The Cellular Target of Histatin 5 on Candida
albicans Is the Energized Mitochondrion*
Eva J.
Helmerhorst
§,
Pieter
Breeuwer¶,
Wim
van `t
Hof
,
Els
Walgreen-Weterings
,
Lauran C. J. M.
Oomen
,
Enno C. I.
Veerman
,
Arie V. Nieuw
Amerongen
, and
Tjakko
Abee¶
From the
Academic Centre for Dentistry, Department of
Oral Biochemistry, Vrije Universiteit, 1081 BT Amsterdam, ¶ Food
Science Group, Department of Food Technology and Nutritional Sciences,
Wageningen University and Research Center, 6703 HD Wageningen, and the
Department of Biophysics, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands
 |
ABSTRACT |
Histatin 5 is a human basic salivary peptide with
strong fungicidal properties in vitro. To elucidate the
mechanism of action, the effect of histatin 5 on the viability of
Candida albicans cells was studied in relation to its
membrane perturbing properties. It was found that both the killing
activity and the membrane perturbing activity, studied by the influx of
a DNA-specific marker propidium iodide, were inhibited by high salt
conditions and by metabolic inhibitors, like sodium azide. In addition,
exposure to histatin 5 resulted in a loss of the mitochondrial
transmembrane potential in situ, measured by the release of
the potential-dependent distributional probe rhodamine 123. Localization studies using tetramethylrhodamine isothiocyanate-labeled
histatin 5 or fluorescein isothiocyanate-labeled histatin 5 showed a
granular intracellular distribution of the peptide, which co-localized
with mitotracker orange, a permeant mitochondria-specific probe. Like
the biological effects, uptake of labeled histatin 5 was inhibited by
mitochondrial inhibitors and high salt conditions. Our data indicate
that histatin 5 is internalized, and targets to the energized mitochondrion.
 |
INTRODUCTION |
It is generally recognized that basic peptides are important
constituents of natural defense systems of most living organisms, including bacteria, plants, insects, and mammals. Examples of such
antibiotic peptides are magainins, secreted by the skin of Xenopus laevis (1), defensins from the human neutrophils
(2), and histatins from human saliva (3-5). Common features of
anti-microbial peptides, which manifest great diversity in primary
structure, are a net positive charge at physiological pH and the
ability to adopt amphipathic
-helix and
-sheet conformations in
hydrophobic solvents (6). It is assumed that the activity of these
peptides is directed against the cellular membranes, mainly the acidic lipid matrices of the target cells (7). In addition, the susceptibility towards some of these peptides is controlled by the metabolic state of
the target cell (8, 9). The mechanism underlying this phenomenon,
however, is not understood. Olson et al. (10) reported the
importance of metabolic activity in the susceptibility of Candida
albicans cells to basic proteins, like protamine, by showing that
uncoupling of oxidative phosphorylation by chemicals or by genetic
mutation, protected the cells against their lethal activity. The
requirement for mitochondrial activity was also found for HNP-1, a
defensin peptide (11). It has been speculated that
energy-dependent processes, e.g.
receptor-mediated uptake, or the maintenance of the cell membrane
electronegative potential would be implicated in the peptide-mediated
killing, but up to now there is no experimental evidence to support
this hypothesis. The aim of the present study was to elucidate the
mechanism of action of the human salivary antifungal peptide, histatin
5. We found that non-respiring yeast cells were protected against
histatin 5 killing activity. In localization studies we identified the energized mitochondrion as the target of histatin 5 in the yeast cell.
The direct association with the mitochondrion and the requirement of
mitochondrial activity provide a new explanation for the
energy-dependent activity of antimicrobial peptides against
C. albicans.
 |
EXPERIMENTAL PROCEDURES |
Yeast Culture and Test Conditions--
C.
albicans (ATCC 10231) was grown for 48 h at 30 °C on
a Sabouraud dextrose agar. For use in killing and permeabilization studies, cells were picked from plate and suspended to 107
colony-forming units/ml in 1 mM potassium phosphate buffer
(PPB),1 pH 7.0. The killing
activity of histatin 5 against C. albicans was investigated
as described previously (12). The killing activity against
non-respiring cells was performed in an anaerobic cabinet, essentially
according to Lehrer et al. (11).
