HindawiPublishingCorporation InternationalJournalofMicrobiology Volume2012,ArticleID713687,26pages doi:10.1155/2012/713687 Review Article Antifungal Resistance and New Strategies to Control Fungal Infections PatrickVandeputte,SeleneFerrari,andAlixT.Coste InstituteofMicrobiology,UniversityofLausanneandUniversityHospital,RueduBugnon48,1011Lausanne,Switzerland CorrespondenceshouldbeaddressedtoAlixT.Coste,[email protected] Received21July2011;Accepted6September2011 AcademicEditor:AriannaTavanti Copyright©2012PatrickVandeputteetal.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttribution License,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperly cited. Despite improvement of antifungal therapies over the last 30 years, the phenomenon of antifungal resistance is still of major concerninclinicalpractice.Inthelast10yearsthemolecularmechanismsunderlyingthisphenomenonwereextensivelyunraveled. In this paper, after a brief overview of currently available antifungals, molecular mechanisms of antifungal resistance will be detailed.Itappearsthatmajormechanismsofresistanceareessentialduetothederegulationofantifungalresistanceeffectorgenes. Thisderegulationisaconsequenceofpointmutationsoccurringintranscriptionalregulatorsoftheseeffectorgenes.Resistance canalsofollowtheemergenceofpointmutationsdirectlyinthegenescodingantifungaltargets.Inadditionwefurtherdescribe newstrategiescurrentlyundertakentodiscoveralternativetherapytargetsandantifungals.Identificationofnewantifungalsis essentiallyachievedbythescreeningofnaturalorsyntheticchemicalcompoundcollections.Discoveryofnewputativeantifungal targetsisperformedthroughgenome-wideapproachesforabetterunderstandingofthehumanpathogenicfungibiology. 1.Introduction at the global level and a summary of clinical treatments availabletodayforclinicians,wewillreviewthemechanisms The fungal kingdom encompasses an enormous diversity underlyinginvitroresistancetoantifungalsinfungalspecies of taxa with varied ecological niches, life-cycle strategies, ofmajorimportanceinhumanmedicine.Lastly,anoverview and morphologies. However, little is known of the true ofongoingresearchundertakentodevelopnewtherapeutic biodiversity of Kingdom Fungi. Of the 1.5 million species strategiestofightagainstfungalinfectionswillbeexposed. estimated to belong to this kingdom, only about 5% were formally classified. Many fungi are parasites for plants, animals, human, and other fungi. Plant pathogenic fungi 2.FungalInfections,ClinicalTreatments, areabletocauseextensivedamageandlossestoagriculture andIncidenceofAntifungalDrugResistance and forestry including the rice blast fungus, Dutch elm disease, and chestnut blight. Some other fungi can cause 2.1.FungalInfections. Atthebeginningofthe20thcentury, serious diseases in humans, several of which may be fatal bacterial epidemics were a global and important cause of if left untreated. These include aspergillosis, candidosis, mortality. In contrast, fungal infections were almost not coccidioidomycosis, cryptococcosis, histoplasmosis, myce- tomas,mucormycosis,andparacoccidioidomycosis.Theso- taken into account. Since the late 1960s when antibiotic called dermatophytic and keratinophilic fungi can attack therapies were developed, a drastic rise in fungal infections eyes,nails,hair,andespeciallyskinandcauselocalinfections was observed, and they currently represent a global health such as ringworm and athlete’s foot. Fungal spores are also threat. This increasing incidence of infection is influenced a cause of allergies, and fungi from different taxonomic by the growing number of immunodeficient cases related groups can provoke allergic reactions. In this paper, after a toAIDS,cancer,oldage,diabetes,cysticfibrosis,andorgan briefpresentationofthemedicalimpactoffungalinfections transplantsandotherinvasivesurgicalprocedures. 2 InternationalJournalofMicrobiology Table1:Characteristicsofmainfungalinfectionsworldwide. Estimatedincidenceof Bodylocation Pathogentype Organ Mostfrequentgenus infection∗ superficial primary Skinandhair Malassezia ∼140,000,000cases/year Trichophyton cutaneous primary Skinandnails Epidermophyton ∼1,500,000,000cases/year Microsporum Vagina, ∼75,000,000cases/year digestivetract, Candida ∼9,500,000cases/year mucosal opportunistic urinarytractand eye Aspergillus,Fusarium ∼1,000,000cases/year Candida ∼300,000cases/year Aspergillus ∼350,000cases/year anyorgan(lungs, Cryptococcus ∼1,000,000cases/year systemic opportunistic brain,bloodstream Histoplasma ∼500,000cases/year etc.) Pneumocystis >200,000cases/year Coccidioidomyces upto300,000cases/year andsoon ∗ adaptedfrom“TheFungalResearchTrust.Howcommonarefungaldiseases?FungalResearchTrust20thAnniversarymeeting.LondonJune18th2011.” These infections are caused by two types of microor- during the 1980s