Aspects of the progesterone response in Hortaea werneckii: Steroid detoxification, protein induction and remodelling of the cell wall

Article abstract of DOI:10.1016/j.steroids.2008.08.004

Progesterone in sublethal concentrations temporarily inhibits growth of Hortaea werneckii. This study investigates some of the compensatory mechanisms which are activated in the presence of progesterone and are most probably contributing to escape from growth inhibition. These mechanisms lead on the one hand to progesterone biotransformation/detoxification but, on the other, are suggested to increase the resistance of H. werneckii to the steroid. Biotransformation can detoxify progesterone efficiently in the early logarithmic phase, with mostly inducible steroid transforming enzymes, while progesterone biotransformation/detoxification in the late logarithmic and stationary phases of growth is not very efficient. The relative contribution of constitutive steroid transforming enzymes to progesterone biotransformation is increased in these latter phases of growth. In the presence of progesterone, activation of the cell wall integrity pathway is suggested by the overexpression of Pck2 which was detected in the stationary as well as the logarithmic phase of growth of the yeast. Progesterone treated H. werneckii cells were found to be more resistant to cell lysis than mock treated cells, indicating for the first time changes in the yeast cell wall as a result of treatment with progesterone.

Full text of DOI:10.1016/j.steroids.2008.08.004

steroids 7 3 ( 2 0 0 8 ) 1465–1474
available at www.sciencedirect.com
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Aspects of the progesterone response in Hortaea werneckii:
Steroid detoxification, protein induction and remodelling of
the cell wall
Lidija Krizˇancˇic´ Bombeka, Ajda Lapornika, Marjeta Ukmara, Maja Matisa,b,c
Bronislava Cresˇnar , Jasna Peter Katalinic´ , Marija Zakelj-Mavricˇ
,
a
b,c
a,∗
ˇ
ˇ
a
Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, SI-1000 Ljubljana, Slovenia
Institute for Medical Physics and Biophysics, Faculty of Medicine, University of Muenster,
b
Robert-Koch Strasse 31, D-48149 Muenster, Germany
c
Institute of Molecular Medicine and Genetics, Medical College of Georgia, 1120 15th Street, Augusta, GA 30912, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Progesterone in sublethal concentrations temporarily inhibits growth of Hortaea werneckii.
This study investigates some of the compensatory mechanisms which are activated in
the presence of progesterone and are most probably contributing to escape from growth
inhibition. These mechanisms lead on the one hand to progesterone biotransforma-
tion/detoxification but, on the other, are suggested to increase the resistance of H. werneckii
to the steroid. Biotransformation can detoxify progesterone efficiently in the early loga-
rithmic phase, with mostly inducible steroid transforming enzymes, while progesterone
biotransformation/detoxification in the late logarithmic and stationary phases of growth is
not very efficient. The relative contribution of constitutive steroid transforming enzymes
to progesterone biotransformation is increased in these latter phases of growth. In the
presence of progesterone, activation of the cell wall integrity pathway is suggested by the
overexpression of Pck2 which was detected in the stationary as well as the logarithmic phase
of growth of the yeast. Progesterone treated H. werneckii cells were found to be more resis-
tant to cell lysis than mock treated cells, indicating for the first time changes in the yeast
cell wall as a result of treatment with progesterone.
Received 15 October 2007
Received in revised form
30 July 2008
Accepted 11 August 2008
Published on line 28 August 2008
Keywords:
Biotransformation
Detoxification
Hortaea werneckii
Cell wall
Yeast proteomics
© 2008 Elsevier Inc. All rights reserved.
1.
Introduction
In contrast to higher organisms microorganisms are capa-
ble of biotransformation and even mineralization of steroids
Steroid hormones are signalling molecules that regulate a host
of organismal functions, exerting their actions by binding to
the plasma membrane or intracellular receptors of the target
cells. Steroids are hydrophobic and relatively stable molecules
which cannot be degraded and used as a source of energy in
higher organisms. After fulfilling their task they are deacti-
vated, transformed into more soluble forms and excreted via
urine and bile into the environment [1].
[2,3]. Numerous microorganisms that inhabit human and ani-
mal tissues, such as the skin and urinary and gastrointestinal
tracts [4,5], come into contact with steroid hormones and can
transform them [6–10]. After their release from the organisms,
steroids are further degraded by mixtures of microorganisms
in the sewage treatment plants and the environment [11–14].