Histatin 5 (DSHAKRHHGYKRKFHEKHHSHRGY), and two unrelated control
peptides, dcysSA (WSPQEEDRIIEGGI) and dcysS (SSSKEENRIIPGGI), were made
by solid phase peptide synthesis procedures as described previously
(13).
Permeabilization Assays--
Permeabilization of whole
C. albicans cells by histatin 5 was studied using the
DNA-staining fluorescent probe propidium iodide (PI) (14).
Permeabilization of the yeast mitochondria in situ by
histatin 5 was studied using the membrane
potential-dependent distributional probe
2-[6-amino-3-imino-3H-xanthen-9-yl]benzoic acid methyl ester
(rhodamine 123) (15). PI and rhodamine 123 were obtained from Molecular
Probes, Inc. (Eugene, OR).
FITC and TRITC Labeling of Peptides--
Tetramethylrhodamine
isothiocyanate (TRITC) or fluorescein isothiocyanate (FITC) were both
purchased from Sigma. Labeling was conducted essentially as described
by Lane et al. (16). In brief, TRITC or FITC were freshly
dissolved in Me2SO to 1 mg/ml, and added to 2 mg/ml of
histatin 5 or dcysS in 50 mM potassium phosphate buffer
(final pH 7.6) to a final concentration of 25 µg/ml. The calculated
molar ratio of TRITC or FITC to histatin 5 was 0.08 and 0.10, respectively. After incubation for 16 h in the dark at 4 °C, 50 mM NH4Cl was added to inactivate the residual TRITC or FITC. The solution was left in the dark for an additional 2 h at 4 °C, and stored in aliquots at
20 °C. Labeling
experiments were performed in 1 mM PPB or in
phosphate-buffered saline (PBS) containing 9 mM
K2HPO4/KH2PO4, pH 7.0, in 150 mM NaCl.
FACS Analysis--
The distribution of PI, rhodamine 123, and TRITC-labeled peptides over the cells was investigated by using a
dual laser fluorescence-activated cell sorter (FACS, Beckton Dickinson,
Franklin Lakes, NJ). The results were analyzed on a MacIntosh computer
using the software package CellQuest provided by Beckton Dickinson.
Co-localization Studies--
The intracellular
localization of FITC-histatin 5 was investigated in a double-labeling
experiment using mitotracker orange CM-H2-TMRos, a
cell-permeant mitochondrion-selective dye (Molecular Probes). C. albicans cells were incubated with 150 nM mitotracker orange in 1 mM PPB, pH = 7.0, washed once in the same
buffer, and subsequently incubated with 17 µM
FITC-histatin 5. After 10 min of incubation at 37 °C, the suspension
was concentrated 10 times, and analyzed by confocal fluorescence microscopy.
Confocal fluorescence microscopy--
Confocal fluorescence
images were obtained on a Leica TCS NT (Leica Microsystems, Heidelberg,
Germany) confocal system, equipped with an argon/krypton laser. Images
were taken using a 100× NA 1.4 objective. Possible cross-talk between
FITC and mitotracker orange, which could give rise to false positive
co-localization of the histatin and mitochondrial signal, was totally
avoided by careful selection of the imaging conditions. The standard
FITC-TRITC filter combination and Kalman averaging were used.
