Ute Zeidler

Marie-Elisabeth Bougnoux

Alexandru Lupan

Olivier Helynck

Antonia Doyen

Zacarias Garcia

Natacha Sertour

Cécile Clavaud

Hélène Munier- Lehmann

Cosmin Saveanu

Christophe D´enfert
email: christophe.denfert@pasteur.fr

Christophe D'enfert

Synergy of the antibiotic colistin with echinocandin antifungals in Candida species

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

Introduction

Invasive fungal infections (IFI) have emerged as a major cause of infectious complications in hospitalized patients with serious underlying diseases, especially those undergoing treatment of haematological malignancies or hospitalized in ICUs. [START_REF] Pfaller | Epidemiology of invasive mycoses in North America[END_REF][START_REF] Perlroth | Nosocomial fungal infections: epidemiology, diagnosis, and treatment[END_REF] Recent and comprehensive epidemiological surveys have reported that invasive candidiasis accounts for approximately 75% of all IFI [START_REF] Azie | The PATH (Prospective Antifungal Therapy) Alliance(R) registry and invasive fungal infections: update 2012[END_REF][START_REF] Horn | Presentation of the PATH Alliance registry for prospective data collection and analysis of the epidemiology, therapy, and outcomes of invasive fungal infections[END_REF] , and Candida species are now recurrently found among the five leading causes of nosocomial bloodstream infections. [START_REF] Pfaller | Epidemiology of invasive candidiasis: a persistent public health problem[END_REF][START_REF] Gudlaugsson | Attributable mortality of nosocomial candidemia, revisited[END_REF] Invasive candidiasis remains associated with a significant mortality, in the range of 40% [START_REF] Pfaller | Epidemiology of invasive candidiasis: a persistent public health problem[END_REF][START_REF] Lass-Florl | The changing face of epidemiology of invasive fungal disease in Europe[END_REF] , even in patients receiving antifungals of the echinocandin class. However, recent studies suggest that early antifungal treatment and the use of echinocandins might be optimal for patient survival and therapeutic success [START_REF] Pfaller | Epidemiology of invasive mycoses in North America[END_REF][START_REF] Garey | Time to initiation of fluconazole therapy impacts mortality in patients with candidemia: a multi-institutional study[END_REF][START_REF] Morrell | Delaying the empiric treatment of Candida bloodstream infection until positive blood culture results are obtained: a potential risk factor for hospital mortality[END_REF][START_REF] Andes | Impact of treatment strategy on outcomes in patients with candidemia and other forms of invasive candidiasis: a patient-level quantitative review of randomized trials[END_REF] . In this respect, it can be postulated that compounds that enhance the efficacy of echinocandins c ould contribute to a faster reduction of the fungal burden and have the potential to provide a breakthrough in the management of invasive candidiasis.

The main objective of the present study was to identify compounds acting synergistically with echinocandins, ie compounds that when combined with echinocandins would have a greater antifungal effect than the sum of the effects of the two individual compounds. [START_REF] Hall | The fractional inhibitory concentration (FIC) index as a measure of synergy[END_REF] We have screened a collection of 38,758 compounds in order to identify those that would increase the antifungal activity of aminocandin (IP960; HMR3270; NXL201) [START_REF] Pasqualotto | New and emerging treatments for fungal infections[END_REF] , a non-marketed echinocandin to which we had easy access. We found that the antibiotic colistin showed synergism with aminocandin and another echinocandin, caspofungin [START_REF] Groll | Caspofungin: pharmacology, safety and therapeutic potential in superficial and invasive fungal infections[END_REF] in all Candida species tested. Colistin (polymyxin E) is a member of the polymyxin family of antibiotics that show efficacy towards Gram-negative bacteria. [START_REF] Newton | The properties and mode of action of the polymyxins[END_REF] Polymyxins are cationic cyclic heptapeptides with a hydrophobic tail that interacts with the bacterial cytoplasmic membrane, therefore changing its permeability and triggering cell death. [START_REF] Newton | The properties and mode of action of the polymyxins[END_REF] Notably, weak antifungal activity of colistin and polymyxin B has been previously reported. [START_REF] Nicholls | Polymyxin sensitivity of Candida tropicalis[END_REF][START_REF] Schwartz | Antifungal properties of polymyxin B and its potentiation of tetracycline as an antifungal agent[END_REF] Moreover, polymyxin B was shown to synergize with polyenes and azole antifungals and it has been proposed that azole-induced changes in the ergosterol content of fungal membranes might enhance the pore-forming activity of polymyxins. [START_REF] Moneib | In-vitro activity of commonly used antifungal agents in the presence of rifampin, polymyxin B and norfloxacin against Candida albicans[END_REF][START_REF] Zhai | Polymyxin B, in combination with fluconazole, exerts a potent fungicidal effect[END_REF][START_REF] Ogita | Synergistic fungicidal activities of polymyxin B and ionophores, and their dependence on direct disruptive action of polymyxin B on fungal vacuole[END_REF] In contrast to azoles and polyenes that impact fungal membranes, echinocandins are lipopeptides that target cell wall biosynthesis through non-competitive inhibition of β-1,3-glucan synthase, the product of the FKS1 gene in C. albicans. [START_REF] Denning | Echinocandin antifungal drugs[END_REF] Moreover, echinocandins are proposed to act at the outer face of the plasma membrane [START_REF] Johnson | Topological and Mutational Analysis of Saccharomyces cerevisiae Fks1[END_REF] making it unlikely that their synergy with colistin has the same basis as that observed with azoles.

Hence, we have further investigated the synergy between echinocandins and colistin with chemogenomic screens and test of individual mutant strains. Our data suggest that echinocandin-mediated alteration of the cell wall might facilitate colistin access to and perturbation of fungal membranes that in turn facilitate echinocandin activity.

Materials and methods

Strains and media

Clinical or reference isolates of C. albicans (SC5314 [START_REF] Gillum | Isolation of the Candida albicans gene for orotidine-5'-phosphate decarboxylase by complementation of S. cerevisiae ura3 and E. coli pyrF mutations[END_REF] ), clinical isolates of Candida lusitaniae (NEM_7104301), Candida parapsilosis (NEM_7103546), Candida glabrata (NEM_7118432), Candida tropicalis (NEM_7114236), Candida krusei (ATCC6258; LGC Standards, Molsheim, France), Yarrowia lipolytica (NEM_7115511) and laboratory isolates of S. cerevisiae (BY4741, BY4743 [START_REF] Brachmann | Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications[END_REF] ) were used in this study. Fluconazole-and caspofunginresistant strains used in this study are listed in Table 1 and were obtained from D. Perlin (PHRI, New Jersey, USA) and D. Sanglard (CHUV, Lausanne, Switzerland). Strains were routinely grown at 30 °C in Yeast Peptone Dextrose medium (YPD; 1% yeast extract, 2% peptone, 2% glucose), or Synthetic Dextrose minimal medium (SD; 0.67% yeast nitrogen base without amino acids (Difco), 2% glucose) and 2% agar for solid media. RPMI 1640 glutamax medium (Invitrogen) buffered with 50 mM Hepes (4-(2-hydroxyethyl)-1piperazineethanesulfonic acid), pH 7 was used for screening compounds that synergize with aminocandin and for chequerboard assays. Alternatively RPMI 1640 glutamax medium buffered with 0.165 M MOPS (3-(N-morpholino)propanesulfonic acid) was used. Aminocandin (a kind gift from Dr. Dominique Lebeller), caspofungin (MSD), colistin sulfate (Sigma-Aldrich, Saint-Quentin Fallavier, France), colistin sodium methanesulfonate (Sigma-Aldrich), hygromycin B (Sigma-Aldrich), nourseothricin (Werner BioAgents, Jena, Germany) and 5-fluorocytosine (Sigma-Aldrich) were dissolved in H 2 O. Fluconazole (Sigma-Aldrich) and amphotericin B (Sigma-Aldrich) were dissolved in DMSO.