These processes are very important because of the effects that
undegraded steroid hormones can have on wildlife and on
∗
Corresponding author. Tel.: +386 1 543 76 45; fax: +386 1 543 76 41.
ˇ
E-mail address: Marija.Zakelj-Mavric@mf.uni-lj.si (M. Zakelj-Mavricˇ).
0039-128X/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2008.08.004

1466
steroids 7 3 ( 2 0 0 8 ) 1465–1474
man [15,16]. However, the environmental fate and the rate of
mineralization of naturally produced steroid hormones are so
far poorly characterized.
(MZKI) was maintained and grown as described [47]. Steroids
were obtained either from Sigma (St. Louis, MO) or from
Steraloids Inc. (Wilton, U.S.A.). (1,2,6,7-3H) progesterone was
supplied by NEN Du Pont (Dreiech, Germany). Lyticase, a ␤-1, 3-
glucanase from Arthrobacter luteus, was obtained from Sigma.
All other chemicals were of analytical grade and obtained from
standard suppliers.
Biotransformation of steroid hormones in the environ-
ment takes place with mixtures of microorganisms, mostly
bacteria, and the contribution of individual species is not
known precisely. The importance of individual microorgan-
isms for the production of commercially important steroids
was first realized in 1952 when Peterson and Murray of the
Upjohn Company patented the process of 11␣-hydroxylation
of steroids by Rhizopus species [17]. Certain of the synthetic
chemistry steps have since been replaced by specific microbial
transformations which are able to achieve stereo- and regio-
specific conversions of the substrates [18–22]. A currently
important area is the use of genetic engineering to improve
the capabilities of microorganisms as steroid-transforming
agents [19,23]. In addition, microorganisms which are adapted
to extreme environments have recently gained great interest
due to their specific characteristics [24].
The primary interest in steroid biotransformation was at
first focused on steroids and the production of commer-
cially valuable products. With time it became evident that
steroid hormones can provoke changes in microorganisms.
Interest in these changes was increased when the levels of
steroid hormones were suggested to affect susceptibility to
fungal infection [25–32]. Experiments with exogenous steroids
have confirmed the effects of steroids on fungal growth
[33–40]. During exposure of microorganisms to steroid hor-
mones, the expression of several members of various gene
families is increased [41–43]. Steroid transforming enzymes
were connected with defence against steroids. Microorgan-
isms are thought to induce enzymes that detoxify steroids
by transforming them into more soluble and less toxic
forms [33]. The contribution of constitutive steroid trans-
forming enzymes to steroid detoxification is presently not
clear [44].
Our studies of progesterone/yeast interaction have been
focused on the halophilic black yeast Hortaea werneckii. The
natural habitat of this eukaryotic microorganism is hyper-
saline water [45] but it is also known as the causative agent of
Tinea nigra, a nonpathologic dermal change of the palms, more
frequently observed in females and children [46]. Recently,
analysis of the H. werneckii proteome suggests the interaction
of progesterone with the cell growth and reproduction sig-
nalling pathways [42]. In the present study we have focused on
progesterone biotransformation and its contribution to pro-
gesterone detoxification in different phases of growth of the
yeast. We provide evidence for the activation of some of the
processes which might, like induction of proteins and remod-
elling of the cell wall, contribute to adaptation of the yeast to
the presence of progesterone and hence its effective escape
from growth arrest.
2.2.
Biotransformation of progesterone and product
identification
H. werneckii was grown at 28 ◦C in YNB growth medium
until early or late logarithmic and stationary growth phase.
Yeast cells were obtained by centrifugation and resuspended
in the growth medium to an optical density of A600 = 2.5.
After the addition of progesterone in N,N-dimethyl formamide
(DMF) to 64 ␮M final conc., biotransformation took place at
28 ◦C for 24 h in the absence or presence of cycloheximide,
5 ␮g/ml final conc. Steroids were extracted with chloroform
and separated by TLC [48]. The Rf values of the biotrans-
formation products were compared to those of standard
steroids. The respective areas were scraped off, eluted with
chloroform/methanol (1:1) and the structures of the biotrans-
formation products confirmed by GC–MS analysis as described
[48].
2.3.
Determination of progesterone biotransformation
profile
To elucidate the progesterone biotransformation profile the
(1,2,6,7-3H) progesterone, 16 kBq/ml final conc., was submit-
ted to biotransformation in the presence of 16 ␮M unlabelled
progesterone, under the same conditions as described above.
