Characterization of Histatin 5 with Respect to Amphipathicity,
Hydrophobicity, and Effects on Cell and Mitochondrial Membrane
Integrity Excludes a Candidacidal Mechanism of Pore Formation*
Eva J.
Helmerhorst,
Wim
van't Hof¶,
Pieter
Breeuwer
,
Enno C. I.
Veerman¶,
Tjakko
Abee
,
Robert F.
Troxler
,
Arie V. Nieuw
Amerongen¶, and
Frank G.
Oppenheim
From the
Department of Periodontology and Oral
Biology, Boston University Goldman School of Dental Medicine, Boston,
Massachusetts 02118, the ¶ Department of Oral Biochemistry,
Academic Center for Dentistry (ACTA), 1081 BT, Amsterdam, The
Netherlands, and the
Department of Food Technology and
Nutritional Sciences, Laboratory of Food Microbiology, 6703 HD,
Wageningen, The Netherlands
Received for publication, September 8, 2000, and in revised form, November 29, 2000
 |
ABSTRACT |
Histatin 5 is a 24-residue peptide from human
saliva with antifungal properties. We recently demonstrated that
histatin 5 translocates across the yeast membrane and targets to the
mitochondria, suggesting an unusual antifungal mechanism (Helmerhorst,
E. J., Breeuwer, P., van`t Hof, W., Walgreen-Weterings, E.,
Oomen, L. C. J. M., Veerman, E. C. I., Nieuw
Amerongen, A. V., and Abee, T. (1999) J. Biol.
Chem. 274, 7286-7291). The present study used specifically
designed synthetic analogs of histatin 5 to elucidate the role of
peptide amphipathicity, hydrophobicity, and the propensity to adopt
-helical structures in relation to membrane permeabilization and
fungicidal activity. Studies included circular dichroism measurements, evaluation of the effects on the cytoplasmic transmembrane potential and on the respiration of isolated mitochondria, and analysis of the
peptide hydrophobicity/amphipathicity relationship (Eisenberg, D. (1984) Annu. Rev. Biochem. 53, 595-623). The 14-residue
synthetic peptides used were dh-5, comprising the functional domain of
histatin 5, and dhvar1 and dhvar4, both designed to maximize
amphipathic characteristics. The results obtained show that the
amphipathic analogs exhibited a high fungicidal activity, a high
propensity to form an
-helix, dissipated the cytoplasmic
transmembrane potential, and uncoupled the respiration of isolated
mitochondria, similar to the pore-forming peptide PGLa
(Peptide with N-terminal Glycine and C-terminal Leucine-amide). In
contrast, histatin 5 and dh-5 showed fewer or none of these features.
The difference in these functional characteristics between histatin 5 and dh-5 on the one hand and dhvar1, dhvar4, and PGLa on the other hand
correlated well with their predicted affinity for membranes based on
hydrophobicity/amphipathicity analysis. These data indicate that the
salivary protein histatin 5 exerts its antifungal function through a
mechanism other than pore formation.
 |
INTRODUCTION |
Throughout living nature, distinct groups of cationic peptides
have been identified that display antimicrobial activity in vitro. Best characterized with respect to their structure-function relationship are magainins from the skin of the frog Xenopus
laevis (1, 2), cecropins from the giant silk moth Hyalophora
cecropia (3), and defensins (
and
) from human leukocytes
and various epithelial sources, respectively (4-6). These peptides
show considerable variation in chain length, hydrophobicity, and charge
distribution; however, they share the common feature of being cationic
in nature and able to adopt 70 ordered amphipathic
-helix or
-sheet conformations in structure-promoting solvents, such as
trifluoroethanol, and in membrane-mimicking liposome vesicles. Their
antimicrobial mode of action is believed to arise from the attraction
to negatively charged surface molecules on the target cell and the
subsequent formation of a membrane-spanning porelike structure, thereby
altering membrane permeability leading to cell lysis.