Screening of a chemical library for synergy with aminocandin

A large-scale screen for antifungal synergy between a sub-inhibitory concentration of aminocandin and a collection of 38,758 chemicals obtained from Prestwick, ChemDiv and the French "Chimiothèque Nationale" [START_REF] Hibert | French/European academic compound library initiative[END_REF] was performed in 96-well plates using a Tecan Freedom EVO 200 platform. C. albicans wild-type cells of strain SC5314 were grown in the presence of 0.0156 mg/L aminocandin and 50 µM (on average) of each tested compound (dissolved in dimethyl sulfoxide, DMSO) in 100µl RPMI medium, starting from an OD 600 of 0.01. Columns 1 and 12 were used for controls: DMSO was used as a negative control to define growth of the cells without compound; 10 mg/L of aminocandin was used as a positive control to kill all cells. Cells were incubated for 24 h at 30°C and cell viability was measured using a resazurin assay [START_REF] Tiballi | Use of a colorimetric system for yeast susceptibility testing[END_REF] through addition of resazurin-sodium salt to a final concentration of 100 mg/L. Plates were incubated for 2 h at room temperature (RT). While resazurin is naturally blue, its oxidation is associated with a change to red colour. To measure the colour change a dual wavelength measurement was performed (reference wavelength 604 nm, measurement wavelength 570 nm) using a Tecan Safire 2 microplate reader. For each plate the Z´factor 26 was calculated and was above 0.55 (Supplemental Figure 1A). The data were normalized using the following formula: % viability = 100 x (sample value -average value of positive controls) / (average of negative controls -average of positive controls). Active compounds were defined as compounds causing <1% viability. Chemical families were defined using clustering methods based on similarity concept using the Tanimoto coefficient and the K-modes clustering algorithm [START_REF] Chatuverdi | K-modes clustering[END_REF][START_REF] Huang | Extensions to the k-means algorithm for clustering large data sets with categorical values[END_REF][START_REF] Willett | Similarity and clustering in chemical information systems[END_REF] . The best compounds were ordered from the different libraries and validated using minimum inhibitory concentration (MIC) assays.

MIC and chequerboard microdilution assays

In order to define the antifungal activity of a compound, MIC assays were performed in RPMI 1640 glutamax medium buffered with 50 mM Hepes. Compounds were diluted two fold in the concentration range 0.1 mg/L -50 mg/L and 1.3x10 5 cells.mL -1 of the C. albicans wild-type strain SC5314 were added. The MIC assays were incubated for 24 h at 30°C and viability of the cells was measured using the resazurin assay as described above.

In order to assess the synergy between two compounds, checkerboad microdilution assays were performed in RPMI 1640 glutamax medium using 96-well plates. [START_REF] Scott | Demonstration of synergy with fluconazole and either ibuprofen, sodium salicylate, or propylparaben against Candida albicans in vitro[END_REF] The tested concentrations were 0.01-1 mg/L for aminocandin and caspofungin, 0.02-1.25 mg/L for fluconazole, 0.4-2.5 mg/L for amphotericin B, 0.008-0.5 mg/L for 5-fluorocytosine, 8-500 mg/L for hygromycin B, 0.08-5 mg/L for nourseothricin and 0.4-25 mg/L for colistin. The starting inoculum was 2.5x10 3 cells/mL. Chequerboards were incubated for 24 h at 30°C and viability of the cells was measured using the resazurin assay as described above. The Fractional Inhibitory Concentration index (FICI) was calculated by using the formula: MIC of drug A tested in combination/MIC of drug A alone + MIC of drug B tested in combination/MIC of drug B alone and compounds were qualified as synergistic when the FICI was below or equal to 0.5. 11

Fitness assay with barcoded S. cerevisiae knock-out collections

Fitness assays were performed with a homozygous and a heterozygous S. cerevisiae knockout collection: 4885 S. cerevisiae mutants from the systematic deletion collection of Giaever et al. (strain BY4741 31 ) were grown in individual wells in 96 deep-well plates at 30°C for 2 days in YPD medium, pooled and aliquots were stored at -80°C. A diploid heterozygous mutant collection (Invitrogen 95401.H4Pool; Life Technologies, Saint Aubin, France) consisting of 5936 mutants was grown for 15 generations in YPD, pooled and aliquots were stored at -80°C.

For each chemogenomics experiment, aliquots were thawed and cells were allowed to recover in YPD medium for 8 h at 30°C, under shaking. Pools of 3x10 6 cells of the homozygous mutants were transferred to 100 ml flasks in YPD and cells were grown for about 11 generations at 30°C under the following conditions: With (1) no compound, (2) 0.00125 mg/L aminocandin, (3) 20 mg/L colistin and (4) the combination of 0.00125 mg/L aminocandin and 5 mg/L colistin. The pool of heterozygous mutants was grown with (1) no compound, (2) 0.002 mg/L aminocandin, (3) 30 mg/L colistin and (4) the combination of 0.002 mg/L aminocandin and 10 mg/L colistin. Subsequently, 7x10 7 cells were recovered by centrifugation, washed once with cold sterile water and stored at -80°C until processing. The growth of individual strains in the different cultures was determined by amplifying, labeling and hybridizing the barcodes on custom barcode microarrays (Agilent G2509F -AMADID N°026035; Agilent, Massy, France).

Briefly, genomic DNA from the collected cells was extracted with phenol-chloroform by extensive vortexing in the presence of glass beads (425-600 nm size). Primers U1 and KU (see Supplemental Table 1 for primer sequences) were used to amplify the upstream barcodes and primers KD and D1 to amplify the downstream barcodes. 25 cycles of PCR with an annealing temperature of 50°C were used. The resulting PCR products were verified by electrophoresis on an agarose gel and used in a labeling PCR reaction with the Cy3 or Cy5 5'labeled oligonucleotides U2comp for the upstream tags and D2comp for the downstream tags and unlabeled U1 and D1 as a control. Only 15 cycles of amplification were used in the labeling step. The labeled PCR products were mixed and precipitated in the presence of linear acrylamide and of a mixture of complementary oligonucleotides (U1, D1, U2block, D2block) in four fold molar excess to avoid binding of the fluorescently labeled oligonucleotides to the microarray probes. Hybridization was performed using the DIG Easy Hyb buffer (Roche Applied Science, Meylan, France), at 24°C, overnight, in a rotating Agilent hybridization chamber. The slides were washed in decreasing concentrations of SSPE buffer (10 mM potassium phosphate (pH 7.4), 150 mM NaCl, 0.5 mM EDTA, 0.05% (w/v) Triton X100) down to 0.2 x SSPE, dried and treated immediately with the Agilent Stabilization and Drying Solution to avoid ozone-induced degradation of the Cy5 fluorophore. Scanning was performed on a Genepix 4200AL scanner (Molecular Devices, Saint Grégoire, France) and the images were analysed using Axon Genepix Pro 7 (Molecular Devices). We filtered the data according to our previous estimates of the reliability of the microarray sig nal. [START_REF] Peyroche | Sodium selenide toxicity is mediated by O2-dependent DNA breaks[END_REF] Filtered data were normalized using the Loess algorithm (R package marray included in Bioconductor [START_REF] Gentleman | Bioconductor: open software development for computational biology and bioinformatics[END_REF] ) separately for signals coming from upstream or downstream barcodes. The average of the values for the upstream barcode and the downstream barcode was calculated.