The steroids were extracted with chloroform and separated
by TLC as above. Radioactive compounds were visualized by
fluorography after spraying the air-dried plates with 10% 2,5-
diphenyloxazolone [48].
In a parallel experiment TLC separated labelled steroids
were scraped off and evaluated quantitatively in a liquid
scintillation counter (LKB 1214 Rackbeta liquid scintillation
counter).
2.4.
Growth inhibition assay
The growth inhibition assay was performed by growing H.
werneckii on YNB agar plates at 26 ◦C in the presence of
selected steroids. Yeast was grown to the logarithmic phase
of growth and diluted to an optical density at 600 nm (OD600
)
of 0.2. Identical volumes of 0.2 OD600 culture, as well as
of 1:10 and 1:100 serial dilutions were spotted onto agar
plates containing 32 and 320 ␮M final conc. of tested steroids.
Tested compounds were dissolved in DMF and controls were
prepared with the appropriate amount of the respective
solvent.
2.
Experimental
2.5.
Water solubility of steroids
2.1.
Strains and materials
Water solubility of steroids expressed as Interactive Analysis
(IA) log W was obtained by the IA LogP and LogW predictor
website http://www.logp.com/.
The B-763 H. werneckii strain from the microbial culture col-
lection of National Institute of Chemistry, Ljubljana, Slovenia

steroids 7 3 ( 2 0 0 8 ) 1465–1474
1467
identified proteins from the logarithmic phase of growth. The
identity of the spots of interest was confirmed using mass
spectrometric analysis as described [42].
2.6.
Lyticase sensitivity assay
Structural changes in the cell wall of H. werneckii were mon-
itored as described [49], using lyticase as the enzyme source.
H. werneckii cells in the early logarithmic phase were exposed
to 320 ␮M steroids (progesterone and other selected steroids),
32 ␮M progesterone, DMF (control) or Calcofluor White in a
100 ␮g/ml final conc. (positive control) for 4 h. After treat-
ment the cells were harvested, washed with TE buffer (10 mM
Tris–HCl, 1 mM EDTA, pH 7.5) and resuspended in 1.5 ml of the
same buffer at OD600 of approximately 0.2. After the addition
of lyticase in 220 U/ml final conc., cell lysis was followed at
room temperature by measuring OD600 of the cell suspensions
every 10 min up to 1 h.
2.10. Isolation of RNA and Northern hybridisation
H. werneckii in the early logarithmic, late logarithmic and sta-
tionary phases of growth was exposed to 320 ␮M progesterone
or mock treated with DMF (control) for 10, 25 or 50 min. Total
RNA was isolated as described [50]. The electrophoresis of
RNA, reversible staining of RNA blots with methylene blue
and hybridisation using 0.5 kb PvuII fragment of cDNA encod-
ing Hsp-1 of fungus Rhizopus nigricans were carried out as
described [43].
2.7.
Protein sample preparation and protein labelling
3.
Results
H. werneckii cultures growing in the stationary phase of growth
were either exposed to 32 ␮M progesterone or mock treated
with DMF (control). After 4 h, progesterone treated cells and
the control were harvested, washed with PGSK buffer (3.8 mM
NaH2PO4, 49.4 mM Na2HPO4, 48.4 mM NaCl, 5 mM KCl, 61 mM
glucose), frozen in liquid nitrogen, pelleted and mechani-
cally disintegrated as described [42]. Powdered frozen lysates
were then resuspended in lysis buffer (4% (w/v) CHAPS, 2 M
thiourea, 7 M urea, 30 mM Tris–HCl, pH 8.5), sonicated and cen-
trifuged at maximum speed for 10 min at 4 ◦C in a tabletop
centrifuge.
3.1.
Progesterone biotransformation profiling
Progesterone biotransformation was followed using radioac-
tively labelled progesterone in the absence and presence of
cycloheximide with H. werneckii cells from the early loga-
rithmic, late logarithmic and stationary phases of growth.
Progesterone was transformed into products with 98% effi-
ciency with H. werneckii cells from the early logarithmic
phase (Fig. 1, line 1). Quantitatively the main product was
11␣-hydroxy-progesterone which represented 34% of all the
Proteins from cell lysates were delipidated and desalted
by chloroform/methanol extraction, washed with methanol
and dissolved in lysis buffer. Protein concentration was deter-
mined with Bradford reagent (BIO-RAD). Proteins were labelled
with CyDye DIGE Fluors Cy3 and with Cy5 (Amersham Bio-
sciences, Upssala, Sweden); the two samples were combined
and analyzed as described [42].
