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Biochemical characterization of terbinafine-resistant

Trichophyton rubrum isolates

BERTRAND FAVRE*%, MAHMOUD A. GHANNOUM$ & NEIL S. RYDER*

*Novartis Research Institute, Vienna, Austria and$Center for Medical Mycology, Depar tment of Dermatology, Case Western

Reserve University and University Hospitals of Cleveland, Cleveland, OH, USA

We investigated the biochemical basis for resistance in six sequential clinical

isolates ofTrichophyton rubrum , from the same patient, which exhibited high-level

primary resistance to terbinafine. Cellular ergosterol biosynthesis was measured by

incorporation of [14C]acetate, and microsomal squalene epoxidase was assayed by

conversion of [3H]squalene to squalene epoxide and lanosterol. Direct comparison

was made with a terbinafine-susceptible reference strain of T. rubrum in which

squalene epoxidase was previously studied. Resistant isolates displayed normal

cellular ergosterol biosynthesis, although slight accumulation of radiolabeled

squalene suggested reduced squalene epoxidase activity. Ergosterol biosynthesis

in the resistant isolates was only inhibited by terbinafine concentrations above 1 mg/ml (IC50 5 mg/ml). In the reference strain, ergosterol biosynthesis was eliminated by

terbinafine at 0.03 mg/ml in accordance with historical data. There was no

significant difference in sensitivity between the six resistant isolates. Squalene

epoxidase from resistant strains was three orders of magnitude less sensitive than

normal enzyme to terbinafine (IC50 of 30 mmol/l and 19 n mol/l respectively). The

epoxidase in the resistant strains was also unresponsive to tolnaftate. Resistance to

terbinafine in these T. rubrum isolates appears to be due to alterations in the

squalene epoxidase gene or a factor essential for its activity.

Keywords dermatophyte, resistance, Terbinafine, Trichophyton

Introduction

The allylamine terbinafine and the related compounds

naftifine and butenafine are selective inhibitors of

fungal squalene epoxidase (SE) [1]. This property is

shared by the thiocarbamates tolnaftate and tolciclate

[2], which are chemically distinct from the allylamines.

Accumulation of squalene appears to be toxic to

filamentous fungi, especially in dermatophytes [3].

Despite the extensive use of terbinafine for the treat-

ment of dermatophytosis and onychomycosis, the first

clinically confirmed case of terbinafine resistance in

dermatophytes was only recently reported [4]. Six

sequential clinical isolates of Trichophyton rubrum

originating from nail of a single patient who failed on

therapy with oral terbinafine, were found to be resistant

to terbinafine, with MIC ]/4 versus 5/0.001 mg/ml for

normal strains. They were cross-resistant to the other

SE inhibitors tested, butenafine, naftifine, tolnaftate

and tolciclate, but normally susceptible to antifungals

with a different mode of action, such as azoles and

griseofulvin [4], as well as amorolfine and amphotericin

B (N. S. Ryder, unpublished data, 2000). These results

suggested a target-specific mechanism of resistance.

The aim of the present study was to determine at the

biochemical level whether resistance was indeed linked

to abnormalities in the ergosterol biosynthesis pathway

and SE enzyme.

Correspondence: Neil S. Ryder, Infectious Diseases Biology, Novartis

Institutes for BioMedical Research Inc., 100 Technology Square,

Cambridge, MA 02139, USA. Tel.: '/1 617 871 3143; Fax: '/1 617

871 7047; E-mail: [email protected]

Received 8 September 2003; Accepted 16 December 2003

% Present address: Laboratory of Dermatology, University Hospital

CHUV, Lausanne, Switzerland.

2004 ISHAM DOI: 10.1080/13693780410001661482

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Materials and methods

Fungal isolates

Six consecutive clinical isolates ofT. rubrum designated

(Novartis Fungal Index) NFI5146 to NFI5151, includ-

ing the baseline isolate NFI5146, were derived from

nail material of a single patient with toenail onycho-mycosis sampled at intervals. Details of the clinical

origin, culturing and mycological testing of these

isolates have been provided previously [4]. For compar-

ison, the reference strain T. rubrum NFI1895 was

tested in parallel as this strain has been studied

extensively in terms of cellular ergosterol biosynthesis

and SE inhibition by terbinafine [5].

Antifungal drugs

Terbinafine (batch 97045) was synthesized at Novartis

(Basel, Switzerland). Tolnaftate was purchased from

Sigma (St Louis, MO, USA, catalogue T-6638) andamorolfine was obtained from Hoffmann La Roche

(Basel, Switzerland).

