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Antimicrobial Agents and Chemotherapy, September 1998, p. 2279-2283, Vol. 42, No. 9
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Identification of Elongation Factor 2 as the
Essential Protein Targeted by Sordarins in Candida
albicans
Juan Manuel
Domínguez and
J. Julio
Martín*
Departamento de Investigación, Glaxo
Wellcome S.A. 28760-Tres Cantos, Madrid, Spain
Received 26 January 1998/Returned for modification 22 April
1998/Accepted 24 June 1998
 |
ABSTRACT |
The target for sordarins in Candida albicans has been
elucidated. Kinetic experiments of sordarin inhibition as well as
displacement experiments showed that the formation of a sordarin-target
complex follows a reversible mechanism. Binding of tritiated drug
to the target is enhanced in the presence of ribosomes. Isolation of the target by classical protein purification methods has allowed us to identify it as elongation factor 2. This is in agreement with the nature of sordarin derivatives as specific inhibitors of the
elongation cycle within protein synthesis in yeasts.
| |
INTRODUCTION |
The appearance of resistance to
current antifungal therapies in recent years has led to the need
for new, effective drugs. In this regard, the finding of sordarins
(11) as new antifungal drugs could be of potential value.
One of the most attractive aspects of this family of compounds lies on
its novel mode of action, inhibition of protein synthesis
(6), an unusual feature in antifungal therapies.
Taking into account the high degree of selectivity of sordarins,
together with the conserved nature of the protein synthesis machinery
within the eukaryotic kingdom, it was interesting to explore the
precise mode of action of these drugs. Once it was established that the
primary target was not the ribosome, we have studied the nature of the
interaction between sordarin and its target on the pathogenic fungus
Candida albicans, analyzing how this interaction is affected
by the individual components of the fungal system. Furthermore, we have
been able to purify the primary target for sordarin and conclusively
identify it as elongation factor 2 (EF-2).
| |
MATERIALS AND METHODS |
Materials.
The microorganisms used in the study
(C. albicans 2005E and Candida parapsilosis
2372E) were obtained from the Glaxo Wellcome culture collection.
Sephadex G-25 (PD-10 prepacked columns) and Q-Sepharose and S-Sepharose
(1.6- by 10-cm prepacked columns) were from Pharmacia (Uppsala,
Sweden). [14C]NAD (9.32 GBq/mmol) was from Amersham
(Little Chalfont, United Kingdom). Sordarin (molecular weight, 492.6)
was provided by the Bioprocessing Group, and [3H]sordarin
(180 GBq/mmol) was synthesized by the Isotope Chemistry Group (both
groups are at Glaxo Wellcome, Stevenage, United Kingdom). Anti-EF-1
and anti-EF-3 antibodies were a generous gift of M. F. Tuite. All
other chemicals and reagents were of the highest quality available. All
procedures were performed at 4°C unless stated otherwise.
Methods. (i) Sordarin binding assays.
Samples were incubated
in the presence of 0.5 µg of [3H]sordarin (180 kBq/ml)
per ml in a final volume of 500 µl at 25°C for 1 h. All
samples were diluted in 30 mM HEPES-KOH (pH 7.4) containing 100 mM
potassium acetate, 2 mM magnesium acetate, and 2 mM
DL-dithiothreitol so that they had a standard protein
concentration (10 mg/ml). Aliquots from chromatography eluates were
previously mixed with 0.2 nmol of C. parapsilosis ribosomes.
In the displacement experiments 100 µg of unlabelled sordarin per ml
was added, and the sample was incubated for an additional 1 h.
After the incubation period, unbound sordarin was removed in all cases
by gel filtration through Sephadex G-25, and the amount of drug
bound to macromolecular components was determined by liquid
scintillation counting.
The binding constants for the C. albicans postribosomal
supernatant (PRS) in the presence or absence of C. parapsilosis ribosomes were determined by equilibrium dialysis
over 16 h at 30°C in a 150-µl final volume.
(ii) Fractionation of PRS.
Soluble factors from the C. albicans PRS were separated by the method described by Uritani and
Miyazaki (33), except that Q-Sepharose and S-Sepharose
columns were used and the last step (chromatography on hydroxyapatite)
was omitted.
(iii) Other methods.
Preparation of C. albicans
and C. parapsilosis cell-free translation systems,
fractionation into ribosomes and PRS, and performance of the
poly(U)-directed in vitro translation assay were done as described
previously (6). ADP ribosylation of samples catalyzed with
diphtheria toxin was carried out as described previously (30). Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) was performed in 10% polyacrylamide gels as
described previously (12). The protein concentration was
determined as described by Bradford (4).
| |
RESULTS |
In order to characterize the interaction between sordarin
and its target, the reversible or irreversible nature of such an interaction was examined. This was initially done by kinetic
experiments, in which we tested the effects of several sordarin
concentrations on the rate of poly(U)-directed
poly-[14C]Phe synthesis in a cell-free in vitro
translation system from C. albicans. As shown in Fig.