2.8.
Protein separation and visualization
Proteins were separated and visualized as described [42].
They were separated in the first dimension on IPG gels of
pIs in the pH 4–7 range on an IPGpfor (Amersham Bio-
sciences, Uppsala, Sweden). After isoelectric focusing the IPG
strips were transferred on to 12% polyacrylamide gels and
electrophoresed in an Ettan DALTtwelve system (Amersham
Biosciences, Uppsala, Sweden) overnight at 2 W per gel at
18 ◦C.
Fig. 1 – Autoradiogram of the progesterone
2D gels were scanned directly between glass plates with
a Typhoon Imager (Amersham Biosciences, Upssala, Sweden)
with the excitation and emission wavelengths for Cy3 and
Cy5. Scanned gels were fixed in 10% methanol, 7% acetic
acid for 2 h, stained in Sypro Ruby stain (BIO-RAD) overnight,
destained (10% methanol, 7% acetic acid) for 1 h and scanned
with the appropriate wavelength for Sypro Ruby stain. Dif-
ferentially expressed proteins were determined as described
[42].
biotransformation products separated by TLC. Lines
represent biotransformation profiles of radiolabelled
progesterone with H. werneckii from the early (1 and 2), late
logarithmic (3 and 4), and stationary (5 and 6) phases of
growth. The biotransformations were performed in the
absence (1, 3 and 5) and presence (2, 4 and 6) of
cycloheximide. 5␣P: 5␣-pregnane-3,20-dione; Ta:
testosterone acetate; P: progesterone; A: androstenedione;
20␣OHP: 20␣-hydroxy-progesterone; T: testosterone;
14OHP: 14-hydroxy-progesterone; 6␣OHP:
2.9.
Mass spectrometry analysis
6␣-hydroxy-progesterone; 11␣OHP:
11␣-hydroxy-progesterone mark the Rf values of the main
identified products.
H. werneckii proteins from the stationary phase of growth were
separated on 2D gels and compared to those of the previously

1468
steroids 7 3 ( 2 0 0 8 ) 1465–1474
Table 1 – Some characteristics of progesterone biotransformation by H. werneckii
Type of biotransformation
Identified progesterone
biotransformation products
Effect of
cycloheximide
Prevailing growth phase
Early logarithmic
I
Hydroxylation
11␣-Hydroxy-progesterone
14-Hydroxy-progesterone
6␣-Hydroxy-progesterone
NSI
NSI
Side chain cleavage
Reduction
Late logarithmic and
stationary
Testosterone acetate
Androstenedione
Testosterone
All phases
4-Pregnen-20␣-ol-3-one
5␣-Pregnane-3,20-dione
Pregnan-3-ol-20-one
Pregnane-3,20-diol
I – significant inhibition of the respective biotransformation detected; NSI – no significant inhibition of the respective biotransformation detected.
recovered steroids. The involvement of inducible steroid
transforming enzymes in progesterone biotransformation
was suspected on the basis of the changed biotransfor-
mation profile in the presence of cycloheximide (Fig. 1,
line 2). The presence of this inhibitor resulted in an
increase of the percentage of untransformed progesterone
from 2% to 35%, the amount of the recovered 11␣-hydroxy-
progesterone was lowered to 8% while the amount of
recovered androstenedione increased to 20% of the recovered
steroids. In contrast, in the late logarithmic and station-
ary phases of growth, progesterone was transformed less
effectively, with only 30% efficiency (Fig. 1, lines 3 and 5).
Androstenedione was found to be the most important prod-
uct, representing 15% of all the recovered steroids, while the
amount of 11␣-hydroxy-progesterone was lowered to 3%. The
presence of cycloheximide did not significantly affect the
biotransformation with H. werneckii cells from the late log-
arithmic or stationary phase of growth (Fig. 1, lines 4 and
6).
Analysis of the respective biotransformation products
revealed three main types of reactions: hydroxylation at
different sites of the steroid nucleus, cleavage of the pro-
gesterone side chain at C17 and reduction of the 3- and/or
20-keto groups and ꢀ4-double bond (Table 1, Fig. 1). Hydrox-
ylation prevailed in the early logarithmic phase of yeast
growth and was catalyzed by inducible enzymes, while
side chain cleavage occurred predominantly in the late log-
arithmic and stationary phases of growth and was not
affected by the presence of cycloheximide (Fig. 1, Table 1).