The log2 of the ratio between the signal obtained for a given mutant growing with and without drug was used as an estimate of the drug's effect on the growth rate of the mutant.

Confirmation of selected S. cerevisiae and C. albicans mutants

In order to confirm the data of the fitness screen we selected several strains of the homozygous and the heterozygous deletion mutant collections (Table 1) and retested them singularly. The respective strains were streaked on YPD plates and single colonies were used to isolate genomic DNA of the strains using the Epicentre MasterPure Yeast DNA purification kit (Tebu-bio, Le Perray en Yvelines, France). PCRs were performed using oligonucleotides binding to the up-stream region of the respective gene and on the KANMX4 cassette, which was used to delete the genes. Confirmed strains, i e those harbouring a correctly integrated marker cassette, were inoculated from an overnight culture to an OD 600 =0.002 in YPD medium and grown with (1) no compound, (2) 0.00125 mg/L aminocandin, (3) 20 mg/L colistin and (4) the combination of 0.00125 mg/L aminocandin and 5 mg/L colistin over night at 30°C (16 h). Subsequently, the OD 600 of the strains was measured to determine the sensitivity of the respective strains. C. albicans mutant strains (Table 1) were tested using the same procedure except that cells were grown with (1) no compound, (2) 0.00125 mg/L aminocandin, (3) 30 mg/L colistin and (4) the combination of 0.00125 mg/L aminocandin and Analysis of cell permeability using Propidium-Iodide staining 2x10 5 CFU/mL of the wild-type strain SC5314 were inoculated from an over-night culture in RPMI and grown for 1 h at 30°C. Subsequently, cells were grown in RPMI with (1) no compound, (2) 0.00125 mg/L aminocandin, (3) 20 mg/L colistin and (4) the combination of 0.00125 mg/L aminocandin and 20 mg/L colistin. After 10, 30, 50 and 70 minutes growth at 30°C, 4x10 6 cells were harvested, washed with 1x PBS, resuspended in 1x PBS and incubated for 3 min with 5µg Propidium Iodide (PI; Sigma-Aldrich) in the dark. Subsequently, the cells were washed with 1x PBS, resuspended in 1x PBS and analysed using a Miltenyi Biotec MACS QUANT FACS machine (Miltenyi, Paris, France). Cells treated with 70% ethanol for 1 h were used as positive control while untreated cells were used as negative control. In order to analyse the data and to calculate the percentage of PI-stained cells, the software FlowJo version 7.6.5 (TreeStar Inc, Oregon, USA) was used. The raw data were analysed using the statistical test Chi2-Mantel-Haenszel.

Mouse model of systemic candidiasis

All animal experiments adhered to the EU Directive 86/609 on the approximation of laws, regulations and administrative provisions of Member States regarding the protection of animals used for experimental and other scientific purposes, and to related national regulations. All experiments were performed according to the guidelines of the European Convention For The Protection Of Vertebrate Animals Used For Experimental And Other Scientific Purposes (ETS No. 123). The protocol was approved by Institut Pasteur Health Center Animal Care Committee (Protocol number:10.455). Recovery of organs was performed following euthanasia of animals, and all efforts were made to minimize suffering.

In vivo interaction between colistin and caspofungin was evaluated using a mouse model of systemic infection. Ten weeks old BALB/c mice (Charles River, L'Arbresle, France) were immuno-supressed with 200mg/kg endoxan (cyclophosphamide; Baxter, Maurepas, France) 4 days before infection and were infected intravenously with 10 4 colony forming units (CFUs) per mouse of C. albicans strain SC5314. Treatment with antifungals was performed intraperitoneally in a volume of 200 µl and started 24 hours after infection. For in vivo studies the less toxic prodrug colistin sodium methanesulfonate was used. Colistin sodium methanesulfonate (Sigma-Aldrich) was injected two times per day at a concentration of 40 mg/kg 34 while Cancidas™ (caspofungin; MSD) was used at a concentration of 0.3 mg/kg and injected once per day [START_REF] Louie | Pharmacodynamics of caspofungin in a murine model of systemic candidiasis: importance of persistence of caspofungin in tissues to understanding drug activity[END_REF] . Mice were kept in groups of 10 for each treatment gro up. One group of mice was treated with colistin alone, one with caspofungin alone and one with caspofungin and colistin, at the given concentrations. The group of mice not treated received 200 µl of sterile saline i.p. At 3 days postinfection (p.i.), mice were euthanized by CO 2 asphyxiation. Kidneys of each animal were removed, weighed and homogenized in 1ml of sterile 0.15 M NaCl. Serial 10-fold dilutions of the homogenates were plated onto SD agar containing 50 mg/L ticarcillin and 10 mg/L gentamycin and incubated for two days at 30°C. The fungal burden was determined by counting the CFUs and analysed using the software GraphPad Prism5. Data shown in Figure 6 represent pooled results of two independent experiments.

Results

Colistin is synergistic with echinocandins

In order to find compounds acting synergistically with the echinocandin aminocandin [START_REF] Pasqualotto | New and emerging treatments for fungal infections[END_REF] , a high-throughput screen was performed on a total of 38,758 molecules with a large diversity of chemical backbones. The compound collection comprised 16,224 commercial compounds [1,120 obtained from Prestwick Chemical (http://www.prestwickchemical.com/index.php? pa=26) and 15,104 obtained from ChemDiv (http://chemistryondemand.com/compoundlibrary)] and 22,534 compounds from the French academic chemical library (Chimiothèque Nationale [START_REF] Hibert | French/European academic compound library initiative[END_REF] , http://chimiotheque-nationale.enscm.fr/). Each compound at a final concentration around 50µM was combined with a sub-inhibitory dose of aminocandin (0.0156 mg/L) and growth of C. albicans SC5314 was recorded after 24h at 30°C using a resazurin assay for measuring cell viability. Data were normalized as percentage of viability relative to positive and negative controls. The Z' factor [START_REF] Zhang | A simple statistical parameter for use in evaluation and validation of high throughput screening assays[END_REF] , which is a statistical test to define the quality of a largescale screen was above 0.55 for all plates, indicating the robustness and reliability of the assay (Supplemental Figure 1A). Five hundred and fifty five compounds were identified whose combination with aminocandin resulted in 100% growth inhibition (Supplemental Figure 1B) and were clustered into chemical families. A hundred and fifty six compounds selected within the different families were reevaluated using MIC tests and chequerboard assays. Among these, 98 showed antifungal activity independently of the addition of aminocandin while 58 had no significant antifungal activity on their own but acted synergistically with aminocandin (data not shown).

Colistin sulfate (referred to below as colistin) was chosen for further study since it exhibited the highest synergy with aminocandin in a chequerboard microdilution assay (FICI=0.14 in RPMI-Hepes, FICI=0.25 in RPMI-MOPS; Table 2). Colistin showed antifungal activity towards C. albicans strain SC5314 at concentrations equal or above 60 mg/L (data not shown). Colistin was also tested in combination with different classes of antifungals using chequerboard-assays and the FIC indices were calculated for each combination (Table 2). We found synergy with the other β-1,3-glucan synthase inhibitor caspofungin (FICI=0.26). With both caspofungin and aminocandin, synergy was observed from a concentration of 0.4 mg/L colistin and C. albicans growth was prevented at all echinocandin concentrations tested in combination with a concentration of 3.1 mg/L colistin and above (data not shown). Our data also indicated synergy of colistin with amphotericin B (FICI=0.27) and fluconazole (FICI=0.47), consistent with previously published data that showed synergy between these antifungals and polymyxin B. [START_REF] Moneib | In-vitro activity of commonly used antifungal agents in the presence of rifampin, polymyxin B and norfloxacin against Candida albicans[END_REF][START_REF] Zhai | Polymyxin B, in combination with fluconazole, exerts a potent fungicidal effect[END_REF] In contrast, colistin did not show synergism with nourseothricin and hygromycin B that act on protein synthesis (FICI=0.55 and data not shown) and 5-fluorocytosine that acts on DNA and RNA synthesis (FICI=1).

