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Antimicrobial Agents and Chemotherapy, September 1999, p. 2256-2262, Vol. 43, No. 9
0066-4804/99/$04.00+0
Histatin 3-Mediated Killing of Candida
albicans: Effect of Extracellular Salt Concentration on Binding
and Internalization
Yanying
Xu,1,
Indu
Ambudkar,1
Hisako
Yamagishi,1,
William
Swaim,2
Thomas J.
Walsh,3 and
Brian C.
O'Connell1,*
Gene Therapy and Therapeutics
Branch1 and Cellular Imaging Core
Facility,2 National Institute of Dental and
Craniofacial Research, and Immunocompromised Host Section,
Pediatric Oncology Branch, National Cancer
Institute,3 Bethesda, Maryland 20892
Received 8 April 1999/Returned for modification 28 May
1999/Accepted 12 July 1999
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ABSTRACT |
Human saliva contains histidine-rich proteins, histatins, which
have antifungal activity in vitro. The mechanism by which histatins are
able to kill Candida albicans may have clinical significance but is currently unknown. Using radiolabeled histatin 3, we show that the protein binds to C. albicans spheroplasts in a manner that is dependent on time and concentration. Binding to the
spheroplasts was saturable and could be competed with unlabeled histatin 3. A single histatin 3 binding site with a
Kd = 5.1 µM was detected. Histatin 3 binding
resulted in potassium and magnesium efflux, predominantly within the
first 30 min of incubation. Studies with fluorescent histatin 3 demonstrate that the protein is internalized by C. albicans
and that translocation of histatin inside the cell is closely
associated with cell death. Histatin binding, internalization, and cell
death are accelerated in low-ionic-strength conditions. Indeed, a low
extracellular salt concentration was essential for cell death to occur,
even when histatin 3 was already bound to the cell. The interaction of
histatin 3 with C. albicans, and subsequent cell death, is
inhibited at low temperature. These results demonstrate that the
candidacidal activity of histatin 3 is not due exclusively to binding
at the cell surface but also involves subsequent interactions with the cell.
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INTRODUCTION |
Histatins are a family of
low-molecular-weight proteins found in abundance in the saliva of
humans and old-world monkeys (1, 2). The histatin family
consists of 12 members; the three major histatins (histatins 1, 3, and
5) constitute 70 to 80% of the total amount. Histatins demonstrate a
number of biological activities in vitro, including maintenance of
tooth surface integrity (8, 19), the induction of histamine
release (27), inhibition of proteases (13, 18),
and the potentiation of rabbit chondrocyte growth (16).
However, most attention has been focused on the antimicrobial activity
of histatins. These proteins exhibit fungicidal activity against
several Candida species (including Candida
albicans), Saccharomyces cerevisiae, and
Cryptococcus neoformans at physiological concentrations
(6, 10, 20, 25, 28). In addition, the histatins have modest
bactericidal or inhibitory effects on Streptococcus mutans,
Streptococcus mitis, Porphyromonas gingivalis,
and Actinobacillus actinomycetemcomitans (14,
15).
The mechanism by which histatins can kill or inhibit the germination of
fungi remains unclear. Incubation of C. albicans with histatin 5 is reported to cause structural changes in the cell wall,
membrane, and cytoplasm and the release of intracellular potassium
(20, 26). However, the action of histatins on the cell
surface probably differs from that of larger antimicrobial peptides
because a 14-amino-acid peptide corresponding to the C terminus of
histatin 5 (and the middle of histatin 3) was identified previously as
the active domain (21, 30). Additionally, histatins are
weakly amphipathic and not likely to independently form transmembrane structures (11, 22). It has been suggested elsewhere that the tendency to form an 
helix is an important functional feature of
histatins, though the significance of this motif is not evident from
functional studies (21, 23, 24).
A specific binding site for histatin 5 has been identified on P. gingivalis and C. albicans (7, 17).
Histatins bind to the C. albicans cell membrane but not to
the membrane of a mammalian cell, which is consistent with the
selective killing activity of these proteins (7). Recently,
it has been reported that histatin 5 is targeted to the mitochondrion
and results in a loss of transmembrane potential (9).