The regulated and constitutive expression of such cationic peptides
provides an immediate protection to tissues that are continuously subjected to microbial challenges (7, 8). In the oral cavity, the hard
and soft tissues are constantly exposed to a variety of microorganisms
that can lead to caries and periodontal disease. This condition
requires that antimicrobial components are continually secreted to
provide a permanent line of defense against microbial invasion or at
least to maintain the harmonious relationship between the commensal
microflora and the host (9). Examples of such salivary antimicrobial
systems are histatins. Histatins are a group of histidine-rich cationic
peptides that are secreted by the parotid and the
submandibular/sublingual human salivary glands (10, 11). At least 12 fragments have been identified (12) that derive from the proteolytic
degradation of two gene products, histatin 1 and histatin 3 (13).
Histatin 5 has been shown to be the most potent member with respect to
its fungicidal activity against the oral opportunistic pathogen
Candida albicans, and the domain responsible for this
activity has been located in the C-terminal 14 residues (14,
15). Despite a great interest in understanding the mechanism of
histatin function, the molecular events leading to cell death have so
far been largely elusive (16-20).
Salivary histatins differ from other natural antimicrobial peptides in
a number of respects. First, histatins are enriched in the amino acid
histidine. Histidine has an isoelectric point of 6.5 and, therefore,
modulates the cationicity of the peptide considerably at lower pH
values. In addition to this, histidine side chains are known
participants in metal chelation. Indeed, histatin 5 is able to form
complexes with various metal ions (21), and this adds potential
biological properties to this peptide compared with other basic
peptides. Secondly, although histatins like other antimicrobial
peptides adopt helical conformations in hydrophobic environments (15,
22), the amphipathicity of this helix quantified by the hydrophobic
moment is rather low compared with other antimicrobial peptides, such
as the magainins (16). Because peptide amphipathicity is believed to be
a key factor in governing the cytolytic activity of pore-forming basic antimicrobial peptides (23), it is questionable whether histatin 5 operates by the same mode of action. We recently demonstrated that
histatin 5 translocates across the yeast membrane and shows intracellular targeting to the mitochondria, suggesting an unusual antifungal mechanism (18). The present investigation focuses on
physical peptide parameters, such as amphipathicity, hydrophobicity, and the propensity to adopt helices in relation to these unique functional characteristics of histatin 5. The biophysical and biological results obtained show that histatin 5, in contrast to more
amphipathic peptides employed for comparison, is not a classic pore
former, and these aspects are discussed with respect to its capacity to
translocate across the yeast membrane.
 |
EXPERIMENTAL PROCEDURES |
Antimicrobial Peptides--
Histatin 5, the C-terminal domain
dh-5, and peptides derived from this domain, dhvar1 and dhvar4, were
chemically synthesized as previously described (16, 24). Synthetic
PGLa1
(Peptide with N-terminal Glycine and
C-terminal Leucine-amide) was a kind
gift from H. V. Westerhoff (Dept. of Molecular Cell Physiology,
Vrije Universiteit, Amsterdam).
Circular Dichroism--
CD spectra were recorded under computer
control on an AVIV 62DS CD spectropolarimeter (AVIV Associates, Inc.,
Lakewood, NJ) from 250 to 183 nm at 25 ± 0.5 °C using a 0.1-cm
quartz cuvette. Peptides were dissolved in pure water, in pure
trifluoroethanol (TFE), or in a mixture of both solvents to a final
concentration of 0.04-0.14 mg/ml. Spectra (1-nm intervals, 1 s/interval with 5-15 repeats) were averaged and corrected for
base-line contribution. Molar ellipticity values [
] were
calculated according to Equation 1,
|
(Eq. 1)
|
where
is the displacement from the base-line value for the
full range in degrees, MRW is the mean residue weight of the amino
acids in the protein structure, l is the path length of the
cuvette in cm, and c is the concentration of the protein in g/ml.