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

Taken together, these results showed that colistin, an antibiotic in clinical use, slightly increases the antifungal activity of amphotericin B and fluconazole, two drugs that target fungal membranes, and strongly potentiates the antifungal activity of cell wall β-1,3-glucan synthase inhibitors of the echinocandin family in yeasts.

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

The rising number of caspofungin-and fluconazole-resistant strains occurring in hospitals is of concern. [START_REF] Pfaller | Epidemiology of invasive candidiasis: a persistent public health problem[END_REF] Therefore, we tested whether colistin acted synergistically with caspofungin in C. albicans caspofungin-and fluconazole-resistant strains (Table 1). While caspofungin-resistant strains harboured mutations in the FKS1 gene that encodes β-1,3-glucan synthase targeted by echinocandins [START_REF] Perlin | Resistance to echinocandin-class antifungal drugs[END_REF] , fluconazole-resistant strains had mutations in E R G 11 that encodes cytochrome P450 lanosterol 14-alpha-demethylase targeted by azoles [START_REF] Vanden Bossche | P450 inhibitors of use in medical treatment: focus on mechanisms of action[END_REF] and/or TAC1 that encodes a transcription factor regulating the expression of the drug efflux pump-encoding genes CDR1 and CDR2. [START_REF] Coste | TAC1, transcriptional activator of CDR genes, is a new transcription factor involved in the regulation of Candida albicans ABC transporters CDR1 and CDR2[END_REF] Consistent with results obtained using C. albicans strain SC5314, we found that colistin acted synergistically with caspofungin or fluconazole in all C. albicans wild-type isolates tested (Table 2 and data not shown). Colistin acted synergistically with caspofungin in fluconazole-resistant strains but not in caspofungin-resistant strains (Table 2 and data not shown). This suggested that efficacy of caspofungin towards its target was a prerequisite to observe synergy with colistin. Moreover, it indicated that colistin toxicity was not affected by increased expression of drug efflux proteins including Cdr1 and Cdr2.

Genome-wide fitness profiling of S. cerevisiae knock-out mutants reveals a role of sphingolipid and chitin biosynthesis in tolerance to the aminocandin/colistin combination Collections of molecularly-barcoded heterozygous and homozygous knock-out S. cerevisiae mutants are available [START_REF] Giaever | Functional profiling of the Saccharomyces cerevisiae genome[END_REF] . Exposure of pools of both types of mutants to a given compound allows revealing those strains that show reduced or increased sensitivity to this compound. This strategy has been used successfully to obtain insights in the mode-of-action of antifungals [START_REF] Hillenmeyer | The chemical genomic portrait of yeast: uncovering a phenotype for all genes[END_REF] . Hence, we screened a collection of 4885 barcoded S. cerevisiae haploid KO mutants (excluding genes essential for S. cerevisiae growth) and a collection of 5936 barcoded S. cerevisiae heterozygous diploid KO mutants (including essential genes) for sensitivity to colistin, aminocandin, and the combination of aminocandin and colistin. Pools of deletion mutants were grown in YPD without compound, in the presence of 20 mg/L colistin (that led to a decrease in growth rate of about 10% allowing us to test the sensitivity of S. cerevisiae KO mutants to colistin alone), in the presence of 0.00125 mg/L aminocandin and in the presence of the combination of 0.00125 mg/L aminocandin and 5 mg/L colistin.

Subsequently, cells treated with the different compounds or combination of compounds were compared to non-treated cells in order to identify those mutants that showed increased or decreased fitness in one of the three conditions tested (Supplemental Tables 2 and3). Selected mutants identified in this manner were then tested individually in order to confirm the data of the high throughput experiments (Supplemental Figure 2). [log 2 (treated/untreated) < -0.7] or the combination of the drugs [log 2 (treated/untreated) < -1.0]. Enriched functional annotations for haploid mutants showing hypersensitivity to drugs were evaluated using the generic GO Term Finder (http://go.princeton.edu/cgibin/GOTermFinder). "Glycosphingolipid biosynthetic process" was the only term significantly enriched (corrected p value 0.00119) among the 14 strains most affected by colistin treatment. The genes annotated to this term code for Sur1/Csg1 and Csg2, two proteins that act in a complex to catalyse the formation of mannose-inositol-P-ceramide, one of the sphingolipid components that concentrate with ergosterol in lipid rafts of the plasma membrane. [START_REF] Uemura | Regulation of the transport and protein levels of the inositol phosphorylceramide mannosyltransferases Csg1 and Csh1 by the Ca2+-binding protein Csg2[END_REF][START_REF] Uemura | Csg1p and newly identified Csh1p function in mannosylinositol phosphorylceramide synthesis by interacting with Csg2p[END_REF][START_REF] Dickson | Thematic review series: sphingolipids. New insights into sphingolipid metabolism and function in budding yeast[END_REF] Similarly, the term "Glycosphingolipid biosynthetic process" was enriched when functional annotations for the 20 heterozygous diploid mutants most sensitive to colistin were analysed (p=0.00598). Deletion of one copy of SUR1/CSG1 led to hypersensitivity of the heterozygous diploid strain to colistin as did the deletion of one copy of IPT1, coding for the enzyme that catalyses the next step in this pathway ie synthesis of mannose-(inositol-P)2ceramide from mannose-inositol-P-ceramide [START_REF] Dickson | Synthesis of mannose-(inositol-P)2ceramide, the major sphingolipid in Saccharomyces cerevisiae, requires the IPT1 (YDR072c) gene[END_REF] , and the deletion of SUR4/ELO3 that encodes an elongase involved in sphingolipid biosynthesis [START_REF] Oh | ELO2 and ELO3, homologues of the Saccharomyces cerevisiae ELO1 gene, function in fatty acid elongation and are required for sphingolipid formation[END_REF] (Supplemental Table 3). As shown in Fig. 1 and Table 3, growth of haploid strains with deletions in sphingolipid biosynthesis genes was not altered in the presence of aminocandin. Therefore, the combination of colistin and aminocandin did not aggravate the fitness defect of these strains observed upon treatment with colistin alone even though "Glycosphingolipid biosynthetic process" was among the enriched GO terms (p=0.0085) found upon analysis of the 25 genes whose deletion conferred highest sensitivity to the aminocandin/colistin combination treatment.

