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Fungal Genetics and Biology xxx (2011) xxx–xxx

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Fungal Genetics and Biology

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The structure–function relationship of the Aspergillus fumigatus cyp51A L98Hconversion by site-directed mutagenesis: The mechanism of L98H azole resistance

Eveline Snelders a,b,⇑, Anna Karawajczyk c, Rob J.A. Verhoeven a,b, Hanka Venselaar c, Gijs Schaftenaar c,Paul E. Verweij a,b, Willem J.G. Melchers a,b

a Radboud University Nijmegen Medical Centre, Department of Medical Microbiology, P.O. Box 9101, 6500 HB Nijmegen, The Netherlandsb Nijmegen Institute for Infection Inflammation and Immunity (N4i), P.O. Box 9101, 6500 HB Nijmegen, The Netherlandsc Centre for Molecular and Biomolecular Informatics, Nijmegen Centre for Molecular Life Sciences, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands

a r t i c l e i n f o

Article history:Received 13 May 2011Accepted 18 August 2011Available online xxxx

Keywords:Aspergillus fumigatusMulti-azole resistancecyp51AHomology modellingMolecular dynamics simulations

1087-1845/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.fgb.2011.08.002

⇑ Corresponding author at: Radboud UniversityDepartment of Medical Microbiology, P.O. Box 910Netherlands. Fax: +31 24 3540216.

E-mail address: [email protected] (E. Snel

Please cite this article in press as: Snelders, E.,rected mutagenesis: The mechanism of L98H az

a b s t r a c t

Since 1998, the rapid emergence of multi-azole-resistance (MAR) was observed in Aspergillus fumigatus inthe Netherlands. Two dominant mutations were found in the cyp51A gene, a 34 bp tandem repeat (TR) inthe promoter region combined with a leucine to histidine substitution at codon 98 (L98H). In this study,we show that molecular dynamics simulations combined with site-directed mutagenesis of amino acidsubstitutions in the cyp51A gene, correlate to the structure–function relationship of the L98H substitu-tion conferring to MAR in A. fumigatus. Because of a L98H directed change in the flexibility of the loops,that comprise a gate-like structure in the protein, the capacity of the two ligand entry channels is mod-ified by narrowing the diameter and thereby binding of azoles is obstructed. Moreover, the L98H inducedrelocation of tyrosine 121 and tyrosine 107 seems to be related to the MAR phenotype, without affectingthe biological activity of the CYP51A protein. Site-directed mutagenesis showed that both the 34 bp TRand the L98H mutation are required to obtain the MAR phenotype. Furthermore, the amino acid leucinein codon 98 in A. fumigatus is highly conserved and important for maintaining the structure of the CYP51Aprotein that is essential for azole docking.

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1. Introduction

Invasive aspergillosis (IA) is a disease with a significant mortal-ity rate, with the opportunistic mould Aspergillus fumigatus as theprinciple etiological agent (Maschmeyer et al., 2007). It is a fearedcomplication of immunosuppressive therapy mostly in patientswith haematological malignancies. Patient survival is directly asso-ciated with timely diagnosis and prompt appropriate antifungaltherapy. In medicine three groups of antifungal drugs are mostused: the triazoles, polyenes and echinocandins. The triazoles arethe main antifungal drugs recommended for IA as primary therapyand prophylaxis (Pascual et al., 2008). The use of triazoles in themanagement of Aspergillus diseases may be threatened by theemergence of acquired resistance in A. fumigatus. Modificationsof the target gene of the triazoles, the cyp51A gene, has been de-scribed to be correlated to specific triazole resistance phenotypes.The cyp51A gene encodes for a CYP450-dependent enzyme, the14a-lanosterol demethylase, which removes the 14a-methylgroup from lanosterol. Triazole antifungals inhibit this enzyme

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Nijmegen Medical Centre,1, 6500 HB Nijmegen, The

ders).

et al. The structure–function reole resistance. Fungal Genet. B

by binding with one of the nitrogen atoms of the triazole ring tothe iron atom of the heme group located in the active site of theCYP51 protein and thereby blocking sterol synthesis at the levelof the sterol C-14a demethylase (Van den Bossche et al., 1995).Two different ligand entry channels have been identified althoughit is not clear if certain antifungals exclusively use only one of thechannels (Xiao et al., 2004; Gollapudy et al., 2004). Blocking the ac-tive site of the CYP51 protein leads to the substitution of methyl-ated sterols, ergosterol depletion in the fungal membrane andaccumulation of toxic sterol intermediates all together causinginhibition of fungal cell growth (Van den Bossche et al., 1995). Spe-cific mutations in this target gene of the triazoles have been corre-lated to triazole resistance for a few amino acid substitutions. Forthese substitutions, at glycine 54, methionine 220 and glycine138, different but codon-specific triazole resistance patterns areobserved and all substitutions are located very close to one ofthe two ligand access channels (Snelders et al., 2010).