Antifungal Assay--
C. albicans 315 (ATCC 10231)
was cultured from a glycerol stock on Sabouraud dextrose agar (Difco,
Detroit, MI) and maintained at most for 1 week. Several yeast colonies
were picked from the plate and suspended in 1, 10, 20, or 50 mM potassium phosphate buffer, pH 7.0, to yield a
suspension of 5.2 × 106 cells/ml. From this
suspension, 250 µl was added to 250 µl of a dilution of histatin 5, dhvar1, or dhvar4 in the same buffer. After 0, 6, 12, 18, 24, 30, 40, 60, and 90 min of incubation in a 37 °C water bath, 50-µl samples
were diluted in 9 ml of phosphate-buffered saline and mixed. From
this suspension, 25 µl was plated on Sabouraud dextrose agar and
incubated for 48 h at 30 °C to determine the percentage
reduction in viable counts. Log count reductions were determined by
plating 25 µl from a 10-fold serial dilution series of the
samples in phosphate-buffered saline.
Fluorescence Spectroscopy--
Relative changes in the
cytoplasmic transmembrane potential of C. albicans were
measured using the fluorescent probe
3,3'-dipropyl-2,2'-thiadicarbocyanine iodide (diS-C3-(5))
(Molecular Probes, Inc., Eugene, OR) (25, 26). The measurement was
carried out in a 3-ml fluorescence cuvette connected to a 30 °C
thermostated water bath. Cells were picked from a Sabouraud dextrose
agar plate, suspended in 1 mM potassium phosphate buffer,
pH 7.0, and added to the thermostated cuvette containing 1 mM potassium phosphate buffer to a final cell density of
2.6 × 106 cells/ml. Subsequently,
diS-C3-(5) was added from a stock solution of 0.5 mM in ethanol to a final concentration of 3.3 µM. Changes in the fluorescence intensity were measured
at
ex = 643 ± 10 nm and
em = 666 ± 10 nm with a PerkinElmer LS 50 B spectrofluorimeter supplied with a pulsed xenon light source.
Isolation of Yeast Mitochondria--
Mitochondria were isolated
from C. albicans spheroplasts essentially as described
previously (27). C. albicans (ATCC 10231) were grown to late
log phase in 1 liter of Sabouraud dextrose broth (Difco), washed once
in 1.2 M sorbitol (Sigma), and suspended in 1.2 M sorbitol supplemented with 50 mM potassium
phosphate, pH 7.4. For every milliliter of yeast suspension with an
A620 of 10, 2 µl of
-mercaptoethanol
(Sigma) and 10 units of yeast lytic enzyme (ICN Biomedicals, Costa
Mesa, CA) were added, and the suspension was incubated for 1 h at
37 °C. Spheroplasts were collected by centrifugation in a
Sorvall tabletop centrifuge with a H-1000B rotor at 2100 × g at 4 °C, washed 4 times in 1.2 M sorbitol, suspended in 5-10 ml of 1.2 M sorbitol, and stored at
4 °C for up to 16 h. After centrifugation for 5 min at
2100 × g, spheroplasts were suspended in 15 ml of 0.4 M sorbitol, 0.2% (w/v) bovine serum albumin (fraction V,
Sigma), and 10 mM imidazole (Fisher), pH 6.4, and
homogenized on ice for 10 min using a manual Potter-Elvehjem homogenizer. The homogenate was mixed 1:1 with buffer containing 1 M sorbitol, 25 mM
KH2PO4, 4 mM EGTA (Sigma), 0.2%
(w/v) bovine serum albumin, and 10 mM imidazole, pH 6.4, and centrifuged in a Sorvall RC-5B centrifuge with a SS-34 rotor for 5 min at 1000 × g at 4 °C. The supernatant was
carefully removed and centrifuged using the same rotor for 10 min at
12,000 × g at 4 °C. The reddish pellet containing
the mitochondria was suspended in a small volume (typically 1 ml) of
0.6 M mannitol, 2 mM EGTA, 0.2% bovine serum albumin, and 10 mM imidazole, pH 6.4, and kept on ice. In
each experiment the same volume of the mitochondrial suspension was used for polarographic measurements (final A620
of 0.15-0.25).