more than 80% of seropositive patients ganisms: primary and opportunistic pathogens. Primary developed such an infection [3]. Fungal infections, of the pathogensarenaturallyabletoestablishaninfectioninthe eye are also classified as mucosal fungal infections, but are healthy population. In contrast, opportunistic pathogens, caused more frequently by Fusarium or Aspergillus species among them commensal microorganisms of the healthy ratherthanCandidaspecies. population, are able to develop infectious colonization of Theoretically systemic mycoses may involve any part thehumanbodywhenparticularcriteria,suchasimmuno- of the body, and a lot of species formerly considered as suppression,aremet.Fungalpathogenscanbedividedinto nonpathogenicarenowrecognizedopportunisticpathogens two major groups: filamentous fungi and yeasts. Most of responsible for deep-seated mycoses. These infections, with theprimarypathogensarefilamentousfungi,whilemostof symptoms ranging from a simple fever to a severe and the opportunistic pathogens are yeasts and some species of rapid septic shock, are very common in immunocompro- filamentousfungiareincreasinglyidentifiedasopportunistic mised patients and are frequently associated with an ele- pathogens.Itisalsoimportanttonotethatfungalinfections vatedmortalityrate.Themostfrequentpathogensinvolved can be classified in function of the tissue infected (see in systemic fungal infections are Candida, Pneumocystis, Table1). Histoplasma,Aspergillus,Cryptococcus,Mucor,Rhizopus,and Superficialmycoses,suchastineaversicolor,arelimitedto Coccidioidomyces[4–6]. themostexternalpartoftheskinandhair.Theseinfections aremostfrequentlycausedbythespeciesMalasseziaglobosa 2.2.AntifungalAgents. Despiteextensiveresearchdedicated and M. furfur, which are estimated to be carried by 2% to the development of new therapeutic strategies, there are to 8% of the healthy population worldwide but could lead only a limited number of available drugs to fight against to tinea versicolor in some conditions that are still unclear invasivefungalinfections.Indeed,onlyfourmolecularclass- [1]. es that target three distinct fungal metabolic pathways are Cutaneous and subcutaneous mycoses caused by der- currently used in clinical practice to treat essentially sys- matophytes fungi affect keratinized structures of the body. temicfungalinfections:fluoropyrimidineanalogs,polyenes, The most frequently involved dermatophyte genera are azoles, and echinocandins. Several other classes, such as Trichophyton, Epidermophyton, and Microsporum. In most morpholinesandallylaminesareonlyusedastopicalagents cases, cutaneous fungal infections require a challenge of due to either poor efficacy, or severe adverse effects when immune system, and their incidence varies depending on administeredsystemically. the site of infection. For example, onychomycoses are very frequentintheglobalpopulation,withanincidencevarying from5to25%[2]. 2.2.1. Fluoropyrimidines. Fluoropyrimidines, of which only Mucosal infections are mostly caused by opportunistic 5-fluorocytosine(5-FC)and5-fluorouracil(5-FU)areused yeasts,andthosebelongingtotheCandidagenusarebyfar in human medicine, are synthetic structural analogs of the the most frequent. Vaginal, esophageal, oropharyngal, and DNAnucleotidecytosine(Figure1). urinary tractcandidiasis areveryfrequentin immunocom- 5-FC was synthesized in 1957 by Duschinsky et al., promised patients. For example, esophageal candidiasis is initiallyasanantitumortherapy[7].In1963,Grunbergand associatedwiththeentryintotheclinicalphaseofAIDSand coworkers discovered its antifungal potential by means of InternationalJournalofMicrobiology 3 NH2 NH2 2.2.2. Polyenes. More than 200 molecules belonging to the chemical class of polyenes have an antifungal activity, most F H ofthembeingproducedbyStreptomycesbacteria.However, N N O N O only three possess a toxicity allowing their use in clinical practice:amphotericinB(AmB),nystatin,andnatamycine. N O N O HN Streptomyces bacteria synthesize polyenes through a H H F geneclusterphylogeneticallyconservedwithinthesespecies. (a) (b) (c) This cluster contains genes coding for several polyke- tide synthases, ABC (ATP-binding cassette) transporters, Figure1:Chemicalstructuresofcytosine(a)andoftwofluoropy- cytochromeP450-dependentenzymes,andenzymesrespon- rimidines,5-fluorocytosine(b),and5-fluorouracil(c). sible for the synthesis and the binding of the mycosamine group [27]. Although it is possible to synthesize polyenes chemically,theyarestillproducedfromStreptomycescultures foreconomicreasons. murinemodelsofcryptococcosisandcandidiasis[8].Several Polyenesarecyclicamphiphilicorganicmoleculesknown years later 5-FC was successfully used for the treatment of as macrolides. Most of them consist of a 20 to 40 carbons systemiccandidiasisandofcryptococcalmeningitis[9]. macrolactone ring conjugated with a d-mycosamine group. 