However, the process by which histatins enter the candidal cell is not
known. In this study, we explore the relationship among the specific
binding of histatin 3 to C. albicans, internalization of
histatin, and cell death. These experiments help to clarify the effects
of the extracellular environment on histatin binding and uptake by
C. albicans and demonstrate that the mechanism of cell death
is distal to the initial binding of histatin to the plasma membrane.
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MATERIALS AND METHODS |
Labeling of histatin 3.
Histatin 3 was synthesized by
GeneMed Synthesis, Inc. (San Francisco, Calif.), and purified by
high-pressure liquid chromatography. Composition of the protein was
verified by mass spectrometry and amino acid analysis. Histatin 3 was
radiolabeled by reductive methylation. Briefly, the protein was
dissolved in 0.1 M HEPES buffer (pH 7.5) at a concentration of 5 mg/ml,
and recrystallized sodium cyanoborohydride was added to a concentration
of 20 mM. [14C]formaldehyde was first passed through a
Dowex 1X-8 column to remove contaminants and then added at a 20-fold
molar excess to the histatin 3. The reaction mixture was incubated at
37°C for 24 h, and then the labeled histatin 3 was separated on
CL2B agarose. The specific activity of the [14C]histatin
3 preparations was from 1.4 × 105 to 2.4 × 105 cpm/µg. In order to assess the anticandidal activity
of methylated histatin 3, the above reaction was performed with
unlabeled formaldehyde. Methylation was confirmed by amino acid
analysis. The methylated histatin 3 was used in the standard
Candida killing assay (see below).
Fluorescent histatin 3 (F-histatin 3) was made by using a fluorescein
conjugate at the N terminus of the protein. The protein was purified by
high-pressure liquid chromatography, and its composition was verified
by mass spectrometry.
C. albicans spheroplast preparation.
A clinical
isolate of C. albicans, CA8621, was used in this study
(29). The MICs of amphotericin B, fluconazole, and
flucytosine for CA8621 are 0.125, 0.5, and 1.0 mg/ml, respectively.
Sabouraud dextrose broth (50 ml) was inoculated with a colony of CA8621 and grown overnight at 37°C. Cells were then centrifuged at
1,500 × g for 10 min, and the pellet was washed with
normal saline. Cells were resuspended in 8 ml of spheroplast buffer (1 M sorbitol, 50 mM sodium phosphate, 0.1% [vol/vol]
2-mercaptoethanol, 100 µg of lyticase per ml) and incubated at 30°C
for 45 min. The spheroplasts were washed twice with normal saline and
once with sodium phosphate buffer (10 mM sodium phosphate, 0.5 M sodium
chloride, pH 7.0). Typically, >90% of cells were lysed by 5% sodium
dodecyl sulfate following lyticase treatment.
Binding of histatin 3 to spheroplasts.
Freshly made
spheroplasts were resuspended at 1.7 × 107 cells/ml
in phosphate-buffered saline (PBS), pH 7.0, and added to 1.5-ml microcentrifuge tubes that were precoated with 1% (wt/vol) bovine serum albumin overnight. The spheroplasts were centrifuged at 1,500 × g for 10 min, and the supernatant was
aspirated. Radiolabeled histatin 3 was added to the spheroplasts in a
total volume of 200 µl of either PBS or PBS diluted to 35 mM sodium
chloride (35 mM PBS). In competition assays, a 100-fold excess of
unlabeled histatin 3 was included in the binding reaction mixture. The
tubes were incubated with shaking at either 0 or 30°C, for up to 120 min. After the appropriate incubation time, the cells were washed three
times with 400 µl of PBS and once with spheroplast buffer. The cells
were resuspended in 200 µl of 10 mM sodium phosphate, which was then
added to 4 ml of liquid scintillation fluid. The tubes were rinsed
twice more to remove all of the spheroplasts, and the radioactivity
associated with the spheroplasts was determined. In order to calculate
the unbound histatin 3, the total counts per minute added to the tube
was measured, and the background level of histatin binding to the tube
was also determined.