We could not find any significant enrichment for a common annotation for the most affected haploid or heterozygous diploid strains from the screen using aminocandin alone. In contrast, two GO terms were highly enriched among the 25 haploid strains most sensitive to the combination of aminocandin and colistin: "fungal-type cell wall chitin biosynthetic process" (p=1.35x10 -5 , 4 genes -CHS3, CHS7, CHS6 and SKT5) and the broader category "cellular cell wall organization or biogenesis" (p=1.51x10 -5 , 9 genes, including chitin biosynthesis genes and SLT2, YPK1, FKS1, ROM2 and TUS1). As shown in Fig. 1, inactivation of these genes also resulted in increased sensitivity to aminocandin alone but to a lesser extent than the sensitivity observed upon treatment with the combination. The observation that chitinsynthesis mutants with a decreased amount of chitin in their cell wall [START_REF] Cid | Molecular basis of cell integrity and morphogenesis in Saccharomyces cerevisiae[END_REF][START_REF] Ziman | Chs6p-dependent anterograde transport of Chs3p from the chitosome to the plasma membrane in Saccharomyces cerevisiae[END_REF][START_REF] Trilla | Chs7p, a new protein involved in the control of protein export from the endoplasmic reticulum that is specifically engaged in the regulation of chitin synthesis in Saccharomyces cerevisiae[END_REF][START_REF] Demarini | A septin-based hierarchy of proteins required for localized deposition of chitin in the Saccharomyces cerevisiae cell wall[END_REF][START_REF] Lam | Palmitoylation by the DHHC protein Pfa4 regulates the ER exit of Chs3[END_REF] have increased sensitivity to aminocandin is consistent with previously published data [START_REF] Hillenmeyer | The chemical genomic portrait of yeast: uncovering a phenotype for all genes[END_REF] and the role of chitin synthesis in protecting yeast cells against the cell wall alterations resulting from β-1,3-glucan synthase inhibition. [START_REF] Walker | Stimulation of chitin synthesis rescues Candida albicans from echinocandins[END_REF] However, the enhanced reduction in fitness observed with the combination of aminocandin and colistin suggested that combined defects in β-1,3-glucan and chitin synthesis might facilitate colistin antifungal activity. Intriguingly, strains deleted for the FKS1 and ROM2 genes, the former encoding one of the two catalytic subunits of the S. cerevisiae β-1,3-glucan synthase and the latter encoding a GDP/GTP exchange protein for the Rho1 regulatory subunit of β-1,3-glucan synthase [START_REF] Ozaki | Rom1p and Rom2p are GDP/GTP exchange proteins (GEPs) for the Rho1p small GTP binding protein in Saccharomyces cerevisiae[END_REF] , were less affected than the average population of mutants by colistin treatment while they were hypersensitive to aminocandin and aminocandin/colistin combination treatments (Fig. 1).

Mutations in sphingolipid and chitin biosynthesis result in increased sensitivity to colistin in C. albicans

Results presented above indicated that defects in sphingolipid biosynthesis increased sensitivity of S. cerevisiae to colistin and the colistin/aminocandin combination while defects in chitin biosynthesis increased sensitivity to this combination and aminocandin alone. In order to test whether this held true in C. albicans, we took advantage of existing C. albicans knock-out mutants in the MIT1, CSG2, IPT1, CHS3, CHS5 and CHS7 genes that encode orthologues of S. cerevisiae SUR1/CSG1, CSG2, IPT1, CHS3, CHS5, a n d CHS7. [START_REF] Mille | Inactivation of CaMIT1 inhibits Candida albicans phospholipomannan beta-mannosylation, reduces virulence, and alters cell wall protein beta-mannosylation[END_REF][START_REF] Prasad | Functional analysis of CaIPT1, a sphingolipid biosynthetic gene involved in multidrug resistance and morphogenesis of Candida albicans[END_REF][START_REF] Noble | Systematic screens of a Candida albicans homozygous deletion library decouple morphogenetic switching and pathogenicity[END_REF][START_REF] Sanz | Candida albicans strains deficient in CHS7, a key regulator of chitin synthase III, exhibit morphogenetic alterations and attenuated virulence[END_REF][START_REF] Bulawa | Attenuated virulence of chitin-deficient mutants of Candida albicans[END_REF] Inactivation of MIT1 and CSG2 did not increase the sensitivity of C. albicans to colistin, aminocandin or their combination (data not shown). In contrast, inactivation of IPT1 resulted in enhanced sensitivity of C. albicans to colistin alone or in combination with aminocandin (Fig. 2A). Furthermore, this increased susceptibility was abolished upon reintroduction of a wild-type allele of IPT1 in the ipt1∆ mutant (Fig. 2A). Data presented in Fig. 2B showed that inactivation of CHS3 increased the sensitivity of C. albicans to the aminocandin/colistin combination while having no significant effect on the susceptibility to the individual drugs.

Inactivation of CHS5 enhanced sensitivity of C. albicans to colistin alone or in combination with aminocandin while inactivation of CHS7 had no effect. Taken together, these results indicated that, as observed in S. cerevisiae, efficient sphingolipid and chitin biosynthesis protected C. albicans against the antifungal activity of colistin and the aminocandin/colistin combination, although the impact of inactivating orthologous genes in S. cerevisiae and C. albicans had somewhat different outcomes.

Colistin treatment increases membrane permeability of aminocandin-treated C. albicans cells

Colistin might achieve its antifungal activity through targeting of biological membranes, as described for its antibacterial activity. [START_REF] Newton | The properties and mode of action of the polymyxins[END_REF] In order to test this hypothesis, we assessed the permeability of colistin-and/or aminocandin-treated C. albicans cells using the fluorescent dye propidium-iodide (PI) that enters cells only when the membrane has been permeabilized. [START_REF] Deere | Flow cytometry and cell sorting for yeast viability assessment and cell selection[END_REF] C. albicans strain SC5314 cells were grown for 1 h at 30°C, treated for different durations with (1) no compound, (2) 0.00125 mg/L aminocandin, (3) 20 mg/L colistin and (4) the combination of 0.00125 mg/L aminocandin and 20 mg/L colistin, and the proportion of PI-stained cells was determined by flow cytometry. We found that a significantly larger amount of cells became stained with PI when treated with aminocandin and colistin relative to aminocandin alone (P <0.0001; Fig. 3). Untreated cells and cells treated with colistin alone were not stained by the dye. Therefore, these results indicated that colistin treatment increased the permeability of C. albicans cells provided that these had been exposed to aminocandin.