Measurement of Mitochondrial Respiratory
Activity--
Mitochondrial oxygen consumption was measured using a
biological oxygen monitor model 5300 equipped with a 5331 standard
oxygen probe (YSI, Inc., Yellow Springs, OH). Measurements were
performed in air-saturated buffer containing 0.65 M
mannitol, 2 mM MgCl2, 16 mM
KH2PO4, 10 mM imidazole, pH 6.4. State 2 or basal rate respiration was measured after the addition of
NADH (Sigma) to a final concentration of 1 mM, a substrate
that can be directly oxidized by yeast mitochondria (28). State 3 respiration was determined after the addition of ADP (Sigma) to a final
concentration of 0.33 mM. State 4 respiration was
determined after all the ADP was used and the respiration had returned
to the basal rate.
 |
RESULTS |
Structural Analysis--
The amino acid sequences, the isoelectric
point, the mean hydrophobicity (H), and the mean hydrophobic
moment (µ) of histatin 5, the fungicidal domain dh-5, the two
substitution analogs dhvar1 and dhvar4, and the two magainin peptides,
magainin 2 and PGLa, are summarized in Table
I. The design of dhvar1 and dhvar4 was based on a helical wheel projection of dh-5 (16). The peptides are all
basic with isoelectric points ranging from 10 to 12. Major differences
were observed in the mean hydrophobicities and mean hydrophobic
moments. Histatin 5 and dh-5 have a relatively low hydrophobic moment
and, therefore, are weakly amphipathic in contrast to dhvar1, dhvar4,
magainin 2, and PGLa, which have a high hydrophobic moment and are
highly amphipathic. These amphipathicities were calculated assuming
pure helix conformation. Previous studies with histatin 5 and a number
of recombinant histatin analogs have shown that these peptides all
adopted helical conformations in pure TFE (29, 30). To assess the
inducibility of
-helix formation and to detect potential differences
between histatin 5, dh-5, dhvar1, and dhvar4, the CD spectra were
recorded in aqueous solvents with a stepwise increase of the TFE
content (Fig. 1). In pure water
(water/TFE ratio of 100/0), the spectra of all peptides showed a
maximum molar ellipticity between 215 and 220 nm and a minimum molar
ellipticity below 200 nm, which is typical for random coil
conformation. At increasing TFE content of the solvent,
-helical
conformations were inducible in all peptides, characterized by two
negative CD bands between 218-222 and 206-208 nm and a maximum molar
ellipticity below 200 nm. The peptides differed in their propensity to
adopt helix conformations. Dh-5 was most reluctant to adopt helices
being only helical in pure TFE, whereas dhvar4 most rapidly adopted
helices being only random coil in pure water. Histatin 5 and dhvar1
showed the spectra consistent with mixtures of random coil and
-helix conformations in mixtures of water and TFE. Although the
tendency to adopt helices at increasing hydrophobicity was predominant,
it is interesting to note that it was repeatedly found that peptide
dhvar1 did not assume the highest degree of
-helix in pure TFE but
did in a 50/50 water/TFE mixture.

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Fig. 1.
CD spectra of histatin 5, dh-5, dhvar1, and
dhvar4. CD spectra were recorded in mixtures (% v/v) of
water/trifluoroethanol: 100/0 ( ), 75/25 ( ), 50/50 ( ), 0/100
( ). Each curve represents the average of 5-15 repeated
measurements on one sample.
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Kinetics of Killing of C. albicans--
To compare the kinetics of
the killing of C. albicans, cells were exposed to histatin
5, dh-5, dhvar1, and dhvar4 for different time intervals after which
the viability of the cells was determined in a colony-forming assay
(Fig. 2). At 8.3 µM,
histatin 5, dhvar1, and dhvar4 caused a 100% reduction in the
viability of the yeast inoculum, which was equivalent to a reduction by
3 log units in viable counts (data not shown). With histatin 5, almost
100% reduction in viability was observed after 20 min of incubation.