Processing images was done on a PC using the software packages
Photoshop (Adobe Systems Inc. Mountain View, CA) and Freelance Graphics (Lotus Development Corp., Cambridge, MA).
 |
RESULTS |
Killing and Permeabilization of C. albicans by Histatin 5--
The
effect of histatin 5 on the membrane integrity was studied by
monitoring the influx of PI, a DNA-staining fluorescent probe, into
C. albicans cells. Fig. 1
shows the results of the FACScan analysis of cells incubated with 4.1 µM histatin 5 or a negative control peptide, in the
presence of 9 µM PI. After 10 min of incubation with
histatin 5, the majority of the cells were fluorescently labeled. To
investigate in more detail the relationship between the effect of
histatin 5 on PI permeabilization and the fungicidal activity of
histatin 5, cells were incubated with varying concentrations of the
peptide, and concomitantly tested for PI labeling (by fluorescence
microscopy) and cell viability (in a killing assay). Fig.
2A indicates that killing and
PI labeling display a similar dose response. These two parameters also
showed a striking similarity with respect to their ionic strength
dependence (Fig. 2B). At phosphate buffer concentrations
exceeding 20 mM, both PI permeability and killing of
C. albicans by histatin 5 were completely abolished.

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Fig. 1.
FACS analysis of histatin 5-treated C. albicans cells stained with the fluorescent dye PI.
C. albicans cells were incubated for 10 min at 37 °C with
4.1 µM negative control peptide, dcysSA (A),
or with 4.1 µM histatin 5 (B) in 1 mM PPB, pH 7.0, the presence of 9 µM PI. The
distribution of cells according to relative fluorescence intensities
are given. Cells treated with histatin 5 were fluorescent, indicating
permeabilization, while dcysSA-treated cells were not.
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Fig. 2.
Killing and PI labeling of C. albicans cells by histatin 5. A, C. albicans cells were incubated for 10 min at 37 °C with a
dilution series of histatin 5 in 1 mM PPB, pH 7.0, in the
absence or presence of 9 µM PI. Viable counts were
determined by plating on Sabouraud dextrose agar (circles).
The percentage of cells permeable to PI was determined by fluorescence
microscopy by counting at least 30 cells in three different fields
(triangles). B, percentages of killed cells and
PI-fluorescent cells after incubation for 10 min at 37 °C with 17 µM histatin 5, at the indicated phosphate buffer
concentrations, pH 7.0.
|
|
Influence of Cellular Respiration on Histatin 5 Activity--
It
was further investigated whether the metabolic state of the yeast cell
influences its susceptibility to histatin 5. For this purpose, cells
were incubated with histatin 5 in the presence of several agents and
conditions that inhibit mitochondrial respiration, including azide,
cyanide, and anaerobic incubation. In a control experiment, it was
found that temporarily blocking respiration by mitochondrial inhibitors
during the time course of the killing experiment (1.5 h) did not
influence the viability of the cells. Fig.
3 shows that blocking of the
mitochondrial respiration protected C. albicans against the
fungicidal activity of histatin 5. A concentration of 5 mM
cyanide, which blocks the conventional respiratory pathway of C. albicans, induced over a 6-fold increase in IC50
value, shifting from 3 µM to 20 µM (Fig.
3A). In buffer containing 5 mM azide, which
blocks both the conventional and the alternative respiratory pathway of
C. albicans cells (17, 18), no histatin 5 killing activity
was observed (IC50 > 65 µM). Another way to block cellular respiration is by creating anaerobic conditions. Cysteine at a concentration of 2.5 mM reduces the redox
potential of a solution to
220 mV, which is the most useful measure
for the degree of anaerobiosis (19). This was verified by the color change of a redox-potential dependent indicator, resazurin, added to a
control vial. Cells incubated in buffer containing cysteine or azide
did not accumulate rhodamine 123 in their mitochondria, indicating that
under these conditions the mitochondrial transmembrane potential was
dissipated (data not shown). In buffer containing cysteine, histatin
5-induced killing was completely abolished (IC50 > 65 µM, Fig. 3B). In Fig. 3C, it is
shown that anaerobic conditions protect C. albicans cells
against histatin 5 (IC50 > 65 µM), although
at high concentrations of peptide 40% killing was observed, probably
due to the presence of trace amounts of oxygen in the anaerobic
cabinet. These results indicate that mitochondrial respiration is a
prerequisite for histatin-induced killing.