Cellular ergosterol biosynthesis

The method for measurement of ergosterol biosynth-

esis inhibition was modified from a previously de-

scribed procedure [6]. Inocula were prepared from

stocks frozen at /808C. Cultures were grown in 500-

ml conical flasks containing 150 ml Sabouraud 2%

dextrose broth pH 6.5, inoculated at 3)/104 to 5)/105

colony forming units (c.f.u.) per ml and incubated on a

rotary shaker (LH Engineering, Stoke Poges, UK) at100 r.p.m. and 308C for 4 to 6 days until adequate

fungal mycelium was obtained. Because of the slow

growth rate of dermatophytes in culture, the organisms

were still in the growth phase when harvested. Cultures

were harvested by gravity filtration on a glass sinter

(porosity 1), washed in K-Na-PO4 buffer, pH 6.5, and

resuspended in incubation medium. Incubation med-

ium consisted of yeast nitrogen base medium without

(NH4)2SO4 and amino acids, containing 2% glucose

(w/v) and 25 mmol/l K-Na-PO4 buffer pH 6.5. Cell

suspensions were adjusted to a density equivalent to

3/4 mg dry weight per ml.

Incubations were performed in 50-ml conical glass

flasks in a shaking water bath at 308C. Inhibitors were

dissolved in DMSO and added at 100-fold final

concentration. Controls received solvent only. Cell

suspension (5 ml) was pre-incubated 10 min with the

test compound and the assay initiated by addition of 25

ml substrate mixture prepared from [U-14C]-acetate

(Amersham Biosciences, Vienna, Austria, 1 mCi/5 ml,

50/62 mCi/mmol), 1 mol/l Na-acetate and distilled

water mixed in the ratio 2:1:7. After an incubation time

of 2 h, assays were terminated and cells harvested by

filtration on Whatman (Maidstone, UK) 2.5 cm GF/A

glass fiber filter discs. The cell mats were transferred to

glass tubes, to which was added 1.5 ml 30% (w/v) KOH

in 90% (v/v) ethanol, 1.5 ml ethanol and 0.3 ml 2%(w/v) 1,2,3-benzenetriol (pyrogallol) in ethanol. Tubes

were closed with screw caps and left overnight in the

dark at room temperature, then incubated 30 min at

808C. After cooling, 1.5 ml water was added and the

non-saponifiable lipids were extracted twice with 2 ml

petroleum ether (bp 40/608C). The extract was washed

with 3 ml distilled water, evaporated to dryness and

dissolved in cyclohexane. The non-saponifiable lipids

were then separated by thin layer chromatography as

described previously [6].

The TLC sheets were analyzed by electronic auto-

radiography using the Instant Imager (Canberra Pack-

ard, Vienna, Austria, with software Packard Image forWindows 2.10). The Instant Imager provided both an

autoradiographic image of the TLC sheet and a

quantitative determination of the distribution of radio-

activity. The non-specific radioactivity which accumu-

lated at the origin of the plates was not included in the

calculations. After automatic background subtraction,

the radioactivity was calculated for each band (squa-

lene, sterols etc.) as a percentage of the sum of the

designated products. These data were transferred from

the Imager results file to a template in Excel 97 for

calculation of percent inhibition of ergosterol biosynth-

esis at each drug concentration and the IC50 value.

Each experiment was performed with duplicate incuba-

tions, and repeated so that each final value was derived

from four separate determinations.

Squalene epoxidase assay

Trichophyton rubrum microsomes (170 000 g pellet)

were prepared from mycelium frozen in liquid nitrogen

[5]. Microsomal SE activity was assayed by measure-

ment of incorporation of [4,8,12,13,17,21-3H]squalene

(American Radiochemicals, St Louis, MO, USA) into

squalene epoxide and lanosterol exactly as previously

described [5], except for the final protein concentration

of 3 mg protein/ml (instead of 2 mg/ml) and the

incubation time of 60 min (instead of 45 min).

Inhibitors were first dissolved in DMSO and then

diluted 100-fold into the assay to achieve the desired

final concentration. Experiments were performed in

duplicate and repeated with each inhibitor twice. IC50values were calculated with the program Origin 6.1

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(OriginLab Corporation, Northampton, MA, USA)

using the curve fitting function.