Incubation for 90 min with 8.3, 3.3, 1.6, and 0.8 µM histatin 5 resulted in a 100, 90, 35, and 15%
reduction in viable counts, respectively. With dh-5 comprising residues
11-24 of histatin 5, very comparable results were obtained. On the
other hand, exposure of the cells to only 1.6 µM dhvar1
or only 0.8 µM dhvar4 resulted in a 100% reduction in
viable counts within 6 min of incubation, demonstrating the higher
activity of these analogs.

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Fig. 2.
Time-dependent killing activity
of histatin 5, dh-5, dhvar1, and dhvar4 against C. albicans.
Cells were incubated with each peptide at four concentrations: 0.8 µM ( ), 1.6 µM ( ), 3.3 µM ( ), and 8.3 µM ( ) in 1 mM potassium phosphate buffer, pH 7.0. At selected time
points, samples were taken, and cell viability was determined in a
colony-forming assay. Values represent the mean and deviation of the
mean of two independent experiments.
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It is well known that the presence of ions affects the antimicrobial
activity of several antimicrobial peptides including histatins (31).
Sensitivity studies to ions with histatin 5, dh-5, dhvar1, and dhvar4
were carried out with equally effective rather than equimolar
concentrations to compensate for the higher molar potencies of dhvar1
and dhvar4 compared with histatin 5 and dh-5. Such concentrations were
derived from experiments as shown in Fig. 2. Under these conditions,
histatin 5, dhvar1, and dhvar4 showed comparable dependence on the
ionic strength (Table II). Because the
peptides are well soluble in all buffers used, these data suggest that
ions interfere with the peptide-cell interaction, either by preventing
electrostatic interactions or by modifying groups on the yeast cell
envelope.
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Table II
Percent viability of C. albicans cells after 1.5-h exposure to equally
effective concentrations of histatin 5, dh-5, dhvar1, and dhvar4
The concentrations that were equally effective in killing C. albicans were 8.3 µM histatin 5, 16.6 µM dh-5, 1.6 µM dhvar1, and 1.6 µM dhvar4.
PPB, potassium phosphate buffer.
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Uncoupling of the Yeast Cytoplasmic Transmembrane
Potential--
Although the initial interaction for histatin 5, dh-5,
dhvar1, and dhvar4 is presumably electrostatic in nature, the ability of these peptides to interact with lipophilic portions of the bilayer
and to insert into the microbial membrane might be significantly different. Such interactions, which may lead to the disruption of
membrane integrity, have often been studied using liposome vesicles as
a model system for microbiological membranes (32, 33). In the present
study, the effect of peptide on aspects of the integrity of whole
C. albicans cells was studied by the measurement of changes
in the cytoplasmic transmembrane potential. For this purpose,
diS-C3-(5), a fluorescent dye with membrane potential-dependent distributional properties, was used.
This probe is fluorescent in solution but autoquenches when it
accumulates intracellularly (26). It should be pointed out that the
accumulation of the dye is driven by the potential across the
cytoplasmic membrane rather than the potential across the mitochondrial
membrane because sodium azide, which dissipates the mitochondrial
transmembrane potential (18), does not prevent the intracellular
accumulation of diS-C3-(5) (data not shown). For this
experiment, relatively high doses of peptide were chosen so that the
dissipating effect could be determined immediately after the addition
of the peptide. Fig. 3 shows that the
exposure of C. albicans cells, loaded with diS-C3-(5), to 18 µM dhvar4 results in an
increase in the fluorescence signal to the level just after adding the
probe to the cells, indicating the complete release of the dye into the
extracellular environment due to cell membrane permeation. At this
concentration, histatin 5 and dh-5 had no effect, and testing at a
6-fold higher concentration to compensate for their lower molar potency
only caused a small increase in fluorescence intensity.

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Fig. 3.