Reduction was a minor reaction and was catalyzed by con-
stitutive enzymes in all phases of growth of H. werneckii
(Table 1).
3.2.
The contribution of different steroid transforming
enzymes to progesterone detoxification
The contribution of the individual biotransformation reac-
tions to progesterone detoxification was estimated by
comparing the inhibitory action of selected progesterone
biotransformation products on the growth of H. werneckii with
that of progesterone. All the tested progesterone derivatives
were found to be less toxic than progesterone. Hydroxylation
of progesterone, e.g., at the 11␣ position, prevails in the
early logarithmic phase (Table 1) and can be considered as
effective detoxification (Table 2). Induction of hydroxylases
in this phase of growth contributes to the defence of the
yeast. The most problematic modification of progesterone
from the viewpoint of detoxification is the cleavage of the
side chain of progesterone. Androstenedione, as one of the
important products of this modification, was found to be
only slightly less toxic than progesterone (Table 2). This
steroid was detoxified when androstenedione was reduced
to non-toxic testosterone in a reaction catalyzed by a consti-
tutive 17␤-hydroxysteroid dehydrogenase (17␤-HSD) from H.
werneckii (Table 2). Since the equilibrium of the reaction with
the 17␤-HSD was found to be shifted towards reduction [51],
constitutive 17␤-HSD contributes to steroid detoxification.
The third type of reaction, e.g., reduction of the 20-keto
group of progesterone, was only slightly less effective than
hydroxylation of the steroid (Table 2).
3.3.
Polarity of progesterone biotransformation
products and their toxicity
Hydroxylation of progesterone was found to increase water
solubility of the steroid, as suggested by the predicted IA log W
values for the hydroxylated derivatives of progesterone. At the
same time, a decrease of toxicity was observed, as shown for
11␣-hydroxy-progesterone (Table 2).
Seven additional progesterone biotransformation products
were identified; most of them were formed with H. werneckii
from the early logarithmic phase. Pregnane-3,6,20-trione,
pregna-4,7-diene-3,20-dione, pregna-4,16-diene-3,20-dione,
methyl-testosterone,
pregnane-3,20-dione,
21-hydroxy-
The connection between the polarity of progesterone
derivatives and their toxicity was further investigated by
growth inhibition assays with a water-soluble form of proges-
terone (progesterone WS) and pregnenolone. Neither steroid,
with higher water solubilities than progesterone, was found
to inhibit H. werneckii growth (Table 2). The same effect was
progesterone and pregnenolone were present in small
amounts and were not expected to contribute significantly
to progesterone detoxification. The presence of very small
amounts of additional steroids in the respective TLC areas
(Fig. 1) cannot be excluded.

steroids 7 3 ( 2 0 0 8 ) 1465–1474
1469
at C20 or C17. Therefore a steroid with the same functional
groups from the estrane series, 4-estrene-3,17-dione, was
tested for its toxicity on the yeast. It was also found to be toxic
(Table 2), suggesting some of the common structural charac-
teristics of steroids toxic to H. werneckii.
Table 2 – Water solubility of progesterone
biotransformation products and other selected steroids
and their potency in inhibiting H. werneckii growth
Steroid
IA log W
H. werneckii
growth
In addition, two steroids of alternative stereochemistry, 5␣-
androstane-3,17-dione and 5␤-androstane-3,17-dione, were
found to inhibit growth of H. werneckii with almost the same
efficiency (Table 2).
inhibition
Substrate
Progesterone
−4.72
I
Biotransformation products
11␣-Hydroxy-progesterone
14-Hydroxy-progesterone
6␣-Hydroxy-progesterone
Testosterone acetate
Androstenedione
−3.44
−3.76
nd
NSI
nd
NSI
nd
I
NSI
i
NSI
NSI
NSI
3.5.