In the Netherlands, the rapid emergence of multi-azole-resistance (MAR) was observed in A. fumigatus since 1998 (Snelderset al., 2008; Howard et al., 2009). MAR A. fumigatus isolates wererecovered from patients with Aspergillus diseases, including IA.Two dominant mutations were found in 94% of MAR isolates, a34 bp tandem repeat (TR) in the promoter region combined witha leucine to histidine substitution at codon 98 (L98H) both in the

lationship of the Aspergillus fumigatus cyp51A L98H conversion by site-di-iol. (2011), doi:10.1016/j.fgb.2011.08.002

http://dx.doi.org/10.1016/j.fgb.2011.08.002

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2 E. Snelders et al. / Fungal Genetics and Biology xxx (2011) xxx–xxx

cyp51A gene (Snelders et al., 2008). The MAR phenotype consists ofresistance to itraconazole and elevated minimum inhibitory con-centrations (MICs) of voriconazole and posaconazole. The MARphenotype was associated with treatment failure and isolates wererecovered from patients with and without previous triazole expo-sure (Snelders et al., 2008). There is increasing evidence that TR/L98H arises through environmental exposure of A. fumigatus todemethylase inhibitors (DMIs) that are commonly used for cropprotection (Verweij et al., 2009). Reduced azole drug efficacyagainst TR/L98H isolates was confirmed in experimental modelsof invasive aspergillosis (Mavridou et al., 2010a, 2010b). The prev-alence of TR/L98H in other countries is largely unknown althoughclinical TR/L98H isolates were recovered in Spain, United Kingdom,Belgium, France and Denmark (Snelders et al., 2008). TR/L98H wasalso found in 8% of soil cultures in Denmark (Snelders et al., 2008;Mortensen et al., 2010).

Using molecular modelling a structural homology model of theCYP51A protein was developed and used to investigate specificamino acid substitutions in the cyp51A gene (Snelders et al.,2010). Mutations previously shown to be correlated with azoleresistance, e.g. in codons G54, M220 or G138, were located in closevicinity to one of the two ligand access channels and are thereforethought to interfere with necessary interactions needed by theazole molecules to dock towards the heme centre of the protein(Snelders et al., 2010). When the L98H substitution was investi-gated in this model, it appeared to be located in a flexible loopnot near to any of the ligand access channels (Snelders et al.,2010). Recombinant experiments have shown however that theTR/L98H substitutions were indeed directly correlated to theMAR phenotype (Mellado et al., 2007). In what way the L98Hmutation contributes to the MAR phenotype has not been de-scribed yet.

The aim of the current study was to investigate the structure–function relationship of the TR/L98H resistance mechanism, byusing molecular dynamics simulations on the leucine to histidinesubstitution in codon 98. In addition, the CYP51A homology modelwas used to design specific amino acid substitutions and to studytheir effect on the structure–function relationship and on theMAR phenotype. Amino acid substitutions were introduced byusing a site-directed mutagenesis system in which any desiredamino acid substitution can be changed in the A. fumigatus cyp51Agene.

Fig. 1. The cyp51A cassette was placed in a pUC57 vector for the possibility of sitedirected mutagenesis by mutagenic primers. To linearize the cyp51A cassette,restriction sites SpeI and MluI were added on both ends of the cyp51A cassette.

2. Materials and methods

2.1. Molecular dynamics simulations

The structure of wild type CYP51A protein of A. fumigatus wasderived from the crystal structure of human lanosterol 14a-demethylase (PDB code: 3I3K) by homology modelling. Both pro-teins share 38% sequence identity. The three-dimensional structurehas been predicted by YASARA’s homology modelling experiment(http://www.yasara.org). The experiment consists of building fourmodels based on different alignment variants. The missing loopswere modelled and optimization of the structure was performedby molecular dynamics (MD). The model with the best Z-scorewas used for the presented studies. The point mutation was intro-duced using Yasara Structure software. The structure was initiallyoptimized in vacuum with all backbone atoms fixed. For all simu-lations the Amber99 force field was used (Ponder and Case, 2003).Model coordinates will be made freely available to the communityfor use (www.cmbi.ru.nl). The MD simulations were performedusing the AMBER package (Pearlman et al., 1995) following thestandard protocol, which consisted of an initial minimization, fol-lowed by gradual heating of the system and equilibration of the

Please cite this article in press as: Snelders, E., et al. The structure–function rerected mutagenesis: The mechanism of L98H azole resistance. Fungal Genet. B

system in the constant temperature of 300 K. Bond lengths involv-ing hydrogens were constrained to their equilibrium values usingthe SHAKE algorithm which allowed adopting time steps of 2 fs.Two independent runs of 25 ns were performed, referred to asMD-1 for wild-type protein and MD-2 for the protein with L98Hmutation introduced. Configurations were saved every 25 ps, yield-ing an ensemble of n = 1000 snapshots per run, after the equilibra-tion period of 1 ns. The analysis of the trajectories was performedusing Ptraj software, that is part of the Amber package, and VMD.