5-FC possesses a broad range of activity. This drug is Their amphiphilic properties are due to the presence of activeagainstCandidaandCryptococcusgenera.5FCactivity several conjugated doublebounds on the hydrophobic side on Phialophora, Cladosporium, and Aspergillus genera is of the macrolactone ring, and to the presence of several much less limited. 5-FC is also active against protozoa hydroxylresiduesontheopposite,hydrophilicside(Figure3) belongingtoLeishmaniaandAcanthamoebagenus[10]. [28]. Due to its high hydrosolubility and small size, 5-FC Polyene drugs target ergosterol, the main sterol com- possesses interesting pharmacokinetic properties, since it ponent of fungal membranes. Their amphiphilic structure diffuses rapidly throughout body even when orally admin- allows them to bind the lipid bilayer and form pores. istered [12]. Generally, it produces negligible side effects, Magnetic nuclear resonance data suggest that eight AmB despite some severe adverse effects, such as hepatotoxicity molecules bind eight ergosterol molecules through their or bone marrow lesions [11, 13–15], occurring in rare hydrophobic moieties, with their hydrophilic sides forming cases [16]. Surprisingly, these side effects are identical to acentralchannelof70–100nmindiameter(Figure4).Pore those observed with 5-FU treatment, despite the fact that formationpromotesplasmamembranedestabilization,and humans do not possess a cytosine deaminase enzyme that channels allow leakage of intracellular components such as is responsible for the conversion from 5-FC into 5-FU in K+ions,responsibleforcelllysis[28]. fungal cells [17, 18]. Some data suggest that the intestinal Whilestructuraldatasuggestthatpolyenestargetergos- microbiome could be responsible for the 5-FU production terol,anddespitethefactthattheirbindingtoergosterolwas andtheobservedsideeffects[19]. experimentally demonstrated [29–31], controversy remains Despite its numerous pharmacological advantages, the over a possible intracellular mode of action. Some research use of 5-FC in clinical practice is decreasing because of has suggested that polyene drugs are able to induce an the frequent occurrence of innate or acquired resistance to oxidative stress (particularly in C. albicans [32, 33]) as well this drug in fungal pathogens. Thus, with few exceptions as their activity seems to be reduced in hypoxic conditions [20], 5-FC is never used as monotherapy but always in [34]. combinationwithanotherantifungal,suchasamphotericin Polyenes possess a lower but non-negligible affinity for B [21, 22]. However, the elevated renal and liver toxicities cholesterol,thehumancounterpartofergosterol.Thisslight ofamphotericinB,thatfurtherincrease5-FChepatotoxicity, affinity for cholesterol explains the high toxicity associated hasledtocombinationtherapyof5-FCmorefrequentlywith with antifungals and is responsible for several side effects azoledrugs. [28]. For this reason, only AmB is given systemically, while 5-FC itself has no antifungal activity, and its fungistatic nystatinandnatamycinareonlyusedlocallyororally.These properties are dependent upon the conversion into 5-FU two last molecules fortunately possess a very limited sys- [16,20,23].Thedrugrapidlyentersthefungalcellthrough temicactivity,sincetheirabsorptiontroughgastrointestinal specific transporters, such as cytosine permeases or pyrim- mucosaisalmostnonexistent[35,36]. idine transporters [24], before it is converted into 5-FU by Forthesereasons,AmBisthemostusedpolyeneantifun- thecytosinedeaminase[16].5-FUitselfisconvertedinto5- gal for systemic infections. Due to its high hydrophobicity fluorouracilmonophosphate(5-FUMP)byanotherenzyme, andpoorabsorptionthroughthegastrointestinaltract,itis uridine phosphoribosyltransferase (UPRT). 5-FUMP can necessary to administer AmB intravenously [28]. However, then be either converted into 5-fluorouracil triphosphate, AmB administration is accompanied with adverse effects, which incorporates into RNA in place of UTP and inhibits mostly at the level of kidneys and liver. New AmB formu- protein synthesis, or converted into 5-fluorodeoxyuridine lations, such as liposomal AmB or lipid AmB complexes, monophosphate, which inhibits a key enzyme of DNA minimizesuchsideeffects[37]. synthesis, the thymidylate synthase, thus inhibiting cell For more than 40 years, AmB was one of the goldstan- replication(Figure2)[16,25,26]. dards for the treatment of systemic fungal infections due 4 InternationalJournalofMicrobiology Intracellular compartment Incorporation Proteinsynthesis 5-FUTP intoRNA inhibition Cytosinepermeases FCY21,FCY2 Uridine Cytosine phosphoribosyl Kinases deaminase transferase FCY1 FUR1 5-FC 