Spectrofluorimetric determination of the
effect of histatin 5, dh-5, and dhvar4 on the cytoplasmic transmembrane
potential of C. albicans cells. To a suspension
of C. albicans in 1 mM potassium phosphate
buffer, pH 7.0, diS-C3-(5) was added at t = 100 s. After internalization of the probe at t = 500 s, histatin 5 (final concentration of 109 µM),
dh-5 (final concentration of 109 µM), or dhvar4 (final
concentration of 18 µM) was added. The graphs
are representative of at least three separate experiments.
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Uncoupling of Respiration in Isolated Yeast
Mitochondria--
Mitochondria were isolated from C. albicans for two purposes. The first purpose was to investigate
the effect of histatin 5 on the respiratory activity of isolated yeast
mitochondria. Interest in this stems from our previous observation that
fluorescein isothiocyanate-labeled histatin 5 associates with C. albicans mitochondria in situ (18). The second purpose
was to assess whether dhvar4, which was shown in the previous
experiment to alter the membrane integrity in whole cells, would in
analogy of magainin peptides (34, 35) act as an uncoupler of the
respiration of isolated mitochondria. In Fig.
4A, the respiratory activity of the mitochondria is shown in the presence of the substrate NADH
(State 2 respiration), after the addition of ADP (State 3), and after
the depletion of ADP (State 4). The respiratory control ratio defined
as the ratio of the respiratory rates in States 3 and 4 was 2.6, indicating that the respiration was coupled to ATP formation. The
increase in respiration upon the addition of ADP is a measure for the
integrity of the mitochondrial inner membrane, which was a prerequisite
to study the uncoupling potential of the peptides. The peptides were
added to mitochondria respiring in State 2. Histatin 5 at a final
concentration of 33 µM inhibited mitochondrial
respiration by 63%. Inhibition of the respiratory chain was verified
by the observation that the addition of the uncoupler CCCP after
histatin 5 did not increase respiration. dh-5 at the same concentration
had no inhibitory effect on respiration, and as expected, the addition
of CCCP after dh-5 increased respiration. In contrast to histatin 5 and
dh-5, dhvar4 increased mitochondrial respiration by a factor of
2.2 ± 0.3 at a concentration of 3.6 µM. For dhvar1
similar results were obtained (data not shown), indicating that both
dhvar1 and dhvar4 act as uncouplers. For comparison, CCCP at a
concentration of 33 µM increased respiration by a factor
of 2.7 ± 0.5 (Fig. 4B). PGLa was even more active than
CCCP because at a concentration of 3.4 µM, a respiratory increase of 3.7 ± 0.5 was found, which is similar to its
previously reported effect on isolated rat liver mitochondria (34).

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Fig. 4.
The effects of antimicrobial peptides on the
respiration of mitochondria isolated from C. albicans.
Respiration was measured in buffer supplemented with NADH (1 mM) as the respiratory substrate. A, addition of
ADP (0.33 mM) increased the respiration showing the coupled
state of the mitochondria. Peptides were added to mitochondria
respiring in State 2 (in the absence of ADP). Histatin 5 and dh-5 were
added in two steps, 3.3 µM and 30 µM,
respectively, followed by 33 µM CCCP. B,
dhvar4, PGLa, and CCCP were added to final concentrations of 3.6, 3.4, and 33 µM, respectively. At the end of
these runs, sodium cyanide at a final concentration of 0.7 mM was added. The graphs are representative of at
least three experiments obtained with different mitochondria
isolations.
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The Coordinates of the Peptides in a Hydrophobicity
Plot--
Attempts to define the hydrophobic properties of a helix
have been made using the hydrophobicity plot developed by Eisenberg (36). In such a plot the vertical axis represents the mean hydrophobic moment (µ), and the horizontal axis represents the mean
hydrophobicity (H) of a given peptide sequence (36).
Empirical data have allowed the assignment of plot domains representing
"surface-seeking," "globular," and "transmembrane" protein
characteristics. For each peptide, the mean hydrophobicity and the
hydrophobic moment were calculated for the most amphipathic 11-residue
sequence spanning three helical turns using the normalized consensus
hydrophobicity scale (36) (Table I) and were plotted as shown in Fig.