Visualization of F-histatin 3 binding to C. albicans was
assessed by using freshly prepared spheroplasts. In coated
microcentrifuge tubes, 3.4 × 106 cells were incubated
with 12 nmol of F-histatin 3 per ml in a total volume of 1.1 ml of PBS
or 35 mM PBS. The cells were incubated at either 0 or 30°C for 10, 45, or 90 min. The cells were then pelleted, and all the supernatant
was aspirated. The spheroplast pellets were resuspended in 100 µl of
water, which was spotted directly onto glass slides. The slides were
dried briefly at 37°C, flamed quickly three times, and covered with
glass coverslips. The slides were kept in the dark at 4°C until they
were examined by plain or confocal microscopy. Plain micrographs were
made at standardized exposures on a Nikon Optiphot microscope, with a fluorescent light source. Confocal images were made on a Leica TCS
microscope, equipped with an argon-krypton laser. Images in each
experiment were made at the same instrument settings.
Candidacidal assay of histatin 3.
Freshly grown colonies of
CA8621 were used to inoculate 50-ml cultures in 2% (vol/vol) Sabouraud
dextrose broth. After overnight growth at 37°C, the cells were
pelleted at 1,500 × g and washed once with normal
saline. The CA8621 cells were resuspended in normal saline to a
concentration of 105 cells/ml. For the Candida
killing assay, there were 104 cells in a total of 2 ml of
PBS or 35 mM PBS, as well as the test concentration of histatin 3 (0 to
200 nmol/ml). After incubation at 0 or 37°C, the cells were serially
diluted in normal saline and plated onto Sabouraud dextrose agar.
Colonies were counted after 48 h, and the percent killing was
calculated as [1 
(number with histatin/number without
histatin)] × 100.
Determination of potassium and magnesium efflux from C. albicans.
C. albicans was incubated overnight in Sabouraud
glucose broth, rinsed, and suspended in saline at a final concentration
of 5 × 105 CFU/ml. Cells were incubated with 100 µM
histatin 3 at 37°C for 5, 15, 30, 60, and 120 min in sterile
deionized double-distilled (SDDS) water. Following this incubation,
tubes were spun at 1,100 × g for 10 min; the
supernatants were collected and analyzed. Supernatants from cells with
and without histatin 3 were collected and analyzed at time zero in
order to establish a baseline. The supernatants were quantitated for
potassium and magnesium concentrations by using an atomic absorption
spectrophotometer (Perkin-Elmer model 2380). Samples were diluted 1:2
with SDDS water before testing. Potassium (40 and 80 mM) and magnesium
(5.0 and 20 mM) standards were run before every five samples to ensure
accuracy. Controls containing SDDS water alone, or with histatin 3, revealed no detectable potassium or magnesium. C. albicans
in this assay is the only source of potassium and magnesium in the
extracellular fluid.
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RESULTS |
Synthetic histatin 3 was radiolabeled by reductive methylation,
since this method causes minimal chemical changes to the protein (12). The functional integrity of the protein was verified
by demonstrating that methylated histatin 3 had the same anticandidal activity as the unmodified protein (data not shown). The labeling reaction yielded specific activities of 568 to 1,480 cpm of
[14C]histatin 3 per pmol. Radiolabeled histatin 3 bound
to spheroplasts of C. albicans (CA8621), and most of the
binding could be eliminated by competition with a 100-fold excess of
unlabeled histatin 3 (Fig. 1A). It is
known that the killing of C. albicans by histatins is highly
dependent on the ionic composition and strength of the assay mixture
(31). In these studies, we used ionic strengths that reflect
two possible environments encountered by histatins: PBS, which is
similar to the subsurface tissue interface, and PBS diluted to 35 mM
sodium chloride (35 mM PBS), which is in the range of human saliva
(4). The binding of [14C]histatin 3 to
spheroplasts increased over 120 min of incubation; however, there was
much higher total binding in 35 mM PBS than in PBS (Fig. 1B). Moreover,
at the higher salt concentration [14C]histatin 3 binding
to C. albicans spheroplasts appeared to proceed in two
phases, an initial association over approximately 20 min followed by a
second phase where the increase in binding was slower.
