though inactivating orthologous genes in S. cerevisiae and C. albi-cans had somewhat different outcomes.

Colistin treatment increases membrane permeabilityof aminocandin-treated C. albicans cells

Colistin might achieve its antifungal activity through the target-ing of biological membranes, as described for its antibacterial ac-tivity.14 In order to test this hypothesis, we assessed thepermeability of colistin- and/or aminocandin-treated C. albicanscells using the fluorescent dye PI, which enters cells only whenthe membrane has been permeabilized.57 C. albicans strainSC5314 cells were grown for 1 h at 308C, treated for differentdurations with (i) no compound, (ii) 0.00125 mg/L aminocandin,(iii) 20 mg/L colistin or (iv) the combination of 0.00125 mg/L ami-nocandin and 20 mg/L colistin, and the proportion of PI-stainedcells was determined by flow cytometry. We found that signifi-cantly larger numbers of cells became stained with PI whentreated with aminocandin and colistin relative to aminocandinalone (P,0.0001; Figure 3). Untreated cells and cells treatedwith colistin alone were not stained by the dye. These resultsindicated that colistin treatment increased the permeability of

C. albicans cells provided that they had been exposed toaminocandin.

The colistin/caspofungin combination reduces fungalburden in kidneys of mice infected with C. albicans

We tested whether the synergistic effect of colistin with echino-candins in vitro could be recapitulated during systemic C. albicansinfection in mice. We could not identify any condition in whichthe combination of CancidasTM, the clinical formulation of caspo-fungin, with the less toxic prodrug colistin sodium methanesulfo-nate allowed improved survival of C. albicans-infected mice (datanot shown). However, when we used a combination of 0.3 mg/kgCancidasTM and 40 mg/kg colistin sodium methanesulfonate, weobserved a slightly but significantly lower fungal burden inkidneys after 3 days of infection relative to the fungal burdenin mice that had been treated with 0.3 mg/kg caspofunginalone (Figure 4; Mann–Whitney test, P,0.05). Taken together,our results indicated that the combination of caspofungin andcolistin might have the potential to decrease the fungal burdenduring early stages of infection.

Discussion

Colistin targets the fungal membrane

In this study, we have found that the antibiotic colistin has weak,if any, antifungal activity against several hemiascomycetousyeasts but that this activity is highly enhanced by combinationwith echinocandins. Colistin is a member of the family of poly-myxin antibiotics, which target bacterial membranes, and is cur-rently used for the treatment of Pseudomonas aeruginosa andAcinetobacter baumannii infections as well as infections causedby multi-resistant bacteria such as the recently emergedNDM-1 Escherichia coli and Klebsiella pneumoniae.58 Weakantifungal activity of colistin and polymyxin B against several dis-tantly related fungal species has already been reported15,16,18,34

and it has been proposed that they act at the fungal membrane.Indeed, colistin could trigger rapid efflux of ATP from germlingsof Mucorales and alterations of membrane structure.34 Moreover,polymyxin B showed synergy with membrane-targeting antifun-gals such as azoles and polyenes,17,18 an observation that wehave extended to colistin in this study. Our results are consistentwith this antifungal mode of action. Indeed, we could show thatunder conditions where antifungal activity against C. albicanscould be manifested, e.g. in combination with the b-1,3-glucan syn-thase inhibitor aminocandin, C. albicans cells became significantlymore permeable to the membrane-impermeable fluorescentdye PI57 than when treated with aminocandin alone. Further-more, fitness profiling experiments performed using S. cerevisiaehaploid and heterozygous diploid knock-out mutants showedthat alteration of sphingolipid biosynthesis results in increasedsusceptibility to colistin. Similarly, a C. albicans ipt1 mutantdefective for one of the enzymes of sphingolipid biosynthesisshowed increased susceptibility to colistin and the aminocan-din/colistin combination. Sphingolipids are essential componentsof the eukaryotic plasma membrane, concentrating togetherwith sterols at so-called lipid rafts, where they have a varietyof functions.42 Notably, S. cerevisiae csg1, csg2 and ipt1 andC. albicans ipt1 mutants, which are impaired in the latest steps

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of mannose-inositol-phosphoceramide (MIPC) and mannose-(inositol phosphate)2-ceramide (M(IP)2C) biosynthesis, showincreased susceptibility to azoles and amphotericin B.39,53 Syner-gism of colistin with these antifungals and mutations that altersphingolipid biosynthesis are therefore consistent with its target-ing of fungal membranes and possibly lipid rafts within thesemembranes.