Results

Reduced sensitivity of cellular ergosterol biosynthesis

Analysis of the labeled non-saponifiable lipids in the

absence of drug revealed that in the resistant isolate

NFI5150 around 25% of the radioactivity was asso-

ciated with squalene (Table 1). Similar results were

observed with the other five resistant isolates (not

shown). By contrast, in the reference strain NFI1895

no significant radioactivity could be detected comigrat-

ing with squalene or other ergosterol precursors (Table

1), in agreement with earlier studies. This difference

indicates that the flux of sterol precursors from

[14C]acetate to ergosterol was not identical in the

susceptible strain NFI1895 and the resistant isolates,

and suggests the possibility of lower SE activity in thelatter. With the reference strain NFI1895, a terbinafine

concentration of 0.004 mg/ml was sufficient to inhibit

cellular ergosterol biosynthesis by 50%, while in the

resistant isolates the IC50 was about 1000-fold higher

(Table 2), confirming that inhibition of ergosterol

biosynthesis underlies the antifungal activity of terbi-

nafine in both susceptible and resistant isolates.

Low sensitivity of squalene epoxidase

A simple way of distinguishing target-based mechan-

isms of resistance from the involvement of membrane

transporters is by testing the sensitivity of the target tothe inhibitor in cell-free conditions, ruling out the

action of transporters [7]. As the cellular studies

revealed no apparent differences in sensitivity between

the resistant isolates, the technically demanding studies

with the SE enzyme were performed in only two of the

resistant isolates. The specific activities of SE measured

under the standard assay conditions were similar in the

three isolates: 0.049/0.01 nmol/h/mg protein (9/SD,n0/5) for reference strain NFI1895, 0.049/0.01 nmol/h/

mg protein (9/SD, n0/4) for NFI5146, and 0.059/0.01

nmol/h/mg protein (9/SD, n0/4) for NFI5150. How-

ever, a dramatic difference was found between the

normal and resistant strains with respect to sensitivity

Table 1 Comparison of radiolabeling patterns of ergosterol and intermediates in cells of Trichophyton rubrum NFI1895 (reference strain) and

NFI5150 (resistant isolate) incubated with [14C]acetate and increasing concentrations of terbinafineaStrain Percent total counts measured in: (g/

ml)

Strain Terbinafine Ergosterol 4a-methyl-sterol Lanosterol Squalene

NFI1895 0 929/1 29/0.5 49/1 29/0.3

NFI1895 0.001 909/2 29/0.4 59/1 39/1

NFI1895 0.003 479/28 29/1 49/2 479/31

NFI1895 0.01 139/8 19/0.3 29/0.2 859/8NFI1895 0.03 59/2 19/0.2 19/0.2 949/3

NFI5150 0 679/3 29/0.4 59/2 259/3

NFI5150 0.01 679/3 39/0.4 69/2 249/4

NFI5150 0.1 649/5 29/0.4 59/2 279/5

NFI5150 1 589/6 39/0.4 69/2 339/6

NFI5150 10 129/2 19/0.3 59/3 819/2

aResults are given as mean9/standard deviation for four data points (two independent experiments each performed with duplicate incubations).

Radiolabeling and analysis of lipids were performed as described in the Methods section.

Table 2 Sensitivity of cellular ergosterol biosynthesis to terbinafine

in reference and resistant strains

Strain Visita Type Terbinafine IC50 (mg/ml)b

NFI1895 / Reference 0.0049/0.002

NFI5146 0 Resistant 5.59/0.9






aClinical history: visit 0 was for screening and culture; therapy with

terbinafine (250 mg once daily) lasting 24 weeks started at visit 1, and

subsequent visits were at 6-week intervals [4]. bMean9/SD for four

separate determinations.

Table 3 Sensitivity of microsomal SE activity from reference and

resistant strains to terbinafine, tolnaftate and amorolfineStrain IC50(mg/ml)a

Strain Terbinafine Tolnaftate Amorolfine

NFI1895 0.006 0.05 9

NFI5146 11 /33 19

NFI5150 9 /33 15

aMean of IC50 values from two separate determinations, each of

which did not vary by more than 30% from the mean value.


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of SE to terbinafine. Microsomal SE activity from

resistant isolates NFI5146 and NFI5150 was 1000-fold

less sensitive to terbinafine than that from the reference

strain NFI1895 (Table 3). A similar difference was

observed with tolnaftate, in agreement with the cross-

resistance of the isolates to thiocarbamates [4]. On the

other hand, amorolfine, which also weakly inhibits T.

rubrum SE [5], showed roughly similar activity against

microsomal SE from reference and resistant strains

(Table 3).