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FIG. 1.
Time course of [14C]histatin 3 binding to
C. albicans in PBS and 35 mM PBS. (A) A total of 2 × 105 spheroplasts (CA8621) were incubated in 2 µM labeled
histatin in PBS (pH 7.0), at 30°C, for up to 120 min. After the cells
were washed, the total amount of bound [14C]histatin 3 was calculated ( ). In order to measure the nonspecific binding of
histatin 3 to C. albicans spheroplasts, the same reaction
mixtures were prepared with the addition of 400 µM unlabeled histatin
3 protein ( ). Hence, the specific binding of
[14C]histatin 3 was calculated to be the total binding
minus the nonspecific binding ( ). (B) Specific binding of
[14C]histatin 3 in 35 mM PBS ( ) and in PBS (). The
data shown are the means of at least two experiments performed in
triplicate ± standard errors of the means.
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To further characterize the binding of histatin to the C. albicans spheroplasts, we determined the kinetic parameters of
[14C]histatin 3 binding under three different conditions,
30°C in PBS, 30°C in 35 mM PBS, and 4°C in PBS (Fig.
2). In these experiments, the initial
binding of histatin following a 10-min incubation was measured. Figure
2A shows the binding of [14C]histatin 3 to the
spheroplasts as a function of the concentration of histatin in the
assay medium. A concentration-dependent increase in binding, which
appeared to saturate around 6 µM when the incubation was carried out
at 30°C in the hypo-osmotic medium, was seen. Further, the level of
binding at each histatin 3 concentration was higher in this medium than
in normal PBS at 30°C or in normal PBS at 4°C. In the latter two
conditions, the binding did not appear to saturate within the
concentration range tested (up to 12 µM; higher concentrations could
not be used due to the relatively low specific activity of the
[14C]histatin). The data were plotted as Lineweaver-Burke
plots (1/bound versus 1/[histatin]). Figure 2B shows the plots of the
data obtained at 30°C with PBS and 35 mM PBS. The plot of the data at
4°C was similar to that of the 30°C-PBS condition and is not shown.
Linear regression of both sets of data demonstrated a dramatic
difference in the slopes of the lines (P < 0.05). The
dissociation constant (Kd) of histatin was
significantly lower in the 35 mM PBS medium than in normal PBS, 2.52 compared with 5.1 µM. The maximal binding of histatin was not
significantly affected by the ionic strength of the medium, 22.85 and
28.9 nM in PBS and 35 mM PBS, respectively. Thus, the apparent affinity
for the binding of histatin to spheroplasts is increased under
hypo-osmotic conditions. This increased affinity is consistent with the
higher level of binding seen in the 35 mM PBS medium (Fig. 1 and Fig.
2A).
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FIG. 2.
Characteristics of initial [14C]histatin 3 binding to C. albicans spheroplasts. A total of 2 × 105 spheroplasts were incubated for 10 min with 0 to 11.25 µM [14C]histatin 3. The assays were performed in PBS at
4°C ( ) and 30°C () and in 35 mM PBS at 30°C ().
Nonspecific binding was determined by the addition of a 100-fold excess
concentration of unlabeled histatin 3. (A) Specific binding under these
conditions (means of three experiments ± standard errors of the
means). (B) Data collected at 30°C are shown as Lineweaver-Burke
plots, from which the dissociation constant and maximal binding were
calculated. The Kd of histatin 3 in PBS and 35 mM PBS was 5.1 and 2.52 µM, respectively. The maximal binding
(Vmax) of histatin 3 was 22.85 and 28.9 nM,
respectively.