5-FC 5-FU 5-FUMP Uridine phosphoribosyl transferase FUR1 Thymidylate Plasma synthase DNAsynthesis membrane 5-FdUMP inhibition inhibition CDC21 Figure2:Intracellularmetabolizationandactionmodeof5-FCinS.cerevisiae,adaptedfrom[11].Inboldareindicatedgenenamesof therespectiveenzymes.5-FC:5-fluorocytosine;5-FU:5-fluorouracil;5-FUMP:5-fluorouridinemonophosphate;5-FUTP:5-fluorouridine triphosphate;5-FdUMP:5-fluorodeoxyribouridinemonophosphate. OH OH OH OH O OH O OH HO O OH OH OH OH O O HO O OH OH OH OH O O OH OH O O O O HO OH HO OH NH2 NH2 (a) (b) H O OH O OH O H OH O O OH O O HO OH NH2 (c) Figure3:ChemicalstructuresofamphotericinB(a),nystatin(b),andnatamycin(c),threemainpolyenedrugs. to the low occurrence of acquired or innate resistance to frequently used for the treatment of cutaneous, vaginal, this drug and also because of its broad range of activity and esophageal candidiasis, and natamycin can be used [38]. Indeed, AmB is active against most yeasts and fil- for the treatment of fungal keratosis or corneal infections amentous fungi. It is recommended for the treatment of [35]. infectionscausedbyCandida,Aspergillus,Fusarium,Mucor, Rhizopus, Scedosporium, Trichosporon, Cryptococcus, and so 2.2.3.Azoles. Azolesarebyfarthemostcommonlyusedanti- on. AmB is also widely used to treat parasitic infections fungals in clinical practice, and consequently, they are also such as leishmaniasis and amibiasis [28]. Natamycin and the mostly studied by the scientific community regarding nystatin are active against fungi belonging to the genera theirmodeofaction,theirpharmacologicalproperties,and Cryptococcus, Candida, Aspergillus, and Fusarium. If nys- the resistance mechanisms developed by microorganisms. tatin is not used to treat molds infections, this drug is Azoleantifungalsarealsolargelystudiedbypharmaceutical InternationalJournalofMicrobiology 5 glucocorticoids production and liver and gastrointestinal complications [50–52]. Lastly, numerous interactions with other drugs were described. For these reasons, the triazoles weredeveloped. Fluconazole became available for use by clinicians in 1990 and provided many advantages over the use of imi- dazoles. Fluconazole is highly hydrosoluble and therefore can be easily injected intravenously. It is almost completely absorbed through the gastrointestinal tract, and it diffuses throughout the whole body, including cerebrospinal fluid [53, 54]. Fluconazole is suitable for the treatment of super- ficialcandidiasis(oropharyngal,esophageal,orvaginal),dis- seminatedcandidiasis,cryptococcalmeningitis,coccidioido- mycosis,andcutaneouscandidiasis.Duetoitsgoodpharma- cokineticpropertiesaswellasitsbroadspectrumofactivity, fluconazolewasthegold-standardtreatmentoffungalinfec- Figure4:3DmodelofporeformedbyamphotericinBintolipid tions during the 1990s. Unfortunately, the overprescription bilayerofthefungalplasmamembrane,adaptedfromBaginskiet of this drug by physicians for prophylaxis or treatment al.[29].AmphotericinB:white(H),green(C),red(O),andblue (N);ergosterol:pink. led to an increase in resistance to azole drugs. Moreover, fluconazoleisalmostineffectiveagainstmostmolds. Itraconazole was approved and made available by the FDA in 1992. This triazole possesses a broad spectrum of companies,whoseektoenhancetheirefficacyandtodevelop activity across fungal species comparable to this of keto- theperfectantifungal. conazole and wider than fluconazole. Moreover, it is less Azolesarecyclicorganicmoleculeswhichcanbedivided toxic than ketoconazole and replaced it for treatment of intotwogroupsonthebasisofthenumberofnitrogenatoms histoplasmosis, blastomycosis, and paracoccidioidomycosis. intheazolering:theimidazolescontaintwonitrogenatoms, Contrary to fluconazole,it is also used for the treatment of and the triazoles contain three nitrogen atoms (Figure5) infectionsduetospeciesbelongingtothegeneraAspergillus [39]. and Sporothrix [55]. However, itraconazole is hydrophobic Azole drugs target the ergosterol biosynthetic pathway andisthusmoretoxicthanfluconazole.Itraconazoleisonly by inhibition of a key enzyme, the lanosterol 14alpha de- indicatedforthetreatmentofonychomycosis,ofsuperficial methylase, encoded be the ERG11 gene. This inhibition infections, and in some cases for systemic aspergillosis occurs through the binding of the free nitrogen atom of [56]. A new itraconazole formulation with an enhanced the azole ring to the iron atom of the heme group of absorption and a decreased toxicity was approved by FDA theenzyme.Theresultingaccumulationandmetabolismof in1997[57].Aninjectableformulationofitraconazolewas 14alpha methylated sterol species leads to the synthesis of madeavailablein2001[58]. toxic compounds, which are unable to successfully replace Fluconazole and itraconazole are still not