The expression of the selected proteins in H.
werneckii in the presence of progesterone
−4.64
−4.40
−4.20
−4.57
−4.84
−4.75
−5.00
We studied the effect of progesterone on the expression of
H. werneckii proteins during the logarithmic and stationary
phases of growth. Protein kinase C-like 2 (Pck2) and prolif-
erating cell nuclear antigen (PCNA) were overexpressed in
the presence of progesterone during the stationary phase of
growth (Fig. 2). This is in accordance with our previous results
showing an increase during logarithmic phase [42]. Hsp70 was
not observed as one of the identified proteins with changed
expression level during stationary phase. Its level did not
change significantly in response to the steroid (Fig. 2), in con-
trast to Hsp70 overexpression in the presence of progesterone
during the logarithmic phase of growth [42].
To further evaluate the changes following application of
progesterone, we determined the H. werneckii Hsp70 tran-
script level by Northern blot analysis. Temporal analyses of
Hsp70 mRNA abundance were carried out during treatment
of the yeast grown to the early logarithmic, late logarith-
mic and stationary phases with progesterone (Fig. 3). The
Hsp70 mRNA level was nearly undetectable when DMF was
added to the growth medium in the early logarithmic and
late logarithmic phases of growth, while the transcript level
of Hsp70 was increased within 10 min of exposure of yeast
to progesterone. In contrast, in the stationary phase of H.
werneckii growth the amount of Hsp70 mRNA is nearly the
same before and after exposure of the yeast to progesterone
(Fig. 3).
Testosterone
4-Pregnen-20␣-ol-3-one
5␣-Pregnane-3,20-dione
Pregnan-3-ol-20-one
Pregnane-3,20-diol
Other selected steroids
Pregnenolone
Progesterone WS
11␤-Hydroxy-androstenedione
5␣-Androstane-3,17-dione
5␤-Androstane-3,17-dione
17␣-Methyl-testosterone
4-Estrene-3,17-dione
−4.17
NSI
NSI
NSI
I
I
i
I
Water soluble
−3.42
−4.43
−4.43
−4.42
−4.02
I – stronger inhibition of H. werneckii growth at 320 ␮M and weak at
32 ␮M concentration of the respective steroid; i – weak inhibition
of H. werneckii growth at 320 ␮M concentration and no significant
inhibition at 32 ␮M concentration of the respective steroid; NSI –
no significant inhibition detected; nd – not determined; IA log W
– predicted water solubility of steroids as obtained by interactive
analysis (IA) predictor website http://www.logp.com/.
observed when the toxicity of androstenedione was compared
to that of 11␤-hydroxy-androstenedione (Table 2). On the other
hand an additional nonpolar methyl group of 17␣-methyl-
testosterone lowered water solubility and resulted in slightly
increased toxicity (Table 2). These results are in accordance
with the above hypothesis that reactions which increase water
solubility of the steroid decrease its toxicity.
3.6.
Changes in the cell wall of H. werneckii in the
A closer look at the products of the second group of reac-
tions, which lead to C19 steroids, shows that the transformed
products are also more water soluble than progesterone,
although the increase is much smaller than that for hydrox-
ylated derivatives of progesterone. Of these derivatives,
androstenedione was still found to inhibit growth of H. wer-
neckii only slightly less efficiently than progesterone.
The reduction reactions of progesterone from the third
group of progesterone biotransformations resulted in prod-
ucts with only slightly increased, as for 4-pregnen-20␣-ol-3-
one, or even decreased water solubility (Table 2). None of
these products was found to be an inhibitor of H. werneckii
growth.
presence of progesterone
Possible structural changes in the cell wall of H. werneckii as
a result of exposure to progesterone were investigated using
the lyticase sensitivity assay. The results indicate decreased
sensitivity to lyticase for H. werneckii cells treated with pro-
gesterone (Fig. 4). The effect was most clearly seen with cells
from the early logarithmic phase of growth, but could only just
be observed with cells from the stationary phase of growth
(data not shown). The effect was found to be specific for the
free form of progesterone, being only just observed with the
water-soluble form of progesterone (Fig. 4A). Exposure of H.
werneckii cells to progesterone resulted in an effect similar
to that on exposure to Calcofluor White and concentration
dependent (Fig. 4B) but was only just observed with the non-
toxic steroid 11␣-hydroxy-progesterone (Fig. 4A). Intra-assay
variability of the results of duplicate experiments was low
(data not shown) while inter-assay/inter-sample variability
was higher. The results suggest some differences in the cell
3.4.
