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Proc Natl Acad Sci U S A. 2001 December 4; 98(25): 14637–14642. Published online 2001 November 20. doi: 10.1073/pnas.141366998. | PMCID: PMC64734 |
Copyright © 2001, The National Academy of Sciences Microbiology The human salivary peptide histatin 5 exerts its antifungal
activity through the formation of reactive oxygen species Eva J. Helmerhorst,* Robert F. Troxler, and Frank G. Oppenheim Department of Periodontology and Oral Biology, Boston University Goldman School of Dental Medicine, 100 East Newton Street, Boston, MA 02118-2392 Received July 17, 2001. Previous studies have shown that the human salivary antifungal
peptide histatin 5 is taken up by Candida albicans cells
and associates intracellularly with mitochondria. The purpose of
the present study was to investigate the biological consequence of this
specific subcellular targeting. Histatin 5 inhibited respiration of
isolated C. albicans mitochondria as well as the
respiration of intact blastoconidia in a dose and time-dependent
manner. A nearly perfect correlation was observed between
histatin-induced inhibition of respiration and cell killing with either
logarithmic- or stationary-phase cells, but stationary-phase cells were
less sensitive. Because nonrespiring yeast cells are insensitive to
histatin 5, the potential mechanistic relationship between histatin 5
interference with the respiratory apparatus and cell killing was
explored by using an oxygen radical sensitive probe (dihydroethidium).
Fluorimetric measurements showed that histatin 5 induced the formation
of reactive oxygen species (ROS) in C. albicans cells as
well as in isolated mitochondria and that ROS levels were highly
correlated with cell death. In the presence of an oxygen scavenger
(l-cysteine), cell killing and ROS formation were
prevented. In addition, the membrane-permeant superoxide dismutase
mimetic 2,2,6,6-tetramethylpiperidine-N-oxyl, abolished
histatin-induced ROS formation in isolated mitochondria. In contrast to
histatin 5, the conventional inhibitors of the respiratory chain,
sodium cyanide or sodium azide, neither induced ROS nor killed yeast
cells. These data provide strong evidence for a comprehensive
mechanistic model of histatin-5-provoked yeast cell death in which
oxygen radical formation is the ultimate and essential step. Histatins constitute a
distinct family of at least 12 low molecular weight, histidine-rich,
cationic, salivary peptides, of which histatin 1, 3, and 5, containing
38, 32, and 24 amino acids, are the most abundant ( 1, 2). Major
interest in histatins stems from the fact that they exhibit cidal
activities against a broad range of pathogenic fungi, including
Candida albicans, Cryptococcus neoformans, and
Aspergillus fumigatus ( 1, 3– 5). In view of this in
vitro activity and the relative abundance of histatins in parotid
and submandibular/sublingual secretions ( 6), these proteins may
represent major components of the nonimmune host defense system
involved in the maintenance of oral health. Understanding of the mechanism of antifungal action is of high
significance when histatins are to be considered as potential
candidates for drug therapy or as templates for drug design ( 7, 8).
Structural analysis has revealed that histatins differ from amphipathic
membrane-active peptides by virtue of their reluctance to rapidly adopt
helical structures in moderately hydrophobic environments and the weak
amphipathicity of their α-helical structures ( 8, 9). The interaction
of histatin 5 with liposome vesicles, mitochondria, or C.
albicans blastoconidia does not result in a massive disintegration
and depolarization of the membranes such as is achieved with strongly
amphipathic peptides ( 9– 11). The general consensus therefore is that
the ultimate events in histatin-provoked cell death are not restricted
to direct membrane effects. Interestingly, conditions that alter cell metabolism strongly influence
cellular sensitivity to histatin 5. For example, low incubation
temperatures and anaerobic incubation conditions prevent killing by
histatin 5 ( 12– 14). Protective effects have, furthermore, been
reported with oxygen scavengers and with the cytochrome oxidase
inhibitors cyanide and azide ( 13– 15). The insensitivity of cells
achieved by anaerobiosis or by respiratory chain inhibitors showed that
cellular respiration is required for cell sensitivity to histatin 5.