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The use of F-histatin 3 facilitated the visualization of histatin
binding to C. albicans. Incorporation of fluorescein at the
N terminus of F-histatin 3 reduced the killing activity of the protein
by less than 10% (data not shown). After 10 min of incubation with
C. albicans spheroplasts, there was little visible binding
of F-histatin 3 (Fig. 3). By 45 min in 35 mM PBS, F-histatin 3 could be seen binding to the spheroplasts with a
patchy distribution, and a few cells appeared to be full of the
protein. In 35 mM PBS, almost all of the spheroplasts contained
abundant F-histatin 3 by 90 min, while those incubated in PBS showed
weak patches of F-histatin 3 binding (Fig. 3). Visualization of
F-histatin 3 with spheroplasts demonstrated that binding is dependent
on time and extracellular ion concentration, which is consistent with
the binding of [14C]histatin 3. Additionally, it seems
that F-histatin 3 is internalized by C. albicans only after
a period of binding to the surface. Exposure of C. albicans
cells to histatin 3 in either PBS or 35 mM PBS for 10 min resulted in
little loss of viability (Fig. 4). Extending the exposure time to 90 min increased cell killing, and the
effect was much more marked in 35 mM PBS (Fig. 4).
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FIG. 3.
Binding of fluorescently labeled histatin 3 to C. albicans. Freshly prepared C. albicans spheroplasts
were incubated at 30°C in the presence of 12 µM F-histatin 3. Progressive binding of histatin to the cells was seen in 35 mM PBS at
10 min (A), 45 min (B), and 90 min (C). Little binding of histatin 3 to
Candida was observed in PBS by the 90-min point (D). The
binding of F-histatin 3 to the cells could be eliminated by competition
with unlabeled histatin 3 (data not shown). The same film exposure was
used for each panel of the figure.
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FIG. 4.
Histatin 3-induced killing of C. albicans.
Whole C. albicans cells were treated with increasing
concentrations of histatin 3 in PBS or 35 mM PBS, for 10 or 90 min, at
37°C. The viability of Candida after treatment was
determined by plating the cells onto Sabouraud dextrose agar and then
counting the number of colonies after 48 h. The percentage of
cells killed was calculated as [1 (number with histatin/number
without histatin)] × 100. Results are shown for cells incubated in
PBS plus 12.5 (), 25 (), and 50 ( ) µM histatin 3 and for
cells incubated in 35 mM PBS plus 12.5 (), 25 (), and 50 ()
µM histatin 3. The data are from a representative experiment
performed in triplicate (means ± standard errors of the means).
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In order to further understand the mechanism of action of histatin 3 on
C. albicans, we investigated the kinetics of potassium and
magnesium efflux from the cells (Fig. 5).
Following incubation of the cells with histatin 3, the potassium and
magnesium concentrations in the extracellular medium were significantly
increased above baseline and controls (P 
0.0001
[analysis of variance]). The largest amount of potassium was released
within the first 30 min of incubation with histatin 3. Extracellular
potassium levels remained significantly elevated at 
200 µM for the
remaining incubation period of 120 min. Similar findings were observed
for the effect of histatin 3 on magnesium efflux. The largest amount of
magnesium also was released into the extracellular fluid within the
first 30 min of incubation. Extracellular magnesium levels remained significantly elevated at 40 µM for the remaining incubation period
of 120 min.
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FIG. 5.
Kinetics of potassium and magnesium efflux from C. albicans. Efflux of potassium (A) and magnesium (B) from C. albicans during incubation with 100 µM histatin 3. Potassium and
magnesium release was significantly increased when cells were incubated
with histatin 3 (), compared with incubation with water only ()
(P 0.0001 [analysis of variance]; values are shown
as means ± standard errors of the means).
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The significance of the extracellular ion concentration on histatin
3-mediated killing of C. albicans was further evaluated in
the following experiment. Cells were first incubated in PBS for 60 min
with 50 µM histatin 3. This served to preload cells with histatin 3 in conditions that do not result in significant cell death. The cells
were then washed and diluted with either PBS or water (final
concentration, approximately 15 mM NaCl), after which the incubation
was continued for 90 min. Figure 6 shows
that only C. albicans cells exposed to histatin 3 and then diluted with water suffered a dramatic loss in viability. These results
clearly imply that even though a low extracellular ionic concentration
can increase binding of histatin 3 to C. albicans, its
significance in cell killing occurs postbinding. In other words,
a low-extracellular-salt environment is important for both the binding of histatin 3 and its subsequent action on the cell.
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FIG. 6.
