Review Article Antifungal Resistance and New Strategies to Control Fungal Infections

26 Pages·2011·1.93 MB·English

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].