Indeed, the genetic modification of C. albicans into
respiratory deficient “petite” mutants rendered the cells
virtually insensitive to histatin 5 ( 16). Thus, cells with
conditionally, chemically, or genetically impaired mitochondrial
metabolism are protected against histatin 5 activity. Localization studies have shown that histatins are able to translocate
across the cytoplasmic membrane. The principles of the uptake process
are as yet unknown, but internalization has now been reported for the
24-residue peptide histatin 5 ( 13, 14) as well as for its 32-residue
precursor molecule histatin 3 ( 12). Once inside the cytosol of the
yeast cell, histatin 5 reaches the mitochondria; these organelles seem
to be the specific intracellular targets ( 13, 14). It is noteworthy
that the mitochondrial targeting of histatin 5 is concomitant with the
dissipation of the mitochondrial transmembrane potential, indicating
detrimental effects on mitochondrial function at some stage in the
fungicidal process by either a direct or an indirect mechanism. Even though several phenomena that occur on exposure of yeast cells to
histatin 5 have been described, it is unclear which of these events are
tangential and which are central in the killing mechanism. As a
consequence, the actual process that connects these phenomena to yeast
cell death has not yet been established. The observations made with
regard to respiration requirements prompted us to investigate in more
detail the interaction of histatin 5 with mitochondria and the
biological consequence of this interaction. This is of particular
interest considering the fact that mitochondria are known to play a key
role in apoptotic as well as necrotic cell death ( 17, 18). Data
obtained in the present investigation indicate that the “point of no
return” in the cascade of events that occur on exposure of C.
albicans to histatin 5 is the generation of oxygen radicals. Antimicrobial Peptides. Histatin 5 (DSHAKRHHGYKRKFHEKHHSHRGY) was obtained from American
Peptide (Sunnyvale, CA). Analysis of this material by HPLC and mass
spectroscopy revealed a purity of >98%. The peptide was dissolved in
10 mM potassium phosphate buffer (PPB; pH 7.0) to a final concentration
of 10 mg/ml. C. albicans Growth Conditions. C. albicans (ATCC no. 10231) cells were grown on Sabouraud
dextrose agar (Difco) and transferred to 100 ml of Sabouraud dextrose
broth (Difco) in a 250-ml of Erlenmeyer flask. After 16 h of
incubation at 30°C, 300 μl from this suspension was subcultured for
5 h in 100 of ml Sabouraud dextrose broth to obtain
logarithmic-phase cultures. Stationary-phase blastoconidia cultures
were obtained after 48 h of incubation under the same conditions.
Germ tube formation was induced by incubating early logarithmic-phase
C. albicans cells for 3 h at 37°C in RMPI medium 1640
containing 25 mM Hepes and 2 mM l-glutamine (GIBCO/BRL),
supplemented with 10 mM N-acetyl-d-glucosamine
(Sigma; pH 7.4). Isolation of C. albicans Mitochondria. Mitochondria were isolated from C. albicans spheroplasts,
essentially as described previously ( 19). Cells were grown to late
logarithmic phase in 1 liter of Sabouraud dextrose broth (Difco),
washed once in deionized water, and suspended in 1 ml of Zymolyase
buffer containing 50 mM Tris, 10 mM MgCl 2, and
1.4 M sorbitol (pH 7.5) per g of yeast pellet. DTT (Sigma) was added to
a final concentration of 30 mM, and the cells were incubated for 15 min
at room temperature. Cells were collected by centrifugation and
suspended in 3 ml of Zymolyase buffer containing 1 mM DDT and 2 mg of
Zymolyase 100 T (Seikagaku America, Rockville, MD) per g of yeast
pellet. After 30 min of incubation, cells were collected by
centrifugation at 1,000 × g and washed twice in
Zymolyase buffer. Cells were homogenized for 5 min on ice in 0.4 M
sorbitol, 0.2% BSA (fraction V, Sigma), and 10 mM imidazole
(Fisher Scientific; pH 6.4), by using a manual Potter–Elvehjem
homogenizer. The homogenate was mixed with an equal volume of 1 M
sorbitol, 25 mM KH 2PO 4, 4
mM EGTA (Sigma), 0.2% BSA, and 10 mM imidazole (pH 6.4) and
centrifuged 5 min at 1,000 × g at 4°C. The
supernatant was carefully removed and centrifuged for 10 min at
12,000 × g at 4°C. The reddish pellet containing the
mitochondria was suspended in 0.6 M mannitol, 2 mM EGTA, 0.2% BSA, and
10 mM imidazole (pH 6.4), to an OD 620 of ≈15,
and kept on ice. Measurement of C. albicans Respiration and Viability. Oxygen consumption was measured by using a biological oxygen monitor
model 5300 equipped with a 5331 standard oxygen probe (Yellow Springs
Instruments). The apparatus consisted of a twin oxygen chamber, which
enabled a control experiment to be conducted at the same time.