Effect of extracellular ion concentration on C. albicans viability after binding of histatin 3. Whole C. albicans cells were first treated with 50 µM histatin 3 for 60 min in PBS. Control cells were incubated without histatin 3. Treated
and control cells were washed once in PBS and then diluted
approximately 10-fold in either PBS or water. Cell viability was
measured immediately after dilution and then measured again 30 and 90 min later. The figure shows the number of CFU ± standard errors
of the means of a representative experiment performed in triplicate.
, histatin 3-treated cells in PBS; , histatin 3-treated cells in
water; , control cells in PBS; , control cells in water.
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In order to further dissect the mechanism of histatin 3 action on
C. albicans, we attempted to inhibit the internalization of
histatin 3. Incubation of cells in the presence of sodium azide, 2-deoxyglucose, or bafilomycin was not able to block the uptake of
[14C]histatin 3 (data not shown). However, when the
binding assay temperature was changed from 30 to 0°C, there was a
substantial decrease in [14C]histatin 3 binding to
spheroplasts (Fig. 7). The reduction in binding at 0°C was especially notable after 20 min, a point at which
the internalization of F-histatin 3 becomes apparent.
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FIG. 7.
[14C]histatin 3 binding to C. albicans is temperature dependent. Binding of
[14C]histatin 3 to spheroplasts was determined at 30 and
0°C. A total of 2 × 105 cells were incubated in 35 mM PBS containing 2 µM labeled histatin 3 for 0 to 120 min. The
specific binding of [14C]histatin 3 was calculated at
0°C () and 30°C (). The data are derived from triplicate
measurements in two experiments.
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The characteristics of F-histatin 3 binding to C. albicans
spheroplasts at 0°C were observed by confocal microscopy (Fig. 8). After 45 min at 0°C in the presence
of F-histatin 3, an occasional cell had detectable protein binding
(Fig. 8C). By 90 min, several cells demonstrated a small point of
F-histatin 3 binding, but very few cells contained histatin
intracellularly (Fig. 8E). These results are consistent with our
interpretation of the [14C]histatin 3 binding experiment;
by reducing the assay temperature, histatin 3 internalization by
C. albicans cells is prevented. In contrast, by 45 min at
30°C, many cells clearly showed the punctate pattern of F-histatin 3 binding, while others had substantial protein within them (Fig. 8D).
When the process continued to 90 min, almost all of the cells were
brightly labeled by F-histatin 3 (Fig. 8F). Optical sections confirmed
that the F-histatin 3 was intracellular, typically concentrated around
the cell periphery and at one cell pole (Fig. 8). Functional studies of
histatin 3 support the significance of temperature in histatin-mediated killing of C. albicans. When cells were incubated with up to
200 nmol of histatin 3 per ml at 0°C, the cell viability remained above 70% of the control (no histatin) level (Fig.
9). However, when cells were treated at
37°C, cell viability was less than 10% of the control. Together,
these data imply that it is not sufficient for histatin to associate
with the Candida cell membrane for killing to occur.
Secondary events, which may include the translocation of histatin to
intracellular compartments, are required for cell death.
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FIG. 8.
Internalization of histatin 3 by C. albicans
is temperature dependent. Whole cells were treated with fluorescently
labeled histatin 3 at either 0 or 30°C, in 35 mM PBS. After rinsing
and fixation of the cells, 0.2-µm optical sections were generated for
each sample. By using a similar instrument gain, the most positive
section representing each experimental condition was selected: 10 min
at 0°C (A), 10 min at 30°C (B), 45 min at 0°C (C), 45 min at
30°C (D), 90 min at 0°C (E), and 90 min at 30°C (F).
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FIG. 9.
C. albicans is resistant to histatin
3-mediated killing at low temperatures. C. albicans cells
were treated with up to 200 µM histatin 3 at 0 or 37°C, and their
viability was determined by measuring colony formation. Control cells
were incubated without histatin. The percentage of viable cells was
calculated as (number with histatin/number without histatin) × 100. The data shown are the means from triplicate measurements in two
experiments ± standard errors of the means.