Discussion

At present, very little is known concerning drug-

resistance mechanisms in dermatophytes. The recent

discovery of the first terbinafine-resistant dermato-

phyte associated with clinical failure [4] raised the

important issue of the molecular mechanism of resis-

tance to this widely used drug. Several mechanisms of

resistance to another class of ergosterol biosynthesisinhibitors, the azoles, have been established in Candida

species [8]. These include modification of the target

enzyme, upregulation of the ergosterol biosynthesis

pathway and, (most commonly) induction of drug

efflux pumps leading to resistance to multiple drugs,

including terbinafine in some cases [9]. We previously

showed that the terbinafine-resistant T. rubrum isolates

were fully cross-resistant to several classes of SE

inhibitors, but displayed normal susceptibility to azoles

and other antifungals [4]. This suggested that resistance

was associated with changes in the target enzyme rather

than with overexpression of efflux pumps. The data

from the present study support this hypothesis. Bothcellular ergosterol biosynthesis and cell-free SE activity

from resistant isolates displayed reduced sensitivity to

terbinafine by several orders of magnitude. The resis-

tant cells possessed a functioning ergosterol biosynth-

esis pathway, but with an unusually high level of

radiolabeled squalene in the control cells, which would

be consistent with a reduced activity of squalene SE

although alternative explanations are possible. Direct

enzyme assays confirmed the existence of a functional

SE in the resistant isolates and its abnormally low

sensitivity to terbinafine. The specific activity of the

resistant microsomal enzyme was similar to that from

reference strain NFI1895, thus contradicting the evi-

dence for reduced SE activity in intact cells. However,

in vitro enzyme assays containing excess concentrations

of cofactors and substrates may not detect differences

in activity within living cells where these factors may be

limiting. Interestingly, the resistant SE was also un-

responsive to tolnaftate suggesting that allylamines and

thiocarbamates may share a common binding site. In

contrast, the sensitivity of the terbinafine-resistant SE

to amorolfine was not very different from that of a

normal enzyme suggesting that amorolfine has a

mechanism of inhibition distinct from that of terbina-

fine and tolnaftate. Amorolfine is a weak inhibitor ofT.

rubrum SE [5], while the principal antifungal targets of

the drug are sterol-D14-reductase and sterol-D7-D8-

isomerase [10]. In the original report of the resistant

isolates [4], there was an apparent further decrease in

susceptibility (detectable only by the broth macro-

dilution assay) over the course of terbinafine treatment.

Since the sensitivity of ergosterol biosynthesis remained

essentially unchanged over the sequence of isolates (see

Table 2), this subtle change must have been due to a

different, as yet unidentified, mechanism.

The six resistant isolates, including one taken at

baseline before start of therapy, were obtained sequen-

tially from the same patient and displayed identical

random amplified polymorphic DNA (RAPD) profiles

[4], indicating that they represent a single genotypicstrain. Several lines of evidence point to a stable genetic

alteration, probably in the SE gene, as the primary

cause of terbinafine resistance in this case. These

include (i) the drastic reduction in enzyme sensitivity,

(ii) the rare occurrence of resistance, and (iii) the stable

nature of the resistance in cultures taken over 24 weeks

of therapy and after subculturing in the absence of the

drug. Previous studies have shown that clinical failure

of terbinafine was not associated with development of

in-vitro resistance during therapy [11]. However, it

appears that strains expressing primary resistance to

the drug occur at a low frequency in the T. rubrum

population. Occasional instances of high terbinafineMIC values have also been reported, but without

clinical information [12]. Fungal SE genes have been

cloned and sequenced from the yeasts Saccharomyces

cerevisiae [13] and Candida albicans [14]. The amino

acid substitution Leu0/Phe251, apparently associated

with a terbinafine-resistant phenotype, was identified in

the S. cerevisiae gene [14], and a second mutation

leading to the substitution Pro0/Ala430 was recently

reported to confer partial loss of sensitivity to inhibi-

tion by terbinafine [15]. Investigations are in progress

to determine whether amino acid substitution(s) are

indeed fully responsible for terbinafine resistance in T.

rubrum, and whether other mechanisms of resistance

can also be detected.

Acknowledgements

We thank Wolfgang Mlineritsch for performing the

cellular sterol biosynthesis assay.


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