Experiments with mitochondria were performed in 1.5 ml of air-saturated
respiration buffer at 30°C containing 0.65 M mannitol, 2 mM
MgCl 2, 16 mM
KH 2PO 4, and 10 mM imidazole
(pH 6.4). In all experiments, mitochondria were diluted from the stock
suspension to a final OD 620 of 0.15 ± 0.05.
State 2, state 3, and state 4 respiration were determined as described
previously ( 9), by using 1 mM NADH and 0.33 mM ADP as substrates.
Polarographic measurements using C. albicans logarithmic- or
stationary-phase blastoconidia were conducted in 1.5 ml of 1 mM PPB.
Cells were added to the oxygen chambers from a concentrated stock
suspension to a final OD 620 of 1.7. The
respiratory rates were determined graphically from the slope of the
tangent line after various time intervals. In the same experimental
setup, the viability of cells incubated with histatin 5 was determined
after the same time intervals by removing a 15-μl aliquot from the
chamber, diluting the cells in 9 ml of PBS and plating 15 μl of the
suspension on Sabouraud dextrose agar. After 48 h of incubation at
30°C, the viability was determined by colony counting and compared
with control experiments without histatin 5. Measurement of C. albicans Reactive Oxygen Species
(ROS) Formation and Viability. The formation of ROS in C. albicans by histatin 5 was
determined by using dihydroethidium (Molecular Probes), which is
rapidly oxidized by ROS into its fluorescent derivative.
Dihydroethidium was added from a stock solution of 2.5 mg/ml in DMSO to
a suspension of logarithmic-phase C. albicans blastoconidia
or germinated cells in 1 mM PPB to a final concentration of 6.7
μg/ml. After 10 min of incubation at 30°C the cells were collected
by centrifugation, and suspended in 1 mM PPB to
OD620 values between 2.0 and 4.0. From these
suspensions, 100 μl was added to 100 μl of a dilution series of
histatin 5, peptide with N-terminal glycine and C-terminal
leucine amide (PGLa), sodium cyanide or sodium azide in 1 mM PPB in a
Microfluor black microtiterplate (Dynex, Chantilly, VA). In
control experiments without cells, 6.7 μg/ml dihydroethidium was
added to a dilution series of histatin 5 in the presence or absence of
5 μg/ml herring sperm DNA (Sigma). Oxidation of the probe was
followed at 2.5-min intervals for 15 min at 30°C at
λex 485 nm and λem 595
nm, and the kinetics of dye oxidation were calculated for each
concentration of histatin 5 (TECAN Spectrofluor Plus fluorimeter and
DELTASOFT 3V2.199SPFL software packet, TECAN,
Männedorf, Switzerland). In addition to kinetic measurements,
endpoint readings were performed after 1 h of incubation of cells
with a dilution series of histatin 5, and the calculation of the
percent of ROS formed was based on the maximum fluorescence intensity
observed. These experiments were also conducted in the presence of 5 mM
of the oxygen scavenger l-cysteine (Sigma).