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DISCUSSION |
The role of salivary antifungal proteins in the control of oral
Candida populations remains an enigma. Though the histatins have long been demonstrated to have quite potent candidacidal activity
in vitro, there are conflicting data with regard to their significance
during candidal overgrowth. One reason that it has been difficult to
assess the effects of histatins on candidal growth is that the basis of
their selective killing of microorganisms remains to be understood.
Hence, there is a need to investigate the nature of histatins'
interactions with Candida (and other fungi) and their
mechanism of action.
This report demonstrates that histatin 3 binds reversibly to a site on
the surface of spheroplasts of C. albicans CA8621. The
dissociation constant for the histatin 3 binding site was dependent on
the salt concentration: 2.52 µM in 35 mM PBS, compared to 5.1 µM in
PBS (Fig. 2). Moreover, the total amount of histatin binding to
C. albicans was greater in a low-salt environment, as shown
by [14C]histatin 3 and fluorescently labeled histatin 3 (Fig. 1B and 3). These findings are consistent with the reported
Kd for 125I-histatin 5 of 0.95 µM
in a 10 mM phosphate buffer and the observation that the candidacidal
activity of histatin 3 is increased at low ionic strength (7,
31). There was no significant difference between spheroplasts and
whole cells in binding [14C]histatin 3 or in
histatin-mediated cell death (data not shown), which supports the
plasma membrane as the presumptive binding site for histatins. This
result concurs with the work of Driscoll and colleagues, who found that
spheroplasts and whole cells were equally sensitive to killing by
histatin 5, but is different from the work of Edgerton and colleagues,
who found that spheroplasts bound histatin 5 poorly and were not
susceptible to killing by histatin 5 (6, 7). The disparity
between these results is likely due to the method by which the
spheroplasts are prepared: lyticase treatment of C. albicans
yields spheroplasts that bind histatins, while Zymolyase renders the
cells insensitive to histatins. Since these enzymes are prepared from
different sources, they can presumably strip different proteins from
the cell surface, which may include the histatin binding site. In
addition, the interaction of histatin 3 with the hyphal form of
C. albicans warrants specific investigation, given the
pathogenic significance of this form of the organism and the lack of
histatin binding data to date.
Binding of histatin 3 to the C. albicans cell is followed by
internalization of the protein, and this internalization is related to
cell death (Fig. 3 and 4). In this study, cell death was observed only
after histatin 3 had bound to the cell and a hypo-osmotic environment
persisted; neither condition alone was sufficient for killing to occur.
There was a delay between the initial binding of histatin 3 to the
cells and the accumulation of protein inside the cells. The amount of
[14C]histatin 3 associated with C. albicans
was noticeably greater in 35 mM PBS than in PBS only after 20 min, and
the difference was progressive after that. These data indicate that
after initial binding to the cell surface, histatin 3 is internalized
more rapidly under low-osmolarity conditions. When cells were preloaded
with histatin 3 and then switched to either PBS or 35 mM PBS, only those in the hypo-osmotic environment suffered significant death (Fig.
6). Likewise, cells which were not exposed to histatin 3 maintained
normal viability in the low-salt environment. Hence, hypo-osmotic
conditions appear to favor binding of histatin 3 to Candida,
internalization of bound histatin 3, and interactions with the cell
that result in cell death.
The above model, which suggests a complex mode of action of histatins
on C. albicans, also supports the theory that histatins do
not act by simply forming pores in the cell membrane, which cause the
cell contents to leak out. It has previously been shown that while
histatin binding may cause cells to lose potassium, they do not become
permeable to the intracellular dye calcein (molecular weight, 622), and
they do not exhibit gross disturbances in intracellular morphology
(7, 20, 26). Rather, after binding histatin 3, C. albicans dies by some mechanism which is osmotically dependent and
may involve the selective loss of small molecules. Here we have shown
that the efflux of potassium from C. albicans begins within
5 min of incubation with histatin 3. Histatin 3-mediated loss of
potassium and magnesium is almost maximal by 30 min, which is the same
time frame as cell death in a very low osmotic medium. These data are
compatible with binding to cell membrane proteins, such as ATPases,
which are responsible for regulating potassium and magnesium
homeostasis. As histatin 3 is a small protein (32 amino acids) and only
weakly amphipathic, the early efflux of potassium and magnesium also
does not suggest a mechanism of classical transmembrane channel
formation observed with peptides such as cecropin and magainins
(5). Since these experiments were performed with synthetic
histatin 3, the results do not support the contention that an earlier
report of potassium efflux from C. albicans was caused by a
contaminant in the histatins purified from human saliva (7,
20).