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Fig. 3.
Effect of blocking respiration on the
susceptibility of C. albicans to histatin 5 killing
activity. A, C. albicans cells were
incubated for 10 min at 37 °C in 1 mM PPB, pH 7.0 containing 5 mM NaCl (open circles),
NaCN (open triangles), or NaN3 (closed
circles) and subsequently added to a dilution series of histatin 5 in 1 mM PPB containing the same inhibitors. After 1.5 h at 37 °C of incubation, the viability of the cells was assessed by
plating. B, C. albicans cells were incubated with
histatin 5 in 1 mM PPB supplemented with 2.5 mM
cysteine (closed circles) or an equimolar amount
of NaCl (open circles). C, incubation
of C. albicans cells with histatin 5 in 1 mM PPB
under anaerobic (closed circles) or aerobic
conditions (open circles).
|
|
Effect of Histatin 5 on Mitochondrial Membrane
Integrity--
C. albicans cells were incubated
with the fluorescent dye rhodamine 123, which accumulates specifically
in intact mitochondria in direct proportion to the mitochondrial
transmembrane potential. Cells preloaded with rhodamine 123 released
the dye upon a 10-min incubation period with 17 µM
histatin 5. Microscopic analysis revealed that the typical granular
appearance of the probe, indicative of a mitochondrial localization,
was abolished upon incubation with histatin 5 (Fig.
4A), but preserved in the
cells treated with the negative control peptide (Fig. 4B).
This was corroborated by FACScan analysis, indicating that histatin 5 reduced the cell-associated rhodamine fluorescence intensity by more
than 90%.

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Fig. 4.
Fluorescence microscopy and FACScan analysis
of rhodamine 123-labeled C. albicans cells. Cells
were incubated for 10 min at 37 °C with 10 µM
rhodamine 123 in 1 mM PPB, pH 7.0, washed, and treated for
10 min at 37 °C with 17 µM histatin 5 (A)
or the negative control peptide dcysSA (B). C,
cells without rhodamine label. The distribution of cells according to
relative fluorescence intensities are given.
|
|
Internalization and Localization of Histatin 5--
The
experiments of Fig. 4 indicated that, upon incubation of C. albicans with histatin 5, the mitochondrial membrane potential was
dissipated. To study whether this was due to a direct association with
the mitochondria, the cellular localization of histatin 5 was
investigated by confocal fluorescence microscopy and FACS using
histatin 5 coupled to FITC or TRITC. The fungicidal activity of
histatin 5 was not affected by the labeling, as was verified in a
killing assay (data not shown). Fig. 5
shows a confocal fluorescence micrograph of cells incubated with
TRITC-histatin 5. A granular, intracellular staining pattern was
observed, indicating the uptake of the peptide into the cell. A similar
fluorescence pattern was obtained with FITC-histatin 5. FACScan
analysis revealed that cells incubated with a TRITC-labeled negative
control peptide or with inactivated TRITC alone, were negative (Fig.
6). Conditions that inhibit histatin 5 killing activity, like the presence of azide, or the relatively high
salt concentration in PBS, prevented the binding and uptake of
TRITC-histatin 5 (Fig. 6). After washing of histatin-labeled cells with
PBS, the cells remained fluorescent, indicating that once histatin 5 has bound, it is not dissociated by a buffer with a relatively high
ionic strength.

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Fig. 5.
Confocal fluorescence microscopy of the
intracellular localization of TRITC-labeled histatin 5. C. albicans cells were incubated for 1 h at
37 °C with 17 µM TRITC-labeled histatin 5 in 1 mM PPB, pH 7.0. Bar = 5 µm.
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Fig. 6.