1A, there was an immediate decrease in
the rate of synthesis, and this decrease was related to the sordarin
concentration. The rate of formation of the sordarin-target complex is
therefore rapid. With the aim of confirming the reversibility of
binding, displacement experiments were carried out by incubating the
C. albicans cell-free system with
[3H]sordarin, followed by the addition of excess
unlabelled sordarin (Fig. 2). As can be
seen, unlabelled sordarin was able to displace tritiated drug, and
therefore, the reaction rates in Fig. 1A were analyzed and fitted to
the following equilibrium binding equation: percent remaining
activity = (100 · Ki/(Ki + [sordarin]),
where Ki is the apparent inhibition constant,
and [sordarin] is the concentration of sordarin. As shown in Fig. 1B,
the experimental data closely fit the predicted values, with a
Ki of 12 nM (5.9 ng/ml).
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FIG. 1.
Kinetics of sordarin inhibition. (A) Poly(U)-directed in
vitro translation was performed as described previously (6)
with a cell-free system from C. albicans in the presence of
several sordarin concentrations. The reaction was stopped by adding
NaOH to 0.5 M each time, and the amount of poly-[14C]Phe
synthesized was measured by trichloroacetic acid precipitation and
liquid scintillation counting. The following symbols correspond to the
indicated sordarin concentrations:  , 3.1 ng/ml;  , 6.2 ng/ml;  ,
12.5 ng/ml;  , 25 ng/ml;  , 50 ng/ml;  ·, 100 ng/ml;  , control (without sordarin). (B) Fitting of experimental
points to the theoretical curve corresponding to the equation for
reversible inhibition (see text).
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FIG. 2.
Displacement of sordarin binding. C. albicans
cell-free lysate was incubated in the presence of 0.5 µg of
[3H]sordarin per ml at 25°C for 1 h. After adding
buffer (A) or unlabelled sordarin in buffer to reach a final
concentration of 100 µg/ml (B), the incubation was continued for an
additional 1 h. (C) Lysate, 0.5 µg of [3H]sordarin
per ml, and 100 µg of unlabelled sordarin per ml were incubated
for 2 h. Subsequent to all the incubations unbound sordarin
was removed by gel filtration, and the amount of bound drug,
excluded as a component of a high-molecular-weight complex, was
measured by liquid scintillation counting.
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With this in mind, isolation of the sordarin-target complex could be
attempted only by fractionation methods under mild conditions that
preserve the target conformation and its interaction with the sordarin
molecule. Thus, it was possible to attempt purification of the target
by conventional methods, taking advantage of the possibility of
detecting the target by means of its ability to specifically bind to
[3H]sordarin. Nevertheless, it is necessary to ascertain
whether the sordarin-binding protein is the real functional target by proving a correlation between sordarin binding and inhibition of
protein elongation. The data in Fig. 3A
suggest that sordarin mostly bound to crude ribosomes from C. albicans, which appears to contradict previous results that
suggested the nonribosomal nature of the sordarin target
(6). To explain this, the following three hypotheses can be
proposed: (i) The target is a ribosomal component. Previous results
could be explained by assuming that resistant species contain a
nonribosomal protein capable of replacing target function when this is
inhibited. (ii) The target is a ribosomal component that interacts with
a soluble factor which, according to its degree of affinity, might
displace (resistance) or not displace (sensitivity) the
sordarin-target interaction. (iii) The target is a soluble
nonribosomal protein that remains attached to ribosomes after the
centrifugation step. The sordarin-binding ability of this protein may
be enhanced by its interaction with the ribosome.
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FIG. 3.
Sordarin binding to fractions from C. albicans (A) or C. parapsilosis (B). PRSs were
separated from ribosomes by centrifugation at 100,000 × g for 4 h; in the case of C. albicans, a
portion of the ribosomes was subsequently washed with 0.5 M KCl.
Afterwards, sordarin binding to each fraction was measured as described
in Materials and Methods. (A) Fractions from C. albicans: 1, whole lysate; 2, PRS; 3, crude ribosomes; 4, KCl-washed ribosomes; 5, KCl-washed ribosomes plus PRS. (B) Fractions from C. parapsilosis: 1, whole lysate; 2, PRS; 3, crude ribosomes; 4, crude C. parapsilosis ribosomes plus C. albicans
PRS.