2.2. Cyp51A site-directed mutagenesis

In order to perform site-directed mutagenesis to introduce ami-no acid substitutions in the cyp51A gene of an A. fumigatus strain, apUC57 plasmid containing the cyp51A gene was synthesized (Gen-Script USA Inc.). As depicted in Fig. 1, in the cyp51A cassette, 1 kb offlanking regions were added at the 50 non-translated region (50

NTR) and 30 non-translated region (30 NTR) to ensure successfultranslation. The Escherichia coli hygromycin B resistance gene(hph) complemented with a TrpC Aspergillus nidulans promoterand terminator was included in the cassette as a dominant select-able marker to select for recombinants independent of azole phe-notype (Carroll et al., 1994). The unique restriction sites SpeI andMluI were introduced on both ends of the full cyp51A cassette tolinearize and release the plasmid for transformation. Sequenceswere obtained from A. fumigatus strain AF293 from the CADREgenomics databank (http://www.cadre-genomes.org.uk/). To intro-duce the 34-bp tandem repeat in the constructed plasmid, a DNAfragment containing the tandem repeat was amplified by PCR withprimers 404.13 and 404.14 (Table 1). Both PCR product and plas-mid were digested with the restriction enzymes BglII and BbrPIand agarose gel purified. The digested PCR product and the di-gested plasmid were ligated with T4 DNA ligase (Roche) duringan overnight incubation and subsequently transformed with XL10-Gold� ultracompetent cells (Stratagene). After incubation, colo-nies were selected and plasmid DNA was sequenced to confirmcorrect incorporation of the 34 bp TR in the cyp51A gene cassettein the pUC57 plasmid.

The QuickChange™ XL Site-Directed Mutagenesis Kit (Strata-gene) was used according to protocol to introduce specific pointmutations in the cyp51A cassette by site-directed mutagenesis.Primers are listed in Table 2. All introduced mutations were


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http://www.cadre-genomes.org.uk/


Table 1Primer sequences.

Primer Sequence (50–30) cyp51A Region

404.13 Forward -GGAATAGACGCCGTTTACCA- TR insertion404.14 Reverse -GCCATACTGCCGGTTAAGAA- TR insertion407.2 Forward -GTAAAACGACGGCCAGT- 50 NTRa

365.12 Reverse -TCTCTGCACGCAAAGAAGAAC- 50 NTR365.1 Forward -ATGGTGCCGATGCTATGG- cyp51A365.2 Reverse -CTGTCTCACTTGGATGTG- cyp51A365.10 Forward -TAGTCCATTGACGACCCC- cyp51A365.20 Forward -ATCTTCCGTTTGGTGCTGG- 30 NTR + hphb

365.19 Reverse -AGATAGGCTAGAAGGAGC- 30 NTR + hph407.4 Forward -AGTCTCCAAGGACATGCCCT- 30 NTR407.3 Reverse -CAGGAAACAGCTATGAC- 30 NTR

a NTR = Non Translating Region.b hph = hygromycin-B resistance marker.

E. Snelders et al. / Fungal Genetics and Biology xxx (2011) xxx–xxx 3

checked by sequencing of the full length pUC57 plasmids contain-ing the cyp51A cassette (Table 1). The A. fumigatus akuBKU80 strainwas used as a recipient strain in the fungal transformation exper-iments (da Silva Ferreira et al., 2006). Transformation by electro-poration was carried out as described for A. nidulans andsubsequently adapted for A. fumigatus (Sanchez and Aguirre,1996; Weidner et al., 1998). Concentration ranging between 0.5and 1.0 lg of linearized cyp51A cassette was used for the transfor-mation of the A. fumigatus akuBKU80 recipient strain, for each muta-tion two recombinants were analyzed that were constructed intwo separate transformation reactions. After selection of recombi-nants amino acid substitutions were again confirmed by sequenc-ing of the cyp51A gene and subsequently recombinants wereinvestigated for their antifungal susceptibility of itraconazole,voriconazole and posaconazole, according to the EUCAST referencemethod (2008). Endpoints were recorded at 48 h by visual inspec-tion and the MIC was defined as the antifungal concentration thatresulted in 100% inhibition of growth. Southern blots were per-formed to confirm size and exclude random genomic integrationin the A. fumigatus akuBKU80 genome. For Southern blot analysis,from all recombinants genomic DNA was digested with eitherEcoRI or EcoRV, size fractionated on a 1% agarose gel and trans-ferred to a Hybond-N membrane (Amersham Biosciences) by grav-itational flow (Sambrook and Russel, 2001). The blot was pre-hybridized in Church buffer followed by hybridization to a cyp51Aspecific probe (Church and Gilbert, 1984). This probe, a PCR frag-ment of the coding region of cyp51A, amplified by primers365.10 and 365.2 (Table 1), was labelled with 32PdATP by usingthe High Prime DNA labelling kit (Roche) and purified with MicroBiospin columns (Biorad). The A. fumigatus akuBKU80 recipientstrain showed the correct size of 5.6 kb for EcoRI and 3.1 kb forEcoRV. All recombinants showed the correct size increase to

Table 2Site-directed mutagenesis primers.