FACScan analysis of C. albicans
cells incubated with TRITC-labeled histatin 5. C. albicans cells in 1 mM PPB, pH 7.0, were
incubated for 1 h at 37 °C with 1.3 µM
NH4Cl-inactivated TRITC (A), with 17 µM TRITC-dcysS (B), or with 17 µM TRITC-histatin 5 (C). Lower
graph, C. albicans cells incubated with 17 µM TRITC-histatin 5 in PBS (D), in 1 mM PPB containing 5 mM NaN3
(E), or in 1 mM PPB followed by PBS
(F).
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The granular staining patterns of FITC- and TRITC-histatin 5, in
combination with the results presented in Figs. 3-6 (summarized in
Table I), suggested a mitochondrial
targeting of histatin 5. To unequivocally establish the mitochondrial
localization of histatin 5, double labeling experiments were performed
using the mitochondria-specific fluorescent probe, mitotracker orange,
in combination with FITC-histatin 5 (Fig.
7). This localization experiment showed
that identical staining patterns were obtained with both labels,
indicating that FITC-histatin 5 was bound to mitochondria.
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Table I
Influence of buffer composition on histatin 5 binding, permeabilization
activity, and killing activity against C. albicans
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Fig. 7.
Confocal fluorescence microscopy of C. albicans cells labeled with FITC-histatin 5 and a
mitochondria specific marker. Cells were incubated for 0.5 h
at 37 °C with 150 nM mitotracker orange
CM-H2-TMRos in 1 mM PPB, pH 7.0, washed once,
and subsequently incubated for 15 min at 37 °C with 17 µM FITC-histatin 5. Microscopic pictures show the
localization of FITC-histatin 5 (A), the localization of
mitotracker orange (B), and the double labeling
(C). There was no cross-talk between the FITC and the
mitotracker orange signal. Bar = 5 µm.
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 |
DISCUSSION |
In this study, we addressed the mechanism of action of histatin 5, a basic amphipathic antifungal peptide from human saliva. In line with
previous observations (20), we found that FITC-histatin 5 is
internalized by C. albicans cells. The uptake is dependent on the metabolic state of the cell, and is directly related to the
fungicidal activity of histatin 5. Non-respiring Candida
cells are protected against histatin 5 killing activity, against its membrane perturbing activity, and do not internalize the peptide.
A direct involvement of the mitochondrion in the killing process is
demonstrated by co-localization of histatin 5 with a
mitochondria-specific probe, revealing that it binds to mitochondria
in situ. In addition, histatin 5 dissipates the
mitochondrial transmembrane potential in situ, monitored by
rhodamine 123 efflux. Together, these results strongly suggest that
killing activity of histatin 5 is directly related to perturbation of
the mitochondrial membrane.
These results imply the following sequence of events: interaction of
histatin 5 with the plasma membrane of the yeast cell, translocation
across the plasma membrane, release into the intracellular compartment,
followed by targeting to and permeabilization of the mitochondria.
Under conditions when histatin 5 was internalized, no labeling of the
plasma membrane by TRITC-histatin 5 could be detected (Fig. 7),
suggesting that the binding to the plasma membrane is a transient
event. Nevertheless, it seems a crucial step in the killing process,
since high ionic strength conditions, which have been reported to
abolish the initial interaction of basic peptides with fungal cellular
membranes (21), prevent both killing and intracellular accumulation of
TRITC-histatin 5 (this study).
It is still unclear by which mechanism histatin 5 is transported across
the fungal membrane into the cytoplasm. It has been suggested that
basic peptides bound to the membrane are subsequently electrophoresed
into the membrane under the influence of an inside-negative transmembrane potential (6). Maduke and Roise (22) described the
potential-dependent import into protein-free phospholipid vesicles of synthetic peptides similar in length to histatin 5. Matsuzaki et al. (23, 24) showed that magainin 2 was
inserted and translocated across artificial phospholipid bilayers to
the inner leaflet. These studies suggest that translocation and
internalization of basic peptides would be possible without the
intervention of membrane-bound permeases. Apparently, from our results,
translocation of histatin 5 is attended with the concomitant influx of
propidium iodide, suggesting that the uptake of histatin 5 leads to
plasma membrane perturbations.