the perfect ergosterol[40]. antifungals, since they have some nonnegligible drug inter- Thefirstazolewassynthesizedin1944byWoolley[41], actions with such drugs that are used in chemotherapy or butitwasnotuntil1958thatscientificcommunitybeganto with AIDS treatment. These interactions can result in a considerazolesaspotentialantifungalagents.Inlate1960s, decrease in azole concentration or even to an increase in clotrimazole, econazole, and miconazole became available toxicity [59]. Furthermore itraconazole and fluconazole are for treatment [42]. However, their use was restricted to ineffective against some emerging pathogens like Scedospo- externalapplicationduetotheirhightoxicitywhenadminis- rium, Fusarium, and Mucorales, and resistance to azoles is tered orally [43, 44]. In 1968, miconazole became the first increasinglyreported[60]. antifungal available for parenteral injection, but due to its So-callednewgenerationtriazoleshavealsobeendevel- toxicity and relatively limited range among fungal species oped.VoriconazoleandposaconazolewereapprovedbyFDA [45],itsusedecreaseduntilitwasnolongercommercialized. in 2002 and 2006, respectively. Ravuconazole is currently In 1981, the Food and Drug Administration (FDA) underclinicaltrialphaseofdrugdevelopment.Theypossess approved a new antifungal, ketoconazole, developed by awiderangeofactivity,sincetheyareactiveagainstCandida, Heeresandhiscoworkers[46].Thisdrugwastheonlyanti- Aspergillus, Fusarium, Penicillium, Scedosporium, Acremo- fungal available for treatment of systemic fungal infections nium, and Trichosporon, and dimorphic fungi, dermato- causedbyyeastsforthefollowingtenyears.However,there phytes, and Cryptococcus neoformans [61, 62]. While new are several drawbacks to this drug. It is poorly absorbed generationtriazoleswereshowntobemoreeffectiveagainst when administered orally, and no ketoconazole form has CandidaandAspergillus[62],comparedtoclassicaltriazoles ever been developed for intravenous injection. Moreover, their side effects and drug interactions are similar to those it cannot cross the cerebrospinal barrier and is less active observed with fluconazole and itraconazole [63]. Likewise, in immunosuppressed patients [42, 47–49]. It causes some fungal isolates resistant to classical triazoles are also cross- severe side effects such as a decrease in testosterone or resistanttonewgenerationtriazoles. 6 InternationalJournalofMicrobiology Cl Cl N Cl N N N Cl O N N Cl O H Cl Cl Cl (a) (b) (c) N N N N N O O O O O O Cl Cl Cl Cl N N N N O O (d) N (e) N N N N N N N N OH OH F F F N N N N N F F (f) (g) N N N N S O N OH N N O O N N N OH N F N N CH3 F F F (h) (i) Figure 5: Chemical structures of the main azole antifungals, four imidazoles: clotrimazole (a), econazole (b), miconazole (c), and ketoconazole (d), two triazoles: itraconazole (e) and fluconazole (f), and three new generation triazoles: voriconazole (g), posaconazole (h),andravuconazole(i). 2.2.4.Echinocandins. Echinocandinsconstitutetheonlynew in2001bytheFDAandin2002bytheEMEA,micafunginin class of antifungals made available for clinicians to fight 2005,andlastlyanidulafunginin2006. invasivefungalinfectionswithinthepast15years[64].Three Echinocandins are synthetic derivatives of lipopeptides echinocandinswerecurrentlyapprovedforclinicalusebythe (Figure6).Theselipopeptidesarenaturallyproducedbysev- FDAinUnitedStatesandlaterbytheEuropeanAgencyfor eral fungal species: Aspergillus rugulovalvus synthesizes cas- theEvaluationofMedicinalProducts(EMEA):caspofungin pofungin B, Zalerion arboricola synthesizes pneumocandin InternationalJournalofMicrobiology 7 OH O OH HO O S OH H N O O H2N NH OH N OOO OH O O N HO NH HN HO OH O HO OH O N H NH H2N HHO HOH CH3 O N NH2 HO NH NHNH H3C O O H H OH CH3 H H H NH (a) OH NH OH H O HO H N NH O O OH H OHH CH3 OH (b) HO H H OH OH HO H OH N NH OH OOH NH NH O O H H HH OH O H HO NH NHOH N H O HHO H OH H (c) Figure6:Chemicalstructureofthethreeechinocandinsusedinclinicalpractice:micafungin(a),caspofungin(b),andanidulafungin(c). B, and Papularia sphaerosperma synthesizes papulacandin. These drugs are poorly absorbed in the gastrointestinal Echinocandins are noncompetitive inhibitors of β(1-3)- tractbecauseoftheirhighmolecularweightsandarethere- glucansynthase,anenzymethatcatalyzesthepolymerization foreonlyusedintravenously.Theirpharmacologicproperties of uridine diphosphate-glucose into β(1-3) glucan, one of are one of the reasons responsible for the approval of the structural components responsible for the maintenance echinocandinsbytheFDAandtheEMEA.Thesemolecules of fungal cell-wall integrity and rigidity [65, 66]. β(1-3)- possess a low toxicity (very rare side effects were reported) glucan synthase consists of an activating and a catalytic and are slowly degraded, and a daily