Primer Sequence (50–30)

404.1 Forward -CTCAACGGCAAGCA404.2 Reverse -CCGCATTGACATCC404.3 Forward -GGGTAGTACCATCA404.4 Reverse -CTTGTAGGGATCAA404.5 Forward -CTCAACGGCAAGCG404.6 Reverse -CCGCATTGACATCC404.7 Forward -CTCAACGGCAAGTA404.8 Reverse -CCTCTTCCGCATTG404.9 Forward -CTCAACGGCAAGAT404.10 Reverse -CCTCTTCCGCATTG404.29 Forward -TTTATTCTCAACGG404.30 Reverse -CTTCCGCATTGACA

Underlining indicates altered nucleotide.


7.3 kb for EcoRI and 4.8 kb for EcoRV which is caused by the addi-tion of the hygromycin selectable marker. CYP51A mRNA levels ofthe recombinant A. fumigatus strains with different cyp51A aminoacid substitutions were determined as well as the akuBKU80 recipi-ent strain. Experimental conditions were performed as describedpreviously (Arendrup et al., 2010). In brief, total RNA was isolatedfrom 16 h cultures in 50 ml of Vogel’s minimal medium at 37 �C at200 rpm in a 5% CO2 humidified chamber. Cultures were homoge-nized with a MagNA Lyser Instrument after liquid nitrogen treat-ment of the harvested mycelia. RNA was extracted from thefungal lysates by a standard phenol–chloroform procedure andlithium chloride precipitation. First-strand cDNA synthesis wasperformed using random hexamer primers. Real-time PCR was per-formed for the cyp51A gene and the Actin gene. The change in geneexpression was determined using the ratio cyp51A/Actin. Experi-mental samples were run in duplicate. All isolates were culturedtwice and from each culture two separate cDNA amplificationswere performed. The standard deviation of the mean value of eachexperiment was calculated (SEM).

3. Results

3.1. Molecular dynamics simulations

Codon 98 is located in a loop that connects helix B and B0 as theonly hydrophobic residue among hydrophilic ones on the surfaceof this part of the protein (Fig. 2). The loop is part of an importantfragment of the CYP51A protein that consists of this loop togetherwith short helix B0 that closely interact with helix F and loop BC(Fig. 2). It is one of the most conserved regions in CYP51 familyof proteins and forms a gate-like structure with open access tothe heme cofactor (Fig. 2) (Podust et al., 2001a). The analysis ofavailable structures of CYP51 and other P450 proteins showed thatall of them contained this structural motif but the opening varieddepending on the protein origin (Lepesheva et al., 2001; Podustet al., 2001a; Wester et al., 2004; Chen et al., 2009). Surprisingly,this opening was never reported in the literature neither its poten-tial function as an entrance to the active centre of the protein. TheL98H substitution takes place rather far from both hypotheticalchannel entries (Fig. 2) and it is not in direct vicinity to the activecentre. The striking variation by altering leucine to histidine in theL98H mutation was the change of hydrophobicity since histidine isa polar residue in contrast to hydrophobic leucine (Fig. 3). In thewild type, the hydrophobic leucine interacts with the opposite res-idue proline 124 located in the BC loop (Fig. 2). These hydrophobicinteractions can no longer occur with the substitution in codon 98from leucine to histidine.

cyp51A alteration

CAAGGATGTCAATGCGG- L98H

TTGTGCTTGCCGTTGAG- L98H

GTTACTGGATTGATCCCTACAAG- G54W

TCCAGTAACTGATGGTACTACCC- G54W

CAAGGATGTCAATGCGG- L98R

TTGCGCTTGCCGTTGAG- L98R

CAAGGATGTCAATGCGGAAGAGG- L98Y

ACATCCTTGTACTTGCCGTTGAG- L98Y

CAAGGATGTCAATGCGGAAGAGG- L98I

ACATCCTTGATCTTGCCGTTGAG- L98I

CAAGCAGAAGGATGTCAATGCGGAAG- L98Q

TCCTTCTGCTTGCCGTTGAGAATAAA- L98Q



Fig. 2. Secondary structure of wild type CYP51A protein with depicted in greenleucine 98 subjected to substitution. The heme cofactor is depicted in black, the twochannels are filled with green and in brown and blue parts of the protein areindicated that are more flexible during the molecular dynamics simulations of L98Hmutated protein. Additionally the brown part represents the important gate of theprotein that undergoes the biggest changes during the molecular dynamicssimulations.