5. The coordinates calculated for
histatin 5 and dh-5 correspond to the values of protein sequences
predominantly found in "globular" protein regions, which do not
show an affinity for membranes. In contrast, the coordinates of dhvar1,
dhvar4, magainin 2, and PGLa correspond to the values of protein
sequences found in association with membrane surfaces, and such
proteins have therefore been labeled as "surface-seeking." The
latter region is hypothesized to contain sequences with an affinity for
a hydrophobic/hydrophilic interphase.

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Fig. 5.
Hydrophobicity plot of histatin 5, dh-5,
dhvar1, dhvar4, magainin 2, and PGLa. Each coordinate
(H, µ) represents the mean hydrophobicity (H)
and the mean amphipathicity (µ) of the most amphipathic 11-residue
segment in the peptide as calculated in Table I.
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 |
DISCUSSION |
The results presented in this manuscript show that there is a
clear difference in the biological activities exerted by histatin 5 and
dh-5 on the one hand and two amphipathic analogs dhvar1 and dhvar4 on
the other hand. These differences include their effect on cell
viability and membrane integrity. Comparison of these biological
activities with physical peptide parameters "mean peptide
hydrophobicity" (H) and "mean peptide hydrophobic
moment" (µ) show that the biological properties of the peptides
used in this study relate well to their coordinates (H, µ) in a
hydrophobicity plot. The peptides with coordinates that fall within the
region that predicts an affinity for membranes display an interaction with membranes leading to permeabilization, as measured by the dissipation of the cytoplasmic transmembrane potential in C. albicans cells, and the uncoupling of respiration in isolated
mitochondria. This shows that the combination of H and µ is a key
determinant for these properties, and it even suggests that this plot
can be used as an approach to predict the potential lytic activity of
basic antimicrobial peptides in biological systems. Histatin 5, of
which the coordinates fall outside the surface-seeking region in the
hydrophobicity plot, showed no such permeabilizing activity either in
whole cells or in isolated mitochondria. This indicates that histatin 5 cannot be considered a classical pore former such as the magainins.
It should be emphasized that for the calculation of peptide
amphipathicity it was assumed that the peptide was in
-helical conformation. Our CD results showed that helices were inducible in all
peptides, but there was a tendency that these conformations were more
rapidly induced in dhvar1 and dhvar4, the peptides which are
biologically more active. As anticipated, analysis of amphipathicity of
the peptides as a function of their predicted structure revealed that
conversion into an
-helical conformation is required for optimizing
their amphipathic characteristics. Because the absolute requirement for
peptide helical conformations to confer biological activity has been
questioned (37) including that for histatins (38), we speculate that
not helix formation per se but rather the ability of
peptides to rapidly adopt an amphipathic structure may be of
importance. The data obtained with the peptides employed in this study
show that membrane activity is determined by both the amphipathicity of
this helix and the hydrophobicity properties of the peptide.