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|
Of the three hypotheses, the first hypothesis seems to be the least
feasible, because it assumes the existence of two genes encoding
the same function, with the second gene being present specifically to
prevent the antifungal effects of sordarin derivatives. Although this
could be valid for genetically modified strains, it is unlikely to
account for the situation in naturally occurring resistant species such
as C. parapsilosis. On the other hand, the third hypothesis
is in good agreement with the results presented in Fig. 3A, which
indicate that KCl-washed ribosomes (devoid of any soluble factors)
(32) have lost their ability to bind to sordarin, while the
addition of PRS partially restores this ability. Moreover, ribosomes
from sordarin-resistant species such as C. parapsilosis were
unable to bind to [3H]sordarin, but when they were
mixed with PRSs of sensitive species (C. albicans), binding of [3H]sordarin was
considerably enhanced (Fig. 3B). Also, the inability of the
ribosomes to bind to [3H]sordarin in the absence
of soluble factors negates the second hypothesis since, according to
that hypothesis, ribosomes from either a resistant or a sensitive
species are expected to bind to [3H]sordarin. The
resulting Kd value, obtained by equilibrium
dialysis, for sordarin in C. albicans PRS was 3.11 µM,
whereas in the mixture of C. albicans PRS and C. parapsilosis ribosomes the Kd value was
0.27 µM; i.e., C. parapsilosis ribosomes increase the
affinity for sordarin 10 times. It is noteworthy that in both
cases a single class of binding sites was found (data not shown). On
the other hand, no binding was detected when soluble factors
from C. parapsilosis were mixed with washed ribosomes
from C. albicans. These results lead us to
conclude that the target is a soluble nonribosomal protein whose
affinity toward sordarin is markedly increased in the presence of
ribosomes, probably due to conformational arrangements in the target
when it is interacting with the ribosome. From this conclusion
good agreement between binding ability and resistance-sensitivity profiles can be stated, and therefore, binding to
[3H]sordarin can be used to detect the sordarin target
when trying to isolate it from the rest of the components of the
C. albicans PRS.
The isolation procedure was essentially the one followed by Uritani and
Miyazaki (33). In the second chromatographic step (Fig.
4B) a single peak was able to bind
sordarin. Western blot analysis with anti-EF-1 and anti-EF-3
antibodies revealed that none of these factors was present in this peak
(data not shown). SDS-PAGE revealed that the major component of the
peak was a protein of 98 kDa that was susceptible to ADP
ribosylation by diphtheria toxin (Fig.
5), which exclusively recognizes EF-2
(3, 21). The elution profile of this protein (determined by
ADP ribosylation [data not shown]) overlaps that of sordarin-binding
activity. On the other hand, minor contaminants present are also
radiolabelled with diphtheria toxin, while further
chromatographic steps did not allow the separation of native EF-2 from
its hydrolyzed contaminants. The bands from lane 9 in Fig. 5A were
excised and sequenced. Figure 6 shows
that the best sequence alignments in any case corresponded to
Saccharomyces cerevisiae EF-2 fragments. From these results it is concluded that the major 98-kDa protein present in the
sordarin-binding fraction is C. albicans
EF-2. Minor contaminants coeluting with it seem to be derived from
EF-2 proteolysis.
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FIG. 4.
Purification of sordarin target from C. albicans PRS. The procedure of Uritani and Miyazaki
(33) was essentially followed. Fractions were analyzed for
their ability to bind to [3H]sordarin in the presence of
C. parapsilosis ribosomes ( ) as described in
Materials and Methods. (A) Q-Sepharose chromatography at pH 7.5 (column
of 1.6 by 10 cm, 0.8 ml/min, 5-ml fractions). (B) S-Sepharose
chromatography at pH 7.5 (column of 1.6 by 10 cm, 0.6 ml/min, 2.5-ml
fractions) of fractions from the chromatography described for panel A
able to bind to [3H]sordarin. Abs, absorbance.
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FIG. 5.
Electrophoresis analysis of S-Sepharose eluate. (A)
SDS-PAGE of fractions from S-Sepharose eluate; lanes 1 and 10, molecular weight markers; lane 2, sample loaded onto the column; lanes
3 to 8, fractions unable to bind to [3H]sordarin; lane 9, fraction with maximum [3H]sordarin binding. (B)
Fluorography of the sample corresponding to lane 9 after treatment with
diphtheria toxin and [14C]NAD (lanes 2 and 3; lanes 1 and
4, molecular weight markers). Both panels are scanned images of the
originals obtained by using Pharmacia Imagemaster; the images were
furtherly processed as a tagged image file format with Microsoft Power
Point.
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FIG. 6.