Fig. 4. The graph shows the average fluctuation of Ca atoms for each residuearound the average structure of the protein. The solid line stands for the L98Hmolecular dynamics trajectory and the dashed line for the wild-type moleculardynamics trajectory. In brown and blue parts of the protein are indicated that aremore flexible during the molecular dynamics simulations of L98H mutated protein.The fluctuations are much larger for the mutated protein compared to the wild-typeprotein specifically for the residues creating the BC and IH loop.


As it was already pointed out, it is important to understand therole of the L98H mutation in relation to its local environment in theCYP51A protein. Further consequences of this mutation were ob-tained from a detailed study of the protein dynamics. In general,the calculated secondary structures of the wild-type and mutatedform of the protein along the molecular dynamics trajectoryshowed that the secondary structure of the protein was stablewithin the simulation time and no drastic modifications were ob-served (data not shown). The fluctuations of the Ca carbons aroundthe average structure are shown in Fig. 4. The most flexible regions,that can be observed with an increase of distance in ångström, cov-ered residues involved in the loop formation. The fluctuations weremuch larger for the mutated protein compared to the wild-typeprotein (Fig. 4) specifically for the residues that created the BCand IH loop, highlighted in blue and brown in Fig. 2. Moleculardynamics simulations showed that the L98H mutation caused dis-ruption of the very fine balance of hydrophobic versus hydrophilic

Fig. 3. Surface representation of hydrophobic (white) versus hydrophilic (red) residues ofat location 98 is depicted in green. In the circled area hydrophobic residues L98 and opprotein this fine balance is disturbed since histidine is hydrophilic, again depicted in circledeeper into the hydrophobic core of protein.


surface (Fig. 3) resulting in a much more flexible BC loop and sub-sequently closure of the first ligand entry channel. This is in agree-ment with evidence that the BC loop determines the opening andclosing of channel 1 (Podust et al., 2001a; Lepesheva et al., 2003;Gollapudy et al., 2004). Another consequence of the L98H mutationis the disruption of the very important interaction of the hemecofactor with the residue tyrosine 121, resulting in a closer posi-tion of the introduced histidine to the carboxyl group of heme(Fig. 5). The crystal structures of the CYP51 family showed a con-served pattern of interaction where the carboxylic groups of hemeðCOO�HEMEÞ interacted with positively charged residues (lysine orarginine) and tyrosine that created hydrogen bonds directly withone of the carboxylic groups or through a water molecule. Therewere salt bridges between residue lysine 132 and ðCOO�HEMEÞ, andresidue arginine 369 and ðCOO�HEMEÞ of the wild-type CYP51A struc-ture of A. fumigatus. Additionally, residues tyrosine 107 and 121were pointing towards the two carboxyl groups of the heme cofac-tor making hydrogen bonds (Fig. 5a). The negatively charged

the CYP51A protein in wild type situation (a) and mutated situation (b). The residueposite to it the hydrophilic phenylalanine 124 are depicted. In the mutated L98Hd area. As a result the only hydrophobic island at the bottom of BC loop tends to fold



Fig. 5. Structural arrangement of heme cofactor bound by residues lysine 132, arginine 369, tyrosine 121 and 107 in (a) the wild-type protein and (b) the L98H mutatedprotein after molecular dynamics simulations. Atoms are indicated in colours by red for oxygen, nitrogen blue, carbon grey and hydrogen white. Location of residue tyrosine107 (c) in relation to the heme, in black, and the two channels, filled green, in the CYP51A protein.


carboxyl group tended to compensate its charge by pulling the his-tidine towards itself. Moreover, the side chains of residue tyrosine107 and 121 were relocated (Fig. 5b). They fit perfectly into ahydrophobic pocket created by residues alanine 102, valine 106,leucine 110, threonine 111, methionine 368, leucine 494 and phen-ylalanine 495. As a consequence, the second ligand entry channelinside the protein became closed. The strong salt bridge of residuearginine 369 with the carboxyl group of heme cofactor remainedunchanged. From crystal structures, it is known that residues tyro-sine 107 and 121 are also involved in the triazole-binding (Podustet al., 2001a). In conclusion, this clearly indicates that the conse-quences of the L98H mutation has a negative effect on exogenousligand binding. On the other hand, the L98H mutation does not dis-rupt the very fine balance of hydrophobicity in the active site of theCYP51A, which is important for binding of lanosterol and proteinactivity. The area above the heme cofactor is divided into twoparts, one of them is hydrophilic and the other hydrophobic. Thelanosterol binds into the hydrophobic part which is penetrated


by only a few hydrophilic residues namely arginine or lysine thatare used to bind the OH group of lanosterol. Therefore only theexogenous ligands that use the access channels seem to be affectedby the L98H mutation, while biological activity remains intact.