In support of a specific action of histatin 3 on C. albicans, it is noteworthy that even low levels of histatin
binding were able to effect cell death under hypo-osmotic conditions.
This was evident when cells were first incubated with histatin 3 in PBS
for 60 min
a condition which was shown to result in modest levels of
histatin binding (Fig. 1B). When the cells were subsequently washed and
incubated in a low-salt environment, almost complete killing of the
cells was observed. This result implies that the amount of histatin 3 bound to the cell was sufficient for effective killing and that
continuous incubation with histatin was not required. In addition,
histatin-mediated killing of C. albicans is known to be pH
dependent, which would be difficult to reconcile with a mechanism
involving direct permeabilization of the cell membrane (3,
31).
Internalization of histatin 3 by C. albicans was inhibited
by lowering the temperature of the cells to 0°C. The initial
association of histatin 3 with the cell was not significantly different
at the lower temperature, though after approximately 20 min the amount of histatin 3 accumulation in the cell decreased dramatically compared
to that in the cells at 30°C (Fig. 7 and 8). Moreover, at 0°C the
amount of [14C]histatin 3 binding to spheroplasts
plateaus at 60 min, whereas at 30°C the binding increases up to 120 min (Fig. 7). This observation suggests that at 0°C binding of
[14C]histatin 3 to the cell membrane becomes saturated,
probably because the histatin does not translocate to the cell
interior. Notably, cell killing was also attenuated at the lower
temperature (Fig. 9). Together, these data support the proposition that
the candidacidal activity of the histatins does not result from
transmembrane channel formation but rather is due to an indirect action
on the cell. Recently, Helmerhorst and colleagues have shown that
histatin 5 localizes to mitochondria and that it is capable of
dissipating the mitochondrial transmembrane potential (9).
Our data are consistent with this view, inasmuch as we have shown that
decreasing the internalization of histatin 3 increases cell survival.
It has been suggested that other molecules can be cointernalized with
histatin 5 into C. albicans, but to date, little is known about the mechanism by which histatin enters cells after binding to its receptor.
Conventional and confocal micrographs indicate clearly that F-histatin
3 binds first to a discrete area of the cell membrane, then
concentrates around the cell periphery, and with increased time of
incubation accumulates within the cells. We did not attempt to localize
the histatin within C. albicans cells, nor is it clear at
this point precisely at which stage of histatin penetration the cells
die. Given the observation that comparatively small amounts of bound
histatin 3 may be lethal to C. albicans, it is possible that
the accumulation of histatin may be a late event that occurs even after
cells have died. Thus, the mechanisms of histatin 3 lethality for
C. albicans may consist of three phases dependent upon
temperature and ionic strength: initial binding to the fungal cell
membrane, increased permeability of the fungal cell membrane, and
intracellular uptake with possible targeting of organelle structures.
Future studies will focus on defining the target for histatin 3 binding
on the plasma membrane, the nature of the initial increase in cell
permeability, and the intracellular role of histatin during candidal
cell death.
| |
ACKNOWLEDGMENTS |
This work was supported by the National Institute of Dental and
Craniofacial Research (ZO1-00675).
We thank Beverly Handyman, Joanne Peter, and Tin Sein for technical
assistance with these studies.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: School of Dental
Science, Trinity College, Dublin 2, Ireland. Phone: 353-1-612-7312. Fax: 353-1-612-7298. E-mail: BrianO'Connell{at}dental.tcd.ie.
Present address: School of Stomatology, Beijing Medical University,
Haidan District, Beijing 10081, China.
Present address: Department of Pharmacology, Tokyo Dental College,
Mihamaku, Chiba 261-8502, Japan.
| |
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Abstract
Introduction