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

We tested whether the synergistic effect of colistin with echinocandins in vitro could be recapitulated during systemic C. albicans infection in mice. We could not identify any condition where the combination between Cancidas™, the clinical formulation of caspofungin, and the less toxic prodrug colistin sodium methanesulfonate allowed improved survival of C. albicans-infected mice (data not shown). However, when we used a combination of 0.3 mg/kg Cancidas™ and 40 mg/kg colistin sodium methanesulfonate, we observed a slightly but significantly lower fungal burden in kidneys after 3 days of infection relative to the fungal burden in mice that had been treated with 0.3 mg/kg caspofungin alone (Fig. 4; Mann Whitney test, p-value <0.05). Taken together, our results indicated that the combination between caspofungin and colistin might have the potential to decrease fungal burden during 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 towards several hemiascomycetous yeasts but that this activity is highly enhanced through combination with echinocandins. Colistin is a member of the family of polymyxin antibiotics that target bacterial membranes and is currently used for the treatment of Pseudomonas aeruginosa and Acinetobacter baumannii infections as well as infections caused by multiresistant bacteria such as the recently emerged NDM-1 Escherichia coli a n d Klebsiella pneumoniae. [START_REF] Li | Colistin: the re-emerging antibiotic for multidrugresistant Gram-negative bacterial infections[END_REF] Weak antifungal activity of colistin and polymyxin B towards several distantly related fungal species has already been reported [START_REF] Nicholls | Polymyxin sensitivity of Candida tropicalis[END_REF][START_REF] Schwartz | Antifungal properties of polymyxin B and its potentiation of tetracycline as an antifungal agent[END_REF][START_REF] Zhai | Polymyxin B, in combination with fluconazole, exerts a potent fungicidal effect[END_REF][START_REF] Ben-Ami | Antifungal activity of colistin against mucorales species in vitro and in a murine model of Rhizopus oryzae pulmonary infection[END_REF] and it has been proposed that they act at the fungal membrane. Indeed, colistin could trigger rapid efflux of ATP from germlings of Mucorales and alterations of membrane structure. [START_REF] Ben-Ami | Antifungal activity of colistin against mucorales species in vitro and in a murine model of Rhizopus oryzae pulmonary infection[END_REF] Moreover, polymyxin B showed synergy with membrane-targeting antifungals such as azoles and polyenes [START_REF] Moneib | In-vitro activity of commonly used antifungal agents in the presence of rifampin, polymyxin B and norfloxacin against Candida albicans[END_REF][START_REF] Zhai | Polymyxin B, in combination with fluconazole, exerts a potent fungicidal effect[END_REF] , an observation that we have extended to colistin in this study. Our results are consistent with this antifungal mode of action. Indeed, we could show that under conditions where the antifungal activity towards C. albicans could be manifested, e g in combination with the β-1,3-glucan synthase inhibitor aminocandin, C. albicans cells became significantly more permeable to the membrane impermeable fluorescent dye propidium-iodide [START_REF] Deere | Flow cytometry and cell sorting for yeast viability assessment and cell selection[END_REF] , than when treated with aminocandin alone. Furthermore, fitness profiling experiments performed using S. cerevisiae haploid and heterozygous diploid knock-out mutants showed that alteration of sphingolipid biosynthesis results in increased sensitivity to colistin. Similarly, a C. albicans ipt1∆∆ mutant defective for one of the enzymes of sphingolipid biosynthesis showed increased sensitivity to colistin and the aminocandin/colistin combination. Sphingolipids are essential components of the eukaryotic plasma membrane, concentrating together with sterols at so-called lipid rafts where they play a variety of functions. [START_REF] Dickson | Thematic review series: sphingolipids. New insights into sphingolipid metabolism and function in budding yeast[END_REF] Notably, S. cerevisiae csg1, csg2 and ipt1 and C. albicans ipt1 mutants that are impaired in the latest steps of mannose-inositol-phosphoceramide (MIPC) and mannose-(inositol phosphate)2-ceramide (M(IP)2C) biosynthesis show increased sensitivity to azoles and amphotericin B. [START_REF] Hillenmeyer | The chemical genomic portrait of yeast: uncovering a phenotype for all genes[END_REF][START_REF] Prasad | Functional analysis of CaIPT1, a sphingolipid biosynthetic gene involved in multidrug resistance and morphogenesis of Candida albicans[END_REF] Synergism of colistin with these antifungals and mutations that alter sphingolipid biosynthesis are therefore consistent with its targeting of fungal membranes and possibly lipid rafts within these membranes.

Synergy of colistin with echinocandins: a rationale?

We have shown that colistin is synergistic with echinocandins. Analysis of the topology of S. cerevisiae β-1,3-glucan synthase and the location of aminoacids whose changes render the enzyme resistant to echinocandins suggest that these drugs interact with regions of the β-1,3glucan synthase that are exposed to the external milieu or embedded in the outer leaflet of the plasma membrane. [START_REF] Johnson | Topological and Mutational Analysis of Saccharomyces cerevisiae Fks1[END_REF] Therefore, synergy between colistin and echinocandins is unlikely to result from colistin-mediated increased influx of echinocandins into cells. Here, we would like to propose that echinocandin-mediated weakening of the cell wall facilitates colistin targeting of fungal membranes that might in turn reinforce the antifungal activity of echinocandins. As mentioned above, combination of aminocandin with colistin rendered C. albicans cells highly permeable to propidium iodide while colistin-treated cells appeared impermeable to this compound. This suggests that echinocandin treatment facilitated access of colistin to membranes and its membrane-targeted antifungal activity, similar to what has been shown for echinocandin-facilitated entry of antimicrobial peptides (AMP). [START_REF] Harris | Combination of caspofungin or anidulafungin with antimicrobial peptides results in potent synergistic killing of Candida albicans and Candida glabrata in vitro[END_REF] Consistently, synergy between colistin and echinocandins was lost in strains harbouring mutations in the FKS1 gene and rendering C. albicans resistant to echinocandins, suggesting that weakening of the cell wall through inhibition of β-glucan synthase was a prerequisite for colistin antifungal activity. Moreover, we have shown that sensitivity to aminocandin of S. cerevisiae and C. albicans mutants with defects in chitin synthesis was markedly aggravated when colistin was added. Chitin synthesis is triggered upon treatment of yeast cells by echinocandins and contributes to the tolerance to these drugs. [START_REF] Walker | Stimulation of chitin synthesis rescues Candida albicans from echinocandins[END_REF][START_REF] Markovich | Genomic approach to identification of mutations affecting caspofungin susceptibility in Saccharomyces cerevisiae[END_REF][START_REF] Lee | Elevated cell wall chitin in Candida albicans confers echinocandin resistance in vivo[END_REF] Our results are consistent with this observation and suggest that colistin might enhance the activity of aminocandin and the chitin biosynthesis requirement for survival of aminocandin-treated cells. While this might also be explained by the impact of impaired chitin biosynthesis on colistin access to the plasma membrane and its antifungal activity, it is probably not the case, as chitin-defective mutants did not show increased sensitivity to colistin alone. In contrast, S. cerevisiae mutants with defects in β-1,3-glucan synthesis showed intrinsically high tolerance to colistin that might reflect adaptative changes in the cell wall in response to constitutive lower β-1,3-glucan levels [START_REF] Lesage | Analysis of beta-1,3-glucan assembly in Saccharomyces cerevisiae using a synthetic interaction network and altered sensitivity to caspofungin[END_REF] , consequently reducing colistin ability to reach membranes.

Mechanisms by which colistin could enhance the activity of echinocandins have not been investigated here. However, it can be proposed that colistin-induced perturbation of the plasma membrane might impair β-glucan synthase activity, rendering it more susceptible to echinocandins, or might facilitate membrane insertion of echinocandins. Yet, it should be noted that not all membrane-targeting drugs are synergistic with echinocandins. For instance, azoles and polyenes are not generally regarded as synergistic with echinocandins in Candida spp. although some examples of synergy have been reported. [START_REF] Kiraz | Synergistic activities of three triazoles with caspofungin against Candida glabrata isolates determined by time-kill, Etest, and disk diffusion methods[END_REF][START_REF] Oliveira | In vitro interaction of posaconazole and caspofungin against clinical isolates of Candida glabrata[END_REF][START_REF] Roling | Antifungal activities of fluconazole, caspofungin (MK0991), and anidulafungin (LY 303366) alone and in combination against Candida spp. and Crytococcus neoformans via time-kill methods[END_REF] As mentioned above, synergy between cationic AMP and echinocandins has been observed. [START_REF] Harris | Combination of caspofungin or anidulafungin with antimicrobial peptides results in potent synergistic killing of Candida albicans and Candida glabrata in vitro[END_REF][START_REF] Rossignol | Endocytosis-mediated vacuolar accumulation of the human ApoE apolipoprotein-derived ApoEdpL-W antimicrobial peptide contributes to is antifungal activity in Candida albicans[END_REF] Polymyxins are cationic cyclic heptapeptides with a hydrophobic tail suggesting that they share properties with other AMP that could specifically impact fungal membranes and β-glucan synthase activity. Interestingly, we did not observe any synergy between colistin and several cell wall-targeting drugs (Congo red, calcofluor white, nykkomycin Z; data not shown) suggesting that there is specificity in the colistin/echinocandin synergy.