3.2. Cyp51A site-directed mutagenesis

Transformation by electroporation using the cyp51A cassettewas performed successfully in the recipient A. fumigatus akuBKU80

strain. To rule out random integration of the cyp51A cassette andto control for correct homologous recombination of the wild-typecyp51A gene with the cyp51A cassette, southern blots were per-formed. A cyp51A cassette without any mutations was transformedto observe whether exchanging the wild type cyp51A gene for thecyp51A cassette had any effect on azole susceptibility. The recom-binants with the cyp51A cassette showed no changes in azole sus-ceptibility compared to the wild type A. fumigatus akuBKU80

recipient strain (Table 3). As a control to check whether the cyp51A



Table 3Minimal inhibitory concentrations (MIC) of recombinant A. fumigatus strains with different cyp51A amino acid substitutions according to the EUCAST method. For each mutationtwo recombinants were constructed in two separate transformation reactions, MIC susceptibility of one representative isolate for each mutation is shown.

Isolate cyp51A substitutions EUCAST MIC (mg/ml) 48 h

TR Coding gene ITC VCZ POS

akuBKU80 recipient strain � – 0.5 0.25 0.25akuBKU80 cassette � – 0.5 0.25 0.25akuBKU80-G54W � G54W >16 0.25 >16akuBKU80-TR + – 1 2 0.5akuBKU80-L98H � L98H 1 1 0.5akuBKU80-TR L98H + L98H >16 2 0.5akuBKU80-TR L98R + L98R >16 2 0.5akuBKU80-TR L98Y + L98Y >16 1 0.5akuBKU80-TR L98Q + L98Q >16 2 0.5akuBKU80-TR L98I + L98I >16 1 0.5

TR: 34 bp tandem repeat in the promoter region, ITC: itraconazole; VCZ: voriconazole; POS: posaconazole.

Fig. 6. CYP51A mRNA levels of the recombinant A. fumigatus strains with different cyp51A amino acid substitutions as well as the akuBKU80 recipient strain. The change ingene expression was determined using the ratio cyp51A/Actin measured by real time PCR. Isolates containing 34 bp TR in the promoter region are represented by the darkgrey bars.


cassette is able to express the cyp51A gene, a G54W mutation,known from literature to be specifically correlated to itraconazoleand posaconazole resistance but not to voriconazole resistance,was introduced in the cyp51A cassette by site-directed mutagene-sis (Mann et al., 2003). Recombinants showed azole resistance spe-cifically for itraconazole (MIC > 16 mg/l) and posaconazole(MIC > 16 mg/l) while voriconazole remained susceptible (MIC0.25 mg/l) (Table 3), confirming the value of this site-directedmutagenesis system for the analysis of the structure–function rela-tionship of cyp51A mutations.

The 34 bp TR and L98H mutations were introduced separatelyand in combination in the cyp51A cassette. The recombinants witheither one of the mutations showed a slight increase in MIC com-pared to the wild type A. fumigatus akuBKU80 recipient strain; itrac-onazole (MIC 1 mg/l) and voriconazole (MIC 1–2 mg/l) andposaconazole (MIC 0.5 mg/l), similar to that observed by conven-tional homologous recombination experiments as described previ-ously (Mellado et al., 2007). However, only when both mutationswere introduced simultaneously a MAR phenotype was observed;itraconazole (MIC > 16 mg/l) and voriconazole (MIC 2 mg/l) andposaconazole (MIC 0.5 mg/l), again confirming the recombinantexperiments described previously (Mellado et al., 2007). Expres-sion levels of the cyp51A gene were determined by real time PCRexperiments and confirmed that only the recombinants that con-tained the 34 bp TR showed and increased expression comparedto the wild type A. fumigatus akuBKU80 recipient strain (Fig. 6).


The site-directed mutagenesis system was specifically able toalter any amino-acid in the CYP51A protein. By using the CYP51Ahomology model specific point mutations and their effect on azoleresistance were investigated. Four different amino acids were cho-sen to be introduced at codon 98 in combination with the 34 bp TRin the promoter region: arginine (R), tyrosine (Y), glutamine (Q)and isoleucine (I). Arginine is a positively charged amino acid,therefore hydrophilic, and similar to histidine. It was expected tobe attracted by the negatively charged parts of the heme groupin the same way as histidine and therefore the recombinant wasexpected to give a MAR phenotype. The recombinant isolate con-taining the arginine (R) at codon 98 and TR indeed showed a sim-ilar resistance profile to the TR/L98H phenotype; itraconazole(MIC > 16 mg/l), voriconazole (MIC 2 mg/l) and posaconazole(MIC 0.5 mg/l) (Table 3). Tyrosine is a less polar amino acid buthas an aromatic ring similar to histidine, the MAR phenotypewas however expected to be less pronounced as histidine. The re-combinant isolate with amino acid tyrosine in codon 98 and TRwas however similar to the TR/L98H phenotype; itraconazole(MIC > 16 mg/l), voriconazole (MIC 1 mg/l) and posaconazole(MIC 0.5 mg/l). Glutamine is a polar amino acid that, unlike histi-dine, contains no aromatic ring and the MAR phenotype was there-fore expected to be less pronounced as histidine. The recombinantisolate with amino acid glutamine and TR however showed similarazole susceptibilities as the TR/L98H phenotype; itraconazole(MIC > 16 mg/l), voriconazole (MIC 2 mg/l) and posaconazole