Synergy of colistin with echinocandins: a rationale?

We have shown that colistin is synergistic with echinocandins.Analysis of the topology of S. cerevisiae b-1,3-glucan synthaseand the location of amino acids whose changes render theenzyme resistant to echinocandins suggest that these drugsinteract with regions of b-1,3-glucan synthase that areexposed to the external milieu or embedded in the outerleaflet of the plasma membrane.21 Therefore, synergy betweencolistin and echinocandins is unlikely to result from colistin-mediated increased influx of echinocandins into cells. Here, wewould like to propose that echinocandin-mediated weakeningof the cell wall facilitates colistin’s access to and perturbationof fungal membrane, and this effect in turn reinforces the anti-fungal activity of echinocandins. As mentioned above, combin-ation of aminocandin with colistin rendered C. albicans cellshighly permeable to PI while colistin-treated cells appeared im-permeable to this compound. This suggests that echinocandintreatment facilitated access of colistin to membranes and its

membrane-targeted antifungal activity, similar to what hasbeen shown for echinocandin-facilitated entry of antimicrobialpeptides (AMPs).59 Consistently, synergy between colistin andechinocandins was lost in strains harbouring mutations in theFKS1 gene and rendering C. albicans resistant to echinocandins,suggesting that weakening of the cell wall through inhibition ofb-1,3-glucan synthase was a prerequisite for colistin antifungalactivity. Moreover, we have shown that susceptibility to amino-candin of S. cerevisiae and C. albicans mutants with defects inchitin synthesis was markedly aggravated when colistin wasadded. Chitin synthesis is triggered upon treatment of yeastcells with echinocandins and contributes to the tolerance to

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Figure 1. Colistin affects sphingolipid biosynthesis-deficient strains andincreases the susceptibility of chitin synthesis mutants to aminocandin.A pool of S. cerevisiae haploid deletion strains was treated with colistin(20 mg/L) (C), aminocandin (0.00125 mg/L) (A) or the combination of0.00125 mg/L aminocandin and 5 mg/L colistin (C+A) and grown for11 generations in liquid medium. Relative growth rates, estimatedusing barcode-specific microarrays, were normalized and representedas log2 ratios of the signal obtained with treated versus untreatedculture. Mutant strain behaviour in the different conditions is illustratedfor genes that belong to functional categories that were found to besignificantly enriched (sphingolipid biosynthesis, cell wall organizationand biogenesis and chitin synthesis; see text for details).

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Figure 2. Efficient sphingolipid or chitin biosynthesis protects C. albicanscells against the combined antifungal activity of aminocandin and colistin.C. albicans strains with (a) mutations in the IPT1 gene involved insphingolipid biosynthesis (DIPT1, TPIPT1-4; DIPT1+IPT1, TPIPT1-4;Table 1) and (b) in the CHS3, CHS5 and CHS7 genes involved in chitinbiosynthesis (CACB3B-5, DCHS5_ARGplu and DCHS3_ARGplus, respectively;Table 1) and their parent strains (CAI4 and SN250_ARGplus; Table 1)were grown in the absence of drugs (untreated) or in the presence ofaminocandin alone (0.00125 mg/L), colistin alone (30 mg/L) or thecombination of aminocandin (0.00125 mg/L) and colistin (20 mg/L). ODwas recorded after 16 h of growth and is represented relative to the ODobtained for the untreated wild-type cells. Data are the means of twoindependent experiments, with bars indicating the range of values.


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these drugs.50,60,61 Our results are consistent with this observa-tion and suggest that colistin might enhance the activity of ami-nocandin and the chitin biosynthesis requirement for survival ofaminocandin-treated cells. While this might also be explained bythe impact of impaired chitin biosynthesis on colistin access tothe plasma membrane and its antifungal activity, it is probablynot the case, as chitin-defective mutants did not show increasedsusceptibility to colistin alone. In contrast, S. cerevisiae mutants

with defects in b-1,3-glucan synthesis showed intrinsically hightolerance to colistin that might reflect adaptative changes inthe cell wall in response to constitutive lower b-1,3-glucanlevels,62 consequently reducing the ability of colistin to reachmembranes.