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

In this study, we have observed in vitro synergy between colistin and echinocandins in several pathogenic yeasts, namely C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, and C. krusei, as well as in fluconazole-resistant C. albicans strains. In contrast, no synergy could be observed between aminocandin and colistin in A. fumigatus (data not shown). Using a mouse model, we have shown that the colistin/caspofungin combination could possibly reduce fungal burden at early stages of systemic candidiasis relative caspofungin alone. However, this observation did not translate into an improvement of survival of animals suggesting that further demonstration of the efficacy of this drug combination in the treatment of systemic candidiasis will probably require optimizing delivery routes and evaluating pharmacodynamic interactions of caspofungin and colistin sodium methanesulfonate. Moreover, we believe that our results suggest that more emphasis should be put on the search and development of compounds that specifically target fungal membranes and could be synergistic with echinocandins.

Legends to Figures

Figure 1: Colistin affects sphingholipid biosynthesis deficient strains and increases the sensitivity of chitin synthesis mutants to aminocandin. A pool of S. cerevisiae haploid deletion strains was treated with colistin (20 mg/L), aminocandin (0.00125 mg/L) or the combination of 0.00125 mg/L aminocandin and 5 mg/L colistin and grown for 11 generations in liquid medium. Relative growth rates, estimated using barcode-specific microarrays, were normalized and represented as log2 ratios of the signal obtained with treated versus untreated culture. Mutant strain behaviour in the different conditions is illustrated for genes that belong to functional categories that were found to be significantly enriched (sphingolipid biosynthesis, green; cell wall organization and biogenesis, blue and chitin synthesis, red, see text for details). deltaIPT1+IPT1, TPIPT1-4; Table 1) and in the CHS3, CHS5 and CHS7 genes involved in chitin biosynthesis (CACB3B-5, ∆CHS5_ARGplu, ∆CHS3_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 of aminocandin alone (AMC; 0.00125 mg/L), colistin alone (COL; 30 mg/L), and the combination of aminocandin (0.00125 mg/L) and colistin (20

Figure 2 :

 2 Figure 2: Efficient sphingolipid or chitin biosynthesis protects C. albicans cells of the combined antifungal activity of aminocandin and colistin. C. albicans strains with mutations in the IPT1 gene involved in sphingolipid biosynthesis (deltaIPT1, TPIPT1-4;

  mg/L) (AMC + COL). Optical density was recorded after 16 h growth and is represented relative to the optical density obtained for the untreated wild-type cells. Data are mean of 2 independent experiments, with bars indicating the range of values.

Figure 3 :

 3 Figure 3: Colistin increases permeability of echinocandin-treated cells Cells were left untreated or treated with colistin alone (COL, 20 mg/L), aminocandin alone (AMC, 0.00125 mg/L) or with the combination of both drugs (AMC + COL) and the proportion of propidium-iodide stained cells was measured by flow cytometry. The statistical test Chi2-Mantel-Haenszel was used to analyse the difference between cells treated with aminocandin alone and with the combination. At all time points the difference was significant (p < 0.0001).

Figure 4 :Supplemental Figure 2 :

 42 Figure 4: The colistin/caspofungin combination reduces fungal burden in kidneys of C. albicans infected mice. Immuno-supressed female BALB/c mice were infected intravenously with 10 4 CFU/mouse of C. albicans strain SC5314. One group of mice was not treated, one was treated with 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 three days after infection and fungal burden in kidneys of mice was evaluated. * P<0.05. Note that only 7 mice untreated or treated with colistin alone were still alive on day 3 after infection, therefore the CFUs of only 7 kidneys are represented in the graph.

Table 3 lists

 3 S. cerevisiae genes whose corresponding haploid gene deletion strains were most affected by treatment with colistin [log 2 (treated/untreated) < -0.7], aminocandin

Table 1 : Yeast strains used in this study

 1

	Species	Strain	Type of strain	Genotype

Table 2 : Synergistic activity of colistin with echinocandins in different yeast species

 2

	Strain (Phenotype)

Table 3 : Saccharomyces cerevisiae haploid gene deletion strains most affected by treatment with colistin, aminocandin or combination of the drugs.

 3 Selection was done using a threshold value of -0.7 for single drug treatment and -1.0 for the combination.

	ORF	Name Colistin 1

Aminocandin 1 Combined 1 Description 2

  Involved in establishing cell polarity and morphogenesis[START_REF] Pfaller | Epidemiology of invasive mycoses in North America[END_REF] Numbers represent log 2 of the ratio treated over control for each strain 2 Descriptions were retrieved from the Saccharomyces Genome Database 71 and simplified

	YLR372W YPL066W	SUR4	-0.6 0.0	-0.4 -0.7	-1.1 -1.8	Fatty acid and sphingolipid biosynthesis Unknown function
	Golgi function YPL086C	ELP3	0.1	-0.7	-0.4	Subunit of Elongator complex -modifies tRNAs
	YER151C YPL158C	UBP3 AIM44	-1.1 0.1	-0.4 -0.6	-0.9 -1.4	Ubiquitin-specific protease that acts in complex with Bre5 to co-regulate Unknown function
	YGR228W Glucan synthesis YKL126W YPK1 YKR001C VPS1 YFL001W DEG1 YDR372C VPS74 YLR140W RRN5*	0.2 -1.3 -0.9 0.3 -0.8 0.3	-0.2 -1.0 0.0 -0.8 0.0 -1.2	-1.1 -1.3 -0.5 0.1 -0.7 0.0	transport between the ER and Golgi Overlaps SMI1 -involved in the regulation of cell wall synthesis Dynamin-like GTPase required for vacuolar sorting tRNA:pseudouridine synthase Protein kinase that affects FKS1 -sphingolipid mediated cell integrity signalling Protein required for Golgi localization of glycosyltransferases Overlaps RRN5 -involved in pol I transcription of rDNA
	YLR425W YFL025C YBR200W	TUS1 BST1 BEM1	0.1 -0.8 0.7	-0.3 -0.5 -0.2	-1.7 -1.4 -1.7	GEF that modulates Rho1 GTPase GPI inositol deacylase in the ER
	YLR371W YAL026C	ROM2 DRS2	1.1 -0.6	-0.3 0.0	-1.6 -1.3	GDP/GTP exchange protein (GEP) for Rho1p and Rho2p Aminophospholipid translocase (flippase)
	YLR342W YJL183W	FKS1 MNN11	1.3 -0.2	-1.3 -0.6	-1.6 -1.9	Catalytic subunit of 1,3-beta-D-glucan synthase; binds regulatory subunit Rho1 Golgi mannosyltransferase complex component
	Chitin synthesis YOR327C SNC2	0.6	-0.8	0.0	Vesicle membrane receptor protein (v-SNARE)
	YBL062W Aromatic aminoacid metabolism overlaps -0.1	-0.6	-1.4	Overlaps MRM1 -chitin synthesis
	YBR069C	SKT5 TAT1	-1.2	-0.2	-1.0	Aminoacid transporter
	YBR023C YER090W	CHS3 TRP2	0.1 -0.8	-0.6 -0.1	-2.2 -0.5	Chitin synthase Anthranilate synthase -tryptophan biosynthesis
	YBL061C YGL148W	SKT5 ARO2	0.2 -0.8	-0.6 -0.2	-2.6 -0.7	Activator ot Chs3 -chitin biosynthesis Chorismate synthase -aromatic acid biosynthesis
	YHR142W Other roles	CHS7	0.3	-0.4	-2.1	Unknown function, chitin biosynthesis
	YJL099W YOR200W YIR023W PKC pathway YJL095W YKL048C YHR030C YCR044C YOR080W Sphingolipid synthesis CHS6 MRM1* DAL81 BCK1 ELM1 SLT2 PER1 DIA2	0.3 -1.0 -0.8 -0.1 -0.8 0.1 -0.5 -0.3	-0.3 -1.2 0.8 -0.4 -0.3 -0.3 -0.1 -1.0	-1.4 -0.6 0.0 -2.5 -0.6 -1.9 -1.4 0.0	Mediates export or cargo proteins, including Chs3, from Golgi to plasma Overlaps MRM1 -mitochondrial enzyme membrane Positive regulator of genes in multiple nitrogen degradation pathways Protein kinase involved in cellular morphogenesis MAP kinase kinase kinase in the PKC pathway ER protein required for GPI-phospholipase A2 activity MAP kinase in the PKC pathway Origin-binding F-box protein that forms an SCF ubiquitin ligase complex with Skp1p and Cdc53
	YBR036C YDR146C YPL057C YER111C	CSG2 SWI5 SUR1 SWI4	-1.4 -0.2 -1.2 -0.1	0.0 -0.6 0.0 0.0	-1.2 -1.5 -1.0 -1.8	ER membrane protein required for mannosylation of Transcription factor inositolphosphorylceramide Mannosylinositol phosphorylceramide (MIPC) synthase in complex with Csg2 Transcription factor