Common structural characteristics of H. werneckii
inhibitory steroids
Progesterone and androstenedione, both toxic to H. werneckii,
contain a 3-keto-4-ene structure and an additional keto group

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steroids 7 3 ( 2 0 0 8 ) 1465–1474
In our previous study progesterone was found to cause
a transitional arrest of cell growth and a change in gene
expression in H. werneckii [42]. Here, we were interested in
progesterone biotransformation/detoxification by the same
microorganism. The major progesterone biotransformation
products suspected to have the greatest effect on progesterone
detoxification were identified from different phases of H. wer-
neckii growth (Table 1). We compared the amounts of identified
products obtained during biotransformation in the absence
and presence of cycloheximide. In this way we were able to
study the contribution of different types of biotransforma-
tion to progesterone detoxification and suggest the role of
constitutive and inducible steroid transforming enzymes. The
results show that induction of steroid transforming enzymes
by progesterone constitutes an effective part of the defence
of the yeast in the early logarithmic phase of growth. Our
data confirm the hypothesis that reactions with inducible
steroid transforming enzymes, e.g. hydroxylases, increase the
polarity of steroids and effectively contribute to their detoxifi-
cation (Tables 1 and 2). This type of defence is less effective
in the late logarithmic and stationary phases of growth of
H. werneckii. The yield of biotransformation products in these
phases of growth is much lower and some of the products,
e.g. androstenedione, are still toxic (Fig. 1 and Table 2). The
yeast appears not to be able to respond, by increasing the
expression of detoxifying enzymes, to the same extent as in
the early logarithmic phase of growth. In the late logarithmic
and stationary phases the relative contribution of constitu-
tive steroid transforming enzymes, e.g., 17␤-HSD, is increased.
These enzymes are less effective in detoxifying progesterone
and its derivatives. At the same time we could not establish a
clear relationship between the water solubility of biotransfor-
mation products of constitutive steroid transforming enzymes
and their toxicity (Table 2).
Fig. 2 – The effect of progesterone on the expression of
proteins in H. werneckii during the stationary phase of
growth. (A) Overlay of 2D-GE images of control sample
(green) and progesterone-stimulated sample (red). (B)
Enlarged regions of the identified proteins. On the left are
images of gels loaded with control sample and on the right
gels loaded with progesterone-stimulated sample. The
arrows point to heat shock protein 70 (Hsp70), proliferating
cell nuclear antigen (PCNA), and protein kinase C-like 2
(Pck2). (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of
the article.)
Steroids that inhibit growth of H. werneckii, e.g., pro-
gesterone, androstenedione, 4-estrene-3,17-dione, possess
common structural characteristics. Our results show that par-
tial or complete detoxification of progesterone or its derivative
androstenedione can be achieved by e.g., reduction of 20- or
17-keto groups, which appear to be important for the toxic-
ity of the respective steroids. However, the stereochemistry of
steroids has almost no effect on the effectiveness of H. wer-
neckii growth inhibition, as shown for the stereochemically
different 5␣-androstane-3,17-dione and 5␤-androstane-3,17-
dione (Table 2).
Progesterone biotransformation is just one of the processes
that might contribute to the escape of the H. werneckii from
growth arrest. Recently, H. werneckii cells treated with proges-
terone in the logarithmic phase of growth were investigated
and the overexpression of Pck2 was noted [42]. In the present
study we show that progesterone provokes an increase in Pck2
levels in H. werneckii in the stationary phase of growth (Fig. 2).
These results are in accordance with the observation that
quiescent cells are still able to respond to environmental sig-
nals by inducing the expression of genes similar to those in
proliferating cells, despite their increased resistance to envi-
ronmental stress [72,73].
wall composition between the cultures grown for each indi-
vidual experiment (Fig. 4).
4.
Discussion
The biotransformation of progesterone has been studied
extensively during the last decades [3,20,35,52–65]. It was
found to affect the growth of different eukaryotic microorgan-
isms [33–40], provoke the induction of steroid hydroxylating
enzymes [33,66–68], trigger the induction of heat shock pro-
teins [43] and ABC transporters [69–71]. In an expression
profiling of the progesterone response in Saccharomyces cere-
visiae, 163 genes were found to be upregulated and 40
downregulated [41].