injection is sufficient, subunit encoded by FKS genes. In most fungi, two FKS andcontrarytootherantifungals,interactionsbetweenechi- genes are found within the genome. It has been shown nocandinsandotherdrugsarerare[64].Combinedtherapy in the model organism Saccharomyces cerevisiae that FKS1 between echinocandins and AmB or another azole often is expressed during the vegetative growth phase and FKS2 leads to a synergistic effect or at least to an additive effect during sporulation [67]. Echinocandins are able to inhibit [70,71]. both isoforms of the enzyme [68]. Inhibition of β(1-3)- Another reason for which the echinocandins were ap- glucan synthase leads to cell wall destabilization and to the provedistheiractivityspectrum.Indeed,echinocandinsare leakage of intracellular components, resulting in fungal cell active against most fungal species, including Candida and lysis[69]. Aspergillus. For still unclear reasons, these molecules are 8 InternationalJournalofMicrobiology therapy, and three minor ergosterol biosynthesis inhibitors Mannoproteins SS are used as topical antifungals. The allylamines and thio- carbamates, such as terbinafine and tolnaftate, both inhibit the ERG1-encoded enzyme, squalene epoxidase. The mor- pholines such as amorolfine act by inhibiting two different Cellwall enzymes of the pathway, the Δ7,8-isomerase (encoded by β(1,3)andβ(1,6)-glucans ERG24)andtheC14-reductase(encodedbyERG2).Despite chitin their wide spectrum of activity, these antifungal agents are essentially used to treat dermatophyte infections such as tineacapitis,tineapedis,andonychomycosis,becausetheydo Plasma membrane presentnumeroussideeffects. Intracellularcompartment Ciclopirox is also used as a topical antifungal agent, Figure7:SchematicrepresentationofS.cerevisiaecellwall,adapted but its mode of action remains poorly understood in fungi fromStoneetal.[69]. [78, 79]. Another drug, griseofulvin, inhibits mitosis by interferingwithmicrotubulesfunction[80]. fungicidalinCandidabutonlyfungistaticinAspergillus[72, 2.3.IncidenceofInVitroResistanceinFungalInfection. The 73].Moreover,fungicidalactivityofechinocandinsisspecies incidence of fungal infections has drastically increased over andisolatedependentwithintheCandidagenus[74].There thepastthreedecadesandwassimultaneouslyaccompanied existseveralspecieswithinthefungalkingdomforwhichthe by increased acquired and innate resistance to antifungal echinocandins are ineffective. Such species include Crypto- drugs. However, antifungal resistance occurrence has to be coccusneoformans[75]orspeciesbelongingtoTrichophyton considered independently for each antifungal class and for and Fusarium genera. Other species have an intermedi- eachfungalgenus.Moreover,epidemiologicaldataregarding ate susceptibility to echinocandins, such as Scedosporium incidenceofresistanceamongfungalspeciesisnotidentically apiospermum, S. prolificans, and Cladophialophora bantiana distributed worldwide [81–83]. Lastly, clinical resistance, [72]. However, echinocandins constitute a good alternative defined as the treatment failure in the patient, does not to fight against fungal infections and most of treatment of always correlate with in vitro resistance, measured as an infectionsforwhichclassicaltherapywithazolesorpolyenes increase in minimal inhibitory concentration of a drug. failed are successfully managed with echinocandins [64]. In this paper, only in vitro resistance incidence will be Therefore, caspofungin is indicated for the treatment of described. candidemia and invasive candidiasis, for fungal infection prophylaxis, and for the treatment of invasive aspergillosis for which itraconazole, voriconazole, or AmB is ineffective. 2.3.1. 5-Fluorocytosine. 5FC resistance is a very common Micafungin is used for treatment of candidemia and is phenomenon [9, 16, 84]. The development of resistance particularly indicated for fungal infection prophylaxis in can be intrinsic, as is the case for C. tropicalis, or acquired bone-marrow transplant patients. Anidulafungin has no through the selection of resistant mutants after antifungal particular indications, but its main advantage is its slow exposure. Within the Candida genus, 7% to 8% of clinical degradationinthebodywithoutliverorkidneyinvolvement, isolatesareresistantto5FC,andthisfrequencyincreasesto thus it can be used in patients with liver and/or kidney 22%whenonlynonalbicansCandidaspeciesareconsidered. insufficiencies[76]. One to two percent of Cryptococcus neoformans clinical Whatmakesechinocandinsuniqueistheirfungaltarget. isolates are resistant to 5FC [85]. Filamentous fungi such For many years, the fungal cell wall was considered to be a as Aspergillus and dermatophytes are not susceptible to promising target for the development of new antifungal 