The antifungal potencies of histatin 5, dh-5, dhvar1, and dhvar4 were
compared in kinetic experiments using a colony-forming assay to
determine cell viability. In this assay, a 48-h incubation time
separates the kinetic experiment from the actual determination of cell
viability. This commonly used assay is potentially flawed by the
possible continuous effect of adsorbed or internalized peptide. That
this was not the case is evidenced by the fact that 6-min time
intervals of peptide (e.g. histatin 5) exposure resulted in
distinct viability differences. Any residual peptide activity would
have obscured such differences. Furthermore, in a previous study we
simultaneously used a direct cell killing dye exclusion method and the
indirect plating technique and found a perfect correlation between both
assays (18). In the present study, we used the plating technique and
found a clear distinction between the functional characteristics of the
natively occurring sequences of histatin 5 and dh-5 and the non-native,
amphipathic sequences of dhvar1 and dhvar4. First, on a molar
basis, natural histatin 5 is less efficient and less fast in killing
C. albicans than its amphipathic variants. Second, although
the initial interactions of histatin 5, dh-5, and the designed
amphipathic analogs are inhibited by salt, there is a remarkable
difference in their ultimate effect on membrane integrity. It is
interesting to note that even at high concentrations, histatin 5 and
dh-5 displayed only a small dissipating effect on the transmembrane
potential. Other studies showed no effect of histatin 5 on the
cytoplasmic transmembrane potential of C. albicans using the
related fluorescent probe, 3,3'-dipentyloxacarbocyanine iodide
(DiOC5-(3)) (19). A slight disturbance of the transmembrane
potential would be expected because exposure of yeast cells to histatin
5 has been shown to promote the influx of propidium iodide (18) and the
efflux of ATP and other components absorbing at 260 nm (19, 39). These
observations indicate that histatin 5 must alter yeast membrane
integrity to a certain extent in a nonspecific way. The present data
obtained with whole cells together with the inability of histatin 5 to uncouple the respiration of isolated mitochondria, however, strongly argue against a classical pore model for membrane permeabilization.
Unlike histatin 5, dhvar4 causes an immediate dissipation of the
cytoplasmic transmembrane potential upon addition to the cells,
indicating complete membrane permeabilization consistent with the
formation of membrane-spanning pore-like structures as described
previously for magainins (32, 34, 40). Another interesting observation
is that dhvar4, similar to PGLa, has an uncoupling effect on isolated
mitochondria. Such activity indicates that respiration is uncoupled
from ATP production due to the dissipation of the proton gradient
across the mitochondrial inner membrane, whereas the functionality of
the respiratory chain is completely maintained. This supports a
mechanistic model for dhvars and magainins to form membrane-spanning
peptide clusters that coexist with a functional respiratory chain. The
permeabilizing effect assessed here against both cytoplasmic and
mitochondrial membranes is likely to account for the acute cell death
that occurs upon exposure of whole yeast cells to these kinds of
amphipathic peptides.
It has now been well established that histatin 5 associates with the
yeast cytoplasmic membrane and is subsequently internalized (18, 20). A
similar observation has recently been reported for the 32-residue
histatin 3 peptide from which the 24-residue histatin 5 peptide is
derived (41). It has been established that histatin 5 is relatively
reluctant to adopt helical structures and that its helices are only
weakly amphipathic. One might speculate that these features of
histatins, which are different from typical amphipathic pore-forming
peptides, prevent histatins from becoming entrapped within the membrane
and facilitate their ability to enter the cytoplasm. It has furthermore
become clear that the ultimate events leading to cell death are more
complex than hitherto thought based on the fact that the killing of
C. albicans by histatins requires active cell metabolism
(18, 41). This indicates that the cell itself may be actively involved
in its own demise and supports a mechanistic model for killing by
histatins that is not restricted to a direct membrane effect.
 |
ACKNOWLEDGEMENTS |
-We thank I. M. Reijnders for technical
assistance and Dr. M. Walsh for assistance with the interpretation of
circular dichroic spectra.
 |
FOOTNOTES |
*
This project was supported in part by the Dutch Technology
Foundation, and Unilever Research Grant. VTH 44.3302, and by
National Institutes of Health Grants DE05672 and DE07652 (NIDCR).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.
Published, JBC Papers in Press, November 30, 2000, DOI 10.1074/jbc.M008229200
§
To whom correspondence should be addressed: Dept. of Periodontology
and Oral Biology, Boston University, Goldman School of Dental Medicine,
700 Albany St., Boston, MA 02118. Tel.: 617-638-4916; Fax:
617-638-4924; E-mail: helmer@bu.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
PGLa, Peptide with N-terminal Glycine and
C-terminal Leucine-amide;
diS-C3-(5), 3,3'-dipropyl-2,2'-thiadicarbocyanine iodide;
TFE, trifluoroethanol;
CCCP, carbonyl cyanide
p-chlorophenylhydrazone.
 |
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