(MIC 0.5 mg/l). Isoleucine is a hydrophobic amino acid that is sim-ilar to leucine in structure and the L98I recombinant was thereforeexpected to have less or no effect on azole susceptibility. Unex-pectedly, the susceptibility testing of the recombinant isolate withisoleucine and TR showed an increase in azole resistance; itraco-nazole (MIC > 16 mg/l), voriconazole (MIC 1 mg/l) and posaconaz-ole (MIC 0.5 mg/l), similar to the TR/L98H azole phenotype as well.

4. Discussion and conclusion

Although modelling of A. fumigatus CYP51A has been describedpreviously, no study investigated yet the dynamics of specific pointmutations in the CYP51A homology model nor investigated therationale of azole resistance caused by the L98H mutation (Xiaoet al., 2004; Gollapudy et al., 2004). The amino acid changes inthe cyp51a gene previously known from literature to be correlatedto azole resistance, e.g. in codon 54, 138 or 220, have a specific im-pact on the azole susceptibility for certain azoles molecules (Snel-ders et al., 2010). However these mutations are located in thedirect vicinity of the opening of one of the two ligand access chan-nels. The mutations have a direct impact on the docking of theazole molecules which does not apply for the L98H mutation.The TR/L98H mutation appears to have a more indirect effect onazole resistance as described in the current study and thereforerepresents a different mechanism of azole resistance. By usingmolecular dynamics simulations the influence of the L98H substi-tution on the general structure of CYP51A was investigated andprovided evidence for a role of this mutation in the MAR pheno-type. The mutation mainly influences the flexibility of the BC andIH loops of the protein and as a consequence the positions of thetyrosine 121 and tyrosine 107 side chains are changed. This causesboth access channels that are identified in the CYP51A protein tobe modified and prevents the binding of azoles towards the activeheme. We have established that the general secondary structure ofthe protein is not altered due to the mutation but due to the chan-ged flexibility of the BC and IH loops the capacity of the ligand en-try channels is modified by narrowing the diameter. Moreover, therelocation of the tyrosine 121 and tyrosine 107 that are known tobe important for triazole binding, is responsible for the MAR phe-notype although it does not affect the biological activity of theCYP51A protein. This is in line with evidence from the multi-se-quence alignment, where the mutated position is occupied by his-tidine in Ustilago maydis or even by asparagine in pig or rat and theprotein is still functional (Podust et al., 2001b). In A. fumigatus, be-sides the cyp51A gene a cyp51B has been identified. A doubleknockout of the cyp51 genes was never obtained and a cyp51Aknock out showed unchanged ergosterol content and no increasedexpression of cyp51B (Mellado et al., 2005). It has been postulatedthat cyp51B encodes for alternative functions for particular growthconditions or is even functionally redundant (Warrilow et al.,2010). This provides a possible explanation to why mutations inazole-resistant A. fumigatus isolates are predominantly detectedin the cyp51A gene and no mutations in cyp51B have been provento be correlated to azole resistance.

In order to determine whether amino acid substitutions in co-don 98 can be correlated to azole resistance, we have developeda site-directed mutagenesis system that is specifically able to alterany amino acid change of interest in the CYP51A protein in A.fumigatus. The TR mutation, a duplication of a 34 bp sequence inthe promoter region of the cyp51A gene has been shown to in-crease the expression of CYP51A by approximately 2.5–8-fold(Mellado et al., 2007; Arendrup et al., 2010). Tandem repeats inthe promoter region of cyp51 genes are known mechanisms ofazole resistance in phytopathogenic moulds (Verweij et al.,2009). However, the 34 bp TR in the cyp51A gene of A. fumigatus