Mechanisms by which colistin could enhance the activity ofechinocandins have not been investigated here. However, itcan be proposed that colistin-induced perturbation of theplasma membrane might impair b-1,3-glucan synthase activity,rendering it more susceptible to echinocandins, or might facili-tate membrane insertion of echinocandins. Yet it should benoted that not all membrane-targeting drugs are synergisticwith echinocandins. For instance, azoles and polyenes are notgenerally regarded as synergistic with echinocandins in Candidaspecies, although some examples of synergy have beenreported.63–65 As mentioned above, synergy between cationicAMPs and echinocandins has been observed.59,66 Polymyxinsare cationic cyclic heptapeptides with a hydrophobic tail, sug-gesting that they share properties with other AMPs that couldspecifically impact fungal membranes and b-1,3-glucan syn-thase activity. Interestingly, we did not observe any synergybetween colistin and several cell wall-targeting drugs (Congored, calcofluor white, nykkomycin Z; data not shown), suggestingthat there is specificity in the colistin/echinocandin synergy.

Synergism of colistin and echinocandins in diverse fungalspecies and in a mouse model of systemic candidiasis

In this study, we have observed in vitro synergy between colistinand echinocandins in several pathogenic yeasts, namely C. albi-cans, C. glabrata, C. tropicalis, C. parapsilosis and C. krusei, aswell as in fluconazole-resistant C. albicans strains. In contrast,no synergy could be observed between aminocandin and colistinin Aspergillus fumigatus (data not shown). Using a mouse model,we have shown that the colistin/caspofungin combination couldpossibly reduce the fungal burden at early stages of systemiccandidiasis relative to caspofungin alone. However, this observa-tion did not translate into an improvement in the survival of theanimals, suggesting that further demonstration of the efficacy ofthis drug combination in the treatment of systemic candidiasiswill probably require optimizing delivery routes and evaluatingpharmacodynamic interactions of caspofungin and colistinsodium methanesulfonate. Moreover, we believe that ourresults suggest that more emphasis should be put on searchingfor and developing compounds that specifically target fungalmembranes and could be synergistic with echinocandins.

AcknowledgementsWe thank Neil Gow and Carol Munro (University of Aberdeen), David Perlin(University of New Jersey), Dominique Sanglard (Centre HospitalierUniversitaire Vaudois), Guilhem Janbon (Institut Pasteur) and RajendraPrasad (Jawaharlal Nehru University) for providing strains. We thankDominique Lebeller for providing aminocandin. We thank Bruno Didierand Marcel Hibert (Faculte de Pharmacie Strasbourg), Aurelien Lesnardand Sylvain Rault (CERM, Caen), Florence Mahuteau-Betzer and DanielDauzonne (Institut Curie, Paris), Francoise Gueritte and Olivier Pamlard(ICSN, Gif-sur-Yvette) and Yves Janin (Institut Pasteur, Paris) forproviding some of the selected compounds. We thank Jean-Paul Latgefor constant interest in this study.

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Figure 4. Colistin/caspofungin combination reduces fungal burden inkidneys of C. albicans-infected mice. Immunosuppressed female BALB/cmice were infected intravenously with 104 cfu/mouse of C. albicansstrain SC5314. One group of mice was not treated, one was treatedwith colistin alone (40 mg/kg), one with caspofungin alone (0.3 mg/kg),and one with caspofungin and colistin at the given concentrations.Mice were killed 3 days after infection and the fungal burden in thekidneys was evaluated. *P,0.05. Note that only seven mice untreatedor treated with colistin alone were still alive on day 3 after infection;therefore, the cfu of only seven kidneys are represented in the graph.

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FundingThis work was supported by a grant from the ‘Conseil Regionald’Ile-de-France’ (Chemical Library Project, grants I 06-222/R and I09-1739/R) to H. M.-L., a grant (Programme Fungi) from InstitutCarnot—Pasteur Maladies Infectieuses to H. M.-L. and C. d’E. and agrant from ‘Agence Nationale de la Recherche’ (ANR-08-JCJC-0019-01,GENO-GIM project) to C. S. U. Z. and C. C. were the recipients of post-doctoral grants in the framework of the Programme Fungi. A. L. wasthe recipient of a post-doctoral fellowship from the ‘Conseil Regionald’Ile-de-France’.

Transparency declarationsM.-E. B. has received a research grant from Gilead and lecture fees fromAstellas. All other authors: none to declare.

Supplementary dataFigures S1 and S2 and Tables S1, S2 and S3 are available as Supplemen-tary data at JAC Online (http://jac.oxfordjournals.org/).

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