Sequencing results for components from sordarin-binding
peak in S-Sepharose eluate. Protein bands from lane 9 in Fig. 5A were
excised and subjected to N-terminal sequencing. Sequence alignment was
performed by the running BLAST program (1) on the
Swiss-Prot database (Swiss Institute of Bioinformatics). The best
scores were obtained with fragments of the sequence of EF-2 from
S. cerevisiae (ScEF-2), as shown in the figure. aa, amino
acids.
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| |
DISCUSSION |
The extremely high degree of selectivity of sordarins, a
fact that favors their use as antifungal drugs, is a surprising event in light of its cellular target, one of the elongation factors involved
in protein synthesis (6), because these factors are very
well conserved proteins within the eukaryotic kingdom (16). So far, EF-3 is the only elongation factor reported to be present in
yeasts but not in mammals (2, 31), and thus, it is commonly accepted as a suitable target for potential antifungal agents. Nevertheless, the selectivity can also come from subtle structural differences between other more conserved components of the protein synthesis machinery, differences that might be magnified by the complex
interactions which are displayed within the elongation cycle (5,
17).
Purification of the sordarin-binding protein from C. albicans PRS has led to the identification of EF-2 as the target
of this new series of antifungal agents. All major bands present on
SDS-PAGE were ADP ribosylated by diphtheria toxin, and their sequences showed homology with EF-2 from S. cerevisiae
(22). The presence of bands with molecular weights lower
than that of EF-2 is due to proteolysis of the factor, a fact that has
been linked to the regulation of protein synthesis (7-9).
Moreover, the existence of a specific protease for EF-2 has been
described previously (29). With regard to this, it is
noteworthy that, in this pool of C. albicans EF-2, all
cleavage points are Asp-Pro bonds. So far this class of bond has not
been described as a specific cleavage site for any protease, although
selective hydrolysis of Asp-Pro bonds can be achieved under acidic
conditions (13, 23).
As shown in the previous section, the reversible interaction between
sordarin and its target is greatly favored by the presence of
ribosomes. It is well known that interaction with ribosomes promotes
conformational changes on the three elongation factors (10)
and that these changes lead to the appearance of a latent enzyme
activity, i.e., GTPase for EF-1 and EF-2 and ATPase as well as
GTPase for EF-3 (24-26, 28). On the ribosomal complex EF-2
increases its affinity for sordarin 10 times. This fact highlights the
role of the ribosome as a modulator of the interaction between sordarin and its target, and taking into account the great diversity of
macromolecular structures present in the ribosome (14,
15), this might provide a clue to explain the selectivity of this
kind of drug. Nevertheless, differences in single key residues at EF-2 itself may also account for the selectivity that has been found. The
possibility of the sordarin interaction at the interface of the
ribosome and the soluble factor can be ruled out because the PRS itself
is able to bind to sordarin.
EF-2, as well as its prokaryotic counterpart, EF-G, promotes
translocation, i.e., displacement of nascent peptidyl-tRNA from the A site to the P site and movement of the ribosome along the mRNA; all of this is accompanied by a conformational change in the
ribosome from the pretranslocational to the posttranslocational state
(18). According to recent findings (27), GTP
hydrolysis catalyzed by the elongation factor might provide the energy
needed for the process. EF-2 is a highly conserved protein (85%
homology and 66% identity between human EF-2 and S. cerevisiae EF-2) (22). It has been shown both for EF-G
and for EF-2 from different sources that this protein is able to
display different conformations depending on whether it is alone or
interacting with GTP, GDP, or a ribosome in either the pre- or the
posttranslocational state (19, 20). This conformational
flexibility provides its biological properties and may be the reason
that explains how such a conserved protein can be the primary
target of such very selective antifungal drugs as sordarins. This fact
might also lead to a revision of the idea that prediction of the
apparent selectivity of a new antifungal target can be based solely on
its primary structure.
| |
ACKNOWLEDGMENTS |
We thank J. M. Viana for technical assistance. The
sequencing work performed by Edith Magnenat (Geneva Biomedical Research Institute, Geneva, Switzerland) is greatly acknowledged. Special thanks
are given to M. F. Tuite (University of Kent, Canterbury, United
Kingdom) for providing us with antibodies, to our colleagues at Glaxo
Wellcome Research and Development (Stevenage, United Kingdom) for
supplying sordarin, and to Michael S. Marriott for kind revision of
this paper.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Departamento de
Investigación, Glaxo Wellcome S.A., C/Severo Ochoa 2, 28760-Tres
Cantos, Madrid, Spain. Phone: 34 91 8070301. Fax: 34 91 8070595. E-mail: jjmp28182{at}GlaxoWellcome.co.uk.
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Antimicrobial Agents and Chemotherapy, September 1998, p. 2279-2283, Vol. 42, No. 9
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Abstract
Introduction