5FC. molecules [68]. The fungal cell wall contains elements that havenoequivalentsinhuman[77].Itsintegrityisnecessary 2.3.2.Polyenes. Despitethereportedincreaseofpolyenere- for the fungal survival, since it provides a physical barrier sistance,itremainsarelativelyrareeventinclinicalisolatesof againstthehostimmunecellsoragainstothermicroorgan- fungalpathogens[86],probablyinrelationwiththeirmode isms. Cell wall integrity is also responsible for osmolarity of action, and the absence of systematic and standardized homeostasisandthemaintenanceofcellshapeandsize.Cell determination of susceptibility of clinical isolates [87]. The wall is also indispensable to essential enzymatic reactions incidence of strains resistant to polyenes may thus be and as an important role in cell-cell communication. The largely underestimated. Most fungal species are considered internallayerofthecellwalliscomposedofaβ(1-3)-glucans assusceptibletopolyenedrugs.However,someofthemare and chitin web, in which are included some mannopro- intrinsically poorly susceptible to these antifungals, such as teins, while external layer is composed of mannoproteins C. glabrata, Scedosporium prolificans, or Aspergillus terreus (Figure7)[77]. [38].Somespeciesaremorepronetoacquirepolyeneresis- tance.Amongyeasts,onemayciteC.lusitaniae[88,89],C. 2.2.5. Other Antifungal Agents. Considering that the ergos- guilliermondii [88], C. krusei [38], and Trichosporon beigelii terolbiosyntheticpathwayrequiresseveralenzymesthatare and among filamentous fungi Scedosporium apiospermum unique to fungi, they constitute good targets for antifungal andSporothrixschenckii[90,91]. InternationalJournalofMicrobiology 9 Table2:Nature,target,modeofaction,andfungalresistancemechanismsofthemajorantifungaldrugsusedinhumantherapy. Antifungalagent Modeofactionandcellulartarget Mechanismofresistance absenceofergosterol(lossoffunctionmutation polyenes bindingtoergosterol inERG3orERG6) decreaseofergosterolcontentincells inhibitionofcytochromep450function: effluxmediatedbymultidrugtransporters 14α-lanosteroldemethylase(ERG11)sterolΔ22 decreaseofaffinityinErg11pbymutations azoles desaturase(ERG5) upregulationofERG11 alterationsintheergosterolbiosyntheticpathway allylamines inhibitionofsqualeneepoxidase(ERG1) unknown inhibitionofsterolΔ14reductase(ERG24)andthe morpholines unknown Δ7–8isomerase(ERG2) defectincytosinepermease 5-fluorocytosine inhibitionofnucleicacidssynthesis deficiencyorlackofenzymesimplicatedinthe metabolismof5-FC deregulationofthepyrimidinebiosynthetic pathway alterationofaffinityofechinocandinsfor echinocandins inhibitionofβ-1,3glucansynthase(FKS1&2) β(1,3)-glucansynthase 2.3.3. Azoles. The early 1990s was the start of a drastic agents in invasive disease and therefore clinical outcome increase in resistance among fungal clinical isolates. How- [99,100]. ever, the improvement of antifungal therapeutic strategies throughout the last several years has helped to stabilize 3.DrugResistanceMolecularMechanisms resistance frequencies. Increase in azoles use selected less susceptiblespeciesaswellasthoseabletodevelopresistance. Microorganisms develop mechanisms to counteract the Thisledtoashiftin thepathogenic fungalspeciesencoun- fungicidal or fungistatic effects of all antifungals classes teredinclinic. that are based on three major mechanisms, namely, (i) reducing the accumulation of the drug within the fungal 2.3.4. Echinocandins. Echinocandins resistance is a rare cell, (ii) decreasing the affinity of the drug for its target, event [92]. For example, it is estimated that more than and(iii)modificationsofmetabolismtocounterbalancethe 97% of clinical isolates belonging to the Candida genus are drugeffect(Table2).Themolecularmechanismsleadingto susceptible to these drugs [93, 94]. Contrary to acquired azoleresistancehavebeenmoststudiedinyeast,andtaking resistanceinotherfungi,intrinsicechinocandinresistancein them as an example, such mechanisms are divided into Cryptococcusneoformansisnot linked withaFKS1orFKS2 fourcategories(Figure8)[101]:(i)decreaseinazoleaffinity mutation. Indeed, C. neoformans β(1–3)-glucan synthase is for their target, (ii) increase in azole target copy number, inhibited by echinocandins, but this yeast is able to grow (iii)alterationofergosterolbiosyntheticpathwayafterazoles in the presence of high concentrations of these drugs. C. action,and(iv)decreaseinintracellularazoleaccumulation. neoformans resistance to echinocandins seems to be due to Insomehighlyresistantclinicalisolates,sampledfromlong- a particular cell-wall polysaccharides composition in this term treated patients, several mechanisms of resistance are species[95]. oftencombined[102,103].Thisincreaseinresistancealong antifungal treatment is due to the sequential acquisition of 2.3.5. Incidence