Pck2 is a member of the cell wall integrity pathway which
is activated under the conditions that jeopardize cell wall
stability [74]. The mechanism by which progesterone stimu-

steroids 7 3 ( 2 0 0 8 ) 1465–1474
1471
Fig. 3 – Temporal analysis of Hsp70 mRNA levels during treatment of H. werneckii with progesterone. The H. werneckii cells
in early, late logarithmic and stationary phases of growth were exposed to N,N-dimethylformamide (DMF) alone (C) and, for
the indicated times (min), to 320 ␮M progesterone. Total RNA blots were prepared, stained reversibly (lower panels) and
hybridised (upper panels) as described in Section 2.10.
lates the induction of Pck2 and its activation in H. werneckii
is not known. Steroid hormones are most probably, because
of their low solubility, present in aqueous medium as a sus-
pension [75]. During the process of crossing the cell wall and
cell membrane, progesterone and other toxic steroids might
therefore cause an acute stretching of the plasma membrane
and/or otherwise modify the plasma membrane, thus acti-
vating specific cell surface sensor proteins [74]. On the other
hand perturbation of the plasma membrane affects the activ-
ity of the plasma membrane-bound enzymes involved in cell
wall synthesis and indirectly weakens the cell wall [76]. It
has been shown that chitosan, as a plasma membrane per-
turbing substance, which can bind membrane phospholipids,
affects the cell wall integrity pathway [77]. In our study we
were interested in changes that progesterone might provoke
in the plasma membrane of H. werneckii. Measurements of the
fluidity of the H. werneckii membrane showed no detectable
changes following the addition of progesterone (M. Turk and
ˇ
M. Zakelj-Mavricˇ, unpublished results).
Cell wall stress provokes a global transcriptional response
in S. cerevisiae [78]. The cell wall integrity pathway is activated
with the help of specific plasma membrane sensor proteins,
leading finally to alterations in the cell wall [74,78–80]. In
S. cerevisiae and Schizosaccharomyces pombe the components
of the cell wall integrity pathway, e.g. protein kinase C
homologues pkc1 and pck1p/pck2p, respectively, have been
extensively studied and their cell wall targets identified
[74,78,81–85]. The pathway was suggested to be conserved
in several yeasts and filamentous fungi [86–88]. Although in
H. werneckii the cell wall integrity pathway has not yet been
investigated, the overexpression of Pck2, and most probably its
increased activity in the presence of progesterone, suggested
that changes in the cell wall could be one of the possible effects
of progesterone action. In our study we showed for the first
time that structural changes occur in the cell wall of H. wer-
neckii treated with progesterone, treated H. werneckii cells being
more resistant to cell lysis (Fig. 4). The effect is specific for the
free form of progesterone and was not detected with H. wer-
neckii cells exposed to 11␣-hydroxy-progesterone. The effect
of progesterone is comparable to that of Calcofluor White
which has been shown to hinder normal cell wall assem-
bly in S. cerevisiae and consequently activate the cell wall
integrity pathway [89]. Our results suggest that changes in the
H. werneckii cell wall induced by progesterone most probably
contribute to an adaptation of the yeast to the presence of the
steroid.
Fig. 4 – The effect of selected steroids on the susceptibility
of H. werneckii to lytic action by lyticase. Yeast cells from the
early logarithmic phase were treated for 4 h with: (A)
progesterone (P), 11␣-hydroxy-progesterone (11␣OHP),
water soluble form of progesterone (PWS) or
N,N-dimethylformamide (DMF) alone; (B) progesterone (P),
lower concentration of progesterone (Plow), Calcofluor
White (CFW) or DMF alone. The steroid concentrations were
320 ␮M; progesterone Plow was 32 ␮M. The percentage of
the initial absorption A600 after the addition of lyticase
(220 U/ml final conc.) is presented. Data are
Another protein whose biosynthesis is stimulated in the
presence of progesterone in the logarithmic [42] and sta-
tionary phases of growth is proliferating cell nuclear antigen
(PCNA) (Fig. 2). PCNA has an essential role in the nucleic acid
means standard deviations of at least three independent
experiments performed in duplicate.

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steroids 7 3 ( 2 0 0 8 ) 1465–1474
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presence of progesterone suggests the cell wall as one of
the possible targets of progesterone action. The progesterone
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Acknowledgements
This work was supported by the research grant P1-0170-0381
from the Slovenian Research Agency. The instrumentation
used for mass spectrometry analysis of proteins was financed
by the Georgia Research Alliance Grant to the IMMAG, MCG,
GA, USA. We are indebted to Professor R.K. Yu (MCG, GA) for
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ongoing support. The authors thank Dr. Dusˇan Zigon from
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