Acknowledgements

We thank Neil Gow and Carol Munro (University of Aberdeen), David Perlin (University of New Jersey), Dominique Sanglard (Centre Hospitalier Universitaire Vaudois) and Rajendra Prasad (Jawaharlal Nehru University) for providing strains. We thank Bruno Didier and Marcel Hibert (Faculté de Pharmacie Strasbourg), Aurélien Lesnard and Sylvain Rault (CERM, Caen), Florence Mahuteau-Betzer and Daniel Dauzonne (Institut Curie, Paris), Francoise Gueritte and Olivier Pamlard (ICSN, Gif-sur-Yvette) and Yves Janin (Institut Pasteur, Paris) for providing some of the selected compounds. We thank Jean-Paul Latgé for constant interest in this study.

Funding

This work was supported by a grant from the "Conseil Régional d'Ile-de-France" (Chemical Library Project, grants n° I 06-222/R and I 09-1739/ R) to H. M.-L., a grant (Programme Fungi) from Institut Carnot -Pasteur Maladies Infectieuses to H.M.-L. and C.d'E. and a grant 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. was the recipient of a post-doctoral fellowship from the "Conseil Régional d'Ile-de-France". Transparency declaration M.-E.B. has received grant for research from GILEAD and lecture fees from Astellas. All other authors: none to declare.


Franck Poirier
email: franck.poirier@univ-ubs.fr

Mohammed Belatar
email: mohammed.belatar@gmail.com

Some approaches derived from UniGlyph to allow text input on tiny devices such as connected watches

Keywords: Text Input, Virtual Keyboard, Connected Watch, Internet of Things

Introduction

In this paper, we will first recall our approaches of text entry and more particularly Uniglyph, a text input method for handheld devices that used a 4-button keyboard. Secondly, we will describe different adaptations of Uniglyph for connected devices such as smartwatches.

Smartwatches

In fact smartwatches are wearable computers on the wrist that in addition to providing the time give access to some functionalities of the smartphone. For example, the user can directly answer or make calls from his/her wrist, receive a notification of a message, take a snapshot or a short video… It is important to note that an essential functionality is absent from smart watches, currently it is not possible to enter text directly on a virtual keyboard displayed on the watch screen.

You must use the vocal assistant built-in application to communicate (S Voice on Android, Siri on IOS). The problem is that voice communication is not always possible or appropriate to the context of user interaction. Voice communication is provided because there is currently no effective virtual keyboard on smartwatches.

From our point of view, text entry is a major functionality that should be present on all mobile or wearable devices [START_REF] Mackenzie | Text Entry Systems: Mobility, Accessibility, Universality[END_REF].

In this paper, we propose some ways for text entry on smartwatches derived from Uniglyph. We call this approach UniWatch.

Uniglyph text input method

UniGlyph [START_REF] Poirier | UniGlyph: only one keystroke per character on a 4-button minimal keypad for key-based text entry[END_REF] is a text entry method for handheld devices derived from Glyph [START_REF] Poirier | Glyph: A New Stroke-Alphabet for Stylus-Based or Key-Based Text Entry[END_REF], [START_REF] Poirier | Text Entry Methods For Handheld Devices Or For AAC Writing System[END_REF]. The methods of the Glyph family are based on the structure of Latin characters composed by a specific sequence of primitives (curve, stroke, loop…).

For UniGlyph, the set of primitive shapes is reduced to only 3 symbols: (1) diagonal stroke, (2) curve and (3) horizontal or vertical line. Each letter is represented by only one input primitive according, the letter entry follows a very simple rule (figure 1):

-if the capital letter contains a diagonal stroke, then click on the "diagonal" key (1); -otherwise, if it contains a loop or a curving stroke, then click on the "loop" key (2); -otherwise, click on the "straight" key (3).

As there are many more characters than primitives, each primitive corresponds to a set of letters. The expected word is deduced by a linguistic predictor like for all the ambiguous keyboards (T9®, SureType®, iTap®…).

The UniGlyph keypad contains three shape keys and one command key used to jump to the different input modes and to select the expected word.

UniWatch: an adaptation of UniGlyph for tiny connected devices

The smartwatch screen is very small, from 1.2 (Pebble) to 1.6 inches (Galaxy Gear). So, it is impossible to finger tap on a complete keyboard on such a so tiny screen in order to enter text. A smartwatch screen can just contain a small number of keys, buttons or icons. It is conceivable to enter text on a small keyboard only if the keyboard contains just a few buttons.

The UniGlyph approach is a good candidate for text entry on smartwatches because it minimizes the keypad to only 4 keys, even 3 if the commands are entered by a gesture directly applied on the watch. UniWatch, the adapted version of UniGlyph that requires only three keys is especially well suited to input text on a smartwatch.

In this paper, we propose different ways for entering text on the smartwatch screen with UniWatch. The easiest adaptation is to use a 3-key keypad, each key corresponds to one input primitive, the enter key can be replace by a gesture. These keys can be placed on the side of the screen a b c

or in each corner (figure 2-a). Another way is to use the touch screen capability by directly drawing the shape of the input primitives (diagonal stroke, curve or straight line) on the screen (figure 2-b). Finally, it is also possible to use flick or swipe gestures on the screen to enter the input primitives and to control all the process steps of text entry. For example, a flick down-left for diagonal stroke, a flick down for curve stroke, a flick down-right for straight stroke (figure 3-c).

The problem of word prediction

As the input is ambiguous, the expected word is proposed by a linguistic predictor. The simplest way is to use the default predictor of the smartphone linked to the watch (QuickType for Aplle IOS, Next Word Prediction for Android, Sense Input for HTC…).

Assuming that only very simple posts with abbreviations (OK, IDTS, B4, 2C…) will be entered on the screen of a connected watch, it must be preferable to use a specific predictor [START_REF] Agarwal | Context Based Word Prediction for Texting Language[END_REF]].

In the final paper, UniWatch will be presented and the choice of the selected approach will be explained.

Figure 1 :

 1 Figure 1: The UniGlyph character set and the associated input keys.

Figure 2 :

 2 Figure 2: Three approaches for texting: a-one key per input primitive, b-directly drawing the input primitive, c-one flick per input primitive. (Red text and arrows are only for explaining the figure. They are not displayed on the screen of the watch).