of In Vitro Resistance on Patient Care. As different mechanisms [104–106]. In the following section, antifungal in vitro resistance poorly correlates with clinical themolecularbasisoftheresistancemechanismstoantifun- outcome, better attention was needed to define parameters galswillbedescribed. thatproducedreproducibleandreliableintra-andinterlab- oratoryresults.Forthispurpose,twostandardizedmethods 3.1.IncreaseofDrugEfflux forthetestingofyeastandmouldisolates(CLSIandEucast) are recognized as the gold standards for drug susceptibility 3.1.1.ABCTransporters. CDR1andCDR2(Candidadrugre- testing[96–98].Thesestandardizedapproachesproducesus- sistance 1 and 2) from C. albicans are the two major ABC ceptibility results comparable between laboratories, which transporters involved in azole resistance in this species. may help to establish breakpoints for antifungal agents CDR1 and CDR2 can be coordinately upregulated in some (see [96–98] for details). These breakpoints, defined as azole-resistant strains or by exposure to a wide vari- susceptibility ranges, together with pharmacokinetic and ety of chemically unrelated inducers such as terbinafine, pharmacodynamic analyses and identification of resistance amorolfine, fluphenazine, or steroid hormones. Several cis- mechanisms,helptoassesstheinvivoactivityofantifungal acting regulatory elements responsible for the regulation of 10 InternationalJournalofMicrobiology Lanosterol Lanosterol Lanosterol Lanosterol Erg11p 14α-demethylated Azolesexport 14α-demethylated Erg11p sterol sterol Azoles Ergosterol uptake Ergosterol Mutation(s)inERG11 UpregulationofERG11 Lanosterol Lanosterol bAiznodliensg Erg11p 14α-demethylated 14α-methylated sterol sterol Upregulationofmultidrug Erg3p Alterationinergosterol transportergenes Ergosterol 14α-methyl-3,6-diol biosynthesis Azolesusceptibleyeastcell Lanosterol Lanosterol Lanosterol Erg11p 14α-demethylated sterol 14α-demethylated 14α-methylated sterol sterol Ergosterol Erg3p Ergosterol 14α-methyl-3,6-diol Figure8:MechanismsofresistancetoazolecompoundsinC.albicans. thesetwogeneswereidentifiedbyseveralinvestigators[107– CgCDR2(formerlydenotedPDH1)aswellasSNQ2(another 111]. Promoter deletion studies have revealed 5 different ABC transporter coregulated with CgCDR1 and CgCDR2) regulatory elements in the CDR1 promoter including a areupregulatedinazole-resistantclinicalisolatesandpartici- BEE (basal expression element), a DRE (drug responsive pateinazoleresistance[114–118].Allthethreegenescontain element), two SREs (steroid responsive element), and a cis-acting elements in their promoters, so-called PDRE. NRE(negativeregulatoryelement)(seeTable3 fordetails). TheseelementsaresimilartothosedescribedinS.cerevisiae Internal deletions of the BEE and DRE motifs in the CDR1 forPDR5,anABCtransporterinvolvedindrugresistanceof promoter affect basal CDR1 expression and drug-induced S.cerevisiae[119,120].DisruptionsofCgCDR1andCgCDR2 expression,respectively[107].SRE1andSRE2werereported lead to hypersusceptibility to fluconazole, cycloheximide, to be involved in the response to steroid hormones: with and chloramphenicol [115, 117]. In both C. albicans and SRE1 responding only to progesterone and SRE2 to both C. glabrata, CDR1 was shown to be the main contributor progesteroneandβ-oestradiol[108].Finally,thedeletionof inazole-resistanceamongtheABC-transporters[121–123]. theNREmotifleadstoanincreaseinthebasalexpressionof OtherABC-transportersfromC.dubliniensis(CdCDR1and CDR1[110,111].IncontrasttoCDR1,theCDR2promoter CdCDR2)[124,125],C.krusei(ABC1and2)[126,127],C. contains only a DRE motif (Table3) [107]. Among these tropicalis(CDR1-homologue),andC.neoformans(CnAFR1, different cis-acting elements, DRE was the only element AntiFungal Resistance 1) were reportedly upregulated in involved in constitutive high expression and in transient azole-resistantisolates.InA.fumigatus,atrF,andAfuMDR4 upregulationofbothCDR1andCDR2.ThisDREsequence are upregulated in itraconazole-resistant strains [128–130]. wasfunctionallyanalyzedbysystematicmutationeachbase The cis-acting regulatory elements of these genes are still oftheinitiallydescribedDREsequence[107,112].Thedata awaitingin-depthdissectionanalysis.Theoverexpressionof obtained from systematic mutational studies are in agree- ABC-transporters have also been identified as a resistance mentwithChIP-Chipassaysperformedwiththetrans-acting mechanismtoazoleinAspergillusnidulans[131,132]. factor binding to the DRE [113]. In other Candida species, Theidentificationoftrans-actingfactorsregulatingABC- functional homologues to CDR1 and CDR2 were described transporters in pathogenic fungi relied first on the well- as involved in drug resistance. In C. glabrata, CgCDR1 and described S. cerevisiae PDR network as a model [138–142].