is by itself not sufficient for the MAR phenotype, and thereforethe substitution at codon 98 is a key alteration in this resistancemechanism. By introducing site-specific amino acid substitutionsusing the CYP51A homology model it becomes possible to investi-gate the structure–function relationship of amino acid alterationsand, in this case, the effect of TR/L98H mutation on azole suscepti-bility. The constructed recombinant isolates show that the site-di-rected mutagenesis system is sound. Introduction of the cyp51Acassette showed no changes in azole susceptibility and as a controlto check whether the cyp51A cassette, a G54W mutation, wasintroduced. This recombinant isolate showed itraconazole andposaconazole resistance but not voriconazole resistance, confirm-ing the specific G54W azole phenotype known from reported clin-ical isolates and validating the site-directed mutagenesis systemfor the analysis of the structure–function relationship of cyp51Amutations (Mann et al., 2003). In codon 98 four different aminoacid changes other than histidine were introduced simultaneouslywith the 34 bp TR. Arginine was expected to act in a similar way ashistidine and the recombinant confirmed this with a MAR pheno-type. Tyrosine and glutamine are similar to histidine but theMAR phenotype was expected to be less pronounced. The recombi-nants however showed the same level of resistance with tyrosineand glutamine as TR/L98H. Isoleucine is similar to leucine in struc-ture and the L98I recombinant was therefore expected to have lessor no effect on azole susceptibility. In contrast, the susceptibilitytesting of the recombinant isolate showed an increase in azoleresistance similar to the TR/L98H azole phenotype. Unlike the dif-ferent hypotheses of the amino acid changes in codon 98, all alter-ations in codon 98 decreased the azole susceptibility. Thisdiscrepancy could be explained by the fact that the resistant phe-notype is not associated with a single mutation but with a combi-nation of a codon 98 alteration together with the 34 bp TR. It waspreviously shown and confirmed in this study that either mutationalone has limited impact on the azole phenotype, but when bothmutations are introduced simultaneously a synergistic effect withan azole resistant phenotype is observed. When both mutations areintroduced separately the MIC for itraconazole is increased to 1–2 (mg/ml), however combined itraconazole is greater then16 (mg/ml). Therefore the mutations do not show an additive ef-fect on azole resistance but a synergistic effect. The impact of the34 bp insertion in the promoter region of the gene could thereforedominate subtle changes in azole phenotype caused by amino acidalterations introduced at codon 98. Introducing amino acidchanges in conserved regions could possibly have an impact notuntil a 34 bp TR increases the expression of the protein by 5–8-fold(Mellado et al., 2007; Arendrup et al., 2010). Another explanation isthat the current methods available to measure the azole phenotypeare not very accurate. Both CLSI and EUCAST standards allow a 3-log two-step range of MICs corresponding to the best achievableprecision (Arendrup et al., 2009). Using EUCAST methodology 51fluconazole MIC determinations of a single Candida glabrata isolateshowed a range of two-2-fold dilution steps (Arendrup et al.,2009). As a consequence subtle changes in phenotype due to sub-stitution of specific amino acids at codon 98 may not be detected.For A. fumigatus no crystal structure of the CYP51 is available andin this study a homology model was constructed by using the crys-tal structure of human lanosterol 14a-demethylase. In this waymolecular dynamic simulations can be performed and mutationscan be located by pinpointing them in the CYP51 homology model.However, by using this method the model cannot reflect the exactappearance and properties of the A. fumigatus CYP51 protein. Re-sults originating from these studies need to be considered as a pre-diction containing uncertainties. As was shown previously by usingthe CYP51A homology model mutations in the cyp51A gene, knownfrom literature to be correlated to azole resistance, could be lo-cated close to the opening of one of the two ligand access channels.




They are thought to disturb the interactions necessary for stabledocking towards the heme centre. The L98H mutation is howeverlocated far from both openings of the ligand access channels andthe mechanism causing an azole-resistant phenotype must there-fore take place in more subtle and indirect way. As was shown inthe molecular dynamic simulations the L98H mutation changesthe capacity of the ligand access channels by a change in flexibilityand thus, alterations in the protein sequence might affect dynamicproperties of the molecule rather than immediate protein–inhibi-tor contacts as was also discussed by Podust et al. (2001b). Themulti sequence alignment in codon 98 show that the amino acidleucine in codon 98 in A. fumigatus is highly conserved. An align-ment of different fungi showed that 14 of 20 fungi contained theamino acid leucine corresponding with codon number 98 in A.fumigatus. Only 6 fungi contained a different amino acid at codon98 (Podust et al., 2001b). The recombinants showed that all intro-duced amino acids other than leucine in combination with the34 bp TR decreased the azole susceptibility. This confirms theimportance of the presence of the highly conserved leucine in co-don 98 in CYP51A for maintaining the structure and functionalityof the ligand access channels that are used by the azoles.

Amino acid substitutions have previously been introduced inthe cyp51A gene in A. fumigatus by homologous recombination ofPCR products or by expression of the gene by using the yeast Sac-charomyces cerevisiae as a model system (Mellado et al., 2007;Alcazar-Fuoli et al., 2010; Martel et al., 2010). In this study wepresent an efficient transformation system for A. fumigatus inwhich gene functions can be studied by introducing site-directedamino acid substitutions and investigate structural changes orchanges in biological activity in the presence of the full backgroundof an A. fumigatus genome. By introducing a dominant selectablemarker such as hygromycin, recombinants can be selected inde-pendently from any changes introduced in azole phenotype in con-trast to the recombinant experiments of the TR/L98H mutationsperformed previously (Mellado et al., 2007). Thereby no mutationsor physiological changes can be induced during selection underazole pressure and recombinants that did not change in expectedphenotype are as well recovered and available for analysis.

Conflicts of interest

Paul Verweij received research grants from Pfizer, Gilead,Merck, Basilea, Schering-Plough and BioRad. Other authors haveno financial disclosures.

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