With regard to the targeting of histatin 5 to the mitochondria, it is
important to note that histatin 5 shows structural and functional
resemblances with mitochondrial presequences, i.e. terminal
amino acid extensions that target mitochondrial proteins from the
cytosol to the mitochondrion. Like histatin 5 and other basic
antimicrobial peptides, these matrix-targeting sequences of
mitochondrial precursor proteins are basic peptides and adapt amphipathic helical structures in hydrophobic environments (25). It is
believed that the divalent negative phospholipid cardiolipin, which is
abundantly present in the mitochondrial membrane, especially supports
the initial attraction of mitochondrial presequences (25). The same
mechanism may apply for histatin 5.
The insertion process of mitochondrial presequences into the
mitochondrion has been shown to be independent of mitochondrial surface
proteins (26), and to occur in a potential-dependent manner
(22, 27). It has been reported that an inside-negative membrane
potential also enhances the activity of membrane-directed basic
antimicrobial peptides against living bacterial cells (28), against
isolated energized mitochondria (29), and against model membrane
vesicles (23). It is conceivable that factors that diminish the
mitochondrial transmembrane potential will reduce histatin 5 binding
and the cytotoxic consequences. This is in agreement with our results
that the presence of a mitochondrial transmembrane potential is a
prerequisite for the antifungal activity of histatin 5, as revealed by
the protective efficacy of mitochondrial inhibitors or anaerobic
conditions. Previously, Lehrer et al. (11) demonstrated that
respiratory activity was necessary to sensitize C. albicans
blastoconidia to the lethal effects of a defensin peptide HNP-1,
suggesting a mechanism of antifungal action similar to that of histatin
5. Interestingly, it was found recently that cardiolipin, which is
enriched in the mitochondria, plays a key role in the membrane
perturbing activity of rabbit neutrophil defensins (30).
Nicolay et al. (25) demonstrated the destructive effect of
amphipathic peptides derived from the presequence of the cytochrome c oxidase subunit IV on the permeability barrier of isolated
mitochondria. These peptides also display antimicrobial activity (31).
Likewise, Westerhoff et al. (29, 32) showed that magainins
dissipated the membrane potential of isolated rat liver mitochondria
and postulated that this would be the mechanism of antibacterial
activity, because of the similar organization of membrane-linked energy coupling in bacteria and mitochondria. Whether these antimicrobial peptides share other mechanistic properties with mitochondrial presequences, like translocation to the mitochondrial inner membrane and processing by proteolytic enzymes, are interesting subjects for
further study.
Based on our results, we propose that the energized mitochondrion,
which might be considered as an endosymbiont of bacterial origin (33),
is the cellular target of histatin 5. This may offer a unifying
principle for the mechanism of action of basic antifungal peptides that
depend on active cell metabolism.
 |
ACKNOWLEDGEMENTS |
We thank I. M. Reijnders,
W. Jansen, and F. ter Veld for technical assistance, and Dr. J. G. M. Bolscher, Prof. Dr. K. Hellingwerf, and Dr. B. van Rotterdam
for helpful discussions.
 |
FOOTNOTES |
*
This work was supported by Dutch Technology Foundation
Projects VTH44.3302 and 790.43.823 and by Unilever Research, Oral Care, Port Sunlight, United Kingdom.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Dept. of Oral
Biochemistry, Vrije Universiteit, Van der Boechorststraat 7, 1081 BT
Amsterdam, The Netherlands. Tel.: 31-20-4448674; Fax: 31-20-4448685; E-mail: EJ.Helmerhorst.obc.acta{at}med.vu.nl.
 |
ABBREVIATIONS |
The abbreviations used are:
PPB, potassium
phosphate buffer;
PI, propidium iodide;
TRITC, tetramethylrhodamine
isothiocyanate;
FITC, fluorescein isothiocyanate;
FACS, fluorescence-activated cell sorting;
PBS, phosphate-buffered
saline.
 |
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