Immediately after the fluorescence reading, cells were diluted in PBS
and plated on Sabouraud dextrose agar to be able to compare ROS
formation with cell killing. Measurement of ROS Formation in Isolated Mitochondria. Mitochondria were diluted to an OD620 of 1.7 in
respiration buffer supplemented with 1 mM NADH. In a black microtiter
plate, a dilution series of histatin 5 was prepared in respiration
buffer, in respiration buffer supplemented with 3 mM of the
membrane-permeant superoxide dismutase mimetic
2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), and in
respiration buffer supplemented with 10 mM
l-cysteine (50 μl per well). A total of 50 μl
of mitochondria suspension was added to each well, immediately followed
by the addition of 5 μl of dihydroethidium to a final concentration
of 6.7 μg/ml. Plates were incubated for 30 min at 30°C, after which
the fluorescence intensity was determined as described above. Histatin 5 Effect on Mitochondrial Respiration. Mitochondria were isolated from C. albicans by spheroplast
formation and a mild homogenization procedure. To verify the integrity
of the mitochondrial inner membrane, the “respiratory control
rate” and the uncoupling activity of carbonyl cyanide
m-chlorophenylhydrazone (CCCP) were determined for
each mitochondrial preparation; these values ranged between 2.5 and 2.7
( 9), which are normal values for yeast mitochondria ( 19). As described
previously ( 9), histatin 5 at a concentration of 33 μM inhibits state
2 respiration. Data obtained with six independent mitochondrial
preparations showed that histatin 5 also inhibits state 3 respiration
(in the presence of ADP) and the respiration of uncoupled mitochondria
(i.e., in the presence of CCCP; Table 1).
A synthetic control peptide derived from cystatin SA at the same
concentration inhibited respiration by <5% (data not shown). These
results indicate that histatin 5 is an inhibitor of mitochondrial
respiration.
| Table 1Effect of histatin 5 (33 μM) on
mitochondrial respiration |
Histatin 5 Effect on C. albicans Respiration. To assess the biological significance of the observed inhibitory
activity of histatin 5 on isolated mitochondria, similar experiments
were conducted with C. albicans blastoconidia, collected in
the logarithmic growth phase. In accordance with the effects of
histatin 5 on isolated mitochondria, histatin 5 inhibited cellular
respiration. The inhibitory effect was concentration dependent and with
higher histatin concentrations (33 μM) complete within 5 min of
incubation (Fig. ). To investigate
whether there is an interrelationship between inhibition of cell
respiration and killing, a polarographic experiment was conducted in
parallel with a colony counting assay in which the percentage
inhibition of respiration and the percentage loss in cell viability
were determined in the same experimental setup at different time
intervals. A nearly perfect correlation between the two parameters was
found (Fig. ). When logarithmic-phase
cells were used, the detrimental effect of histatin 5 on both cell
viability and respiration was rapid (i.e., minutes) and reached >95%.
When stationary-phase cells were used, also an excellent correlation
between the percent inhibition of respiration and killing was found,
but the stationary-phase cells were overall less sensitive to histatin
5 than were logarithmic-phase cells. With respect to the reduced
sensitivity of stationary-phase cells compared with log-phase cells, it
is of importance to note that the intrinsic respiratory rate (i.e.,
before addition of histatin 5) of stationary- phase cells was
approximately 1/4 of that of logarithmic-phase cells (Table
2). Although there are many differences
between logarithmic- and stationary-phase cells that may explain their
different susceptibilities to histatin 5, the influence of the
intrinsic respiratory rate may be of crucial importance, as previous
studies have shown that cells that do not respire at all are almost
completely protected against histatin 5 killing activity ( 14, 16). In
other words, the sensitivity of the cells to histatin 5 decreases with
a decreasing respiratory rate. Taken together, these observations point
toward the intriguing possibility that the respiratory apparatus itself
may be actively involved in initiating cell death. Therefore, we
hypothesized that histatin 5 exerts its antifungal activity through the
formation of ROS.
| Table 2Candidacidal activity and inhibition of respiration by
histatin5 |
ROS Formation in C. albicans Cells. The fluorescent probe dihydroethidium, suitable for measurement of ROS
formation in yeast cells ( 17) was used to monitor ROS formation in
C. albicans blastoconidia and germinated cells. Fig.
A shows that histatin 5
causes a concentration-dependent and cell-density-dependent increase in
the fluorescence intensity of C. albicans blastoconidia as
well as of germinated C. albicans cells loaded with
dihydroethidium. Histatin 5 alone had no effect on the fluorescence
properties of the probe (data not shown). These data indicate that
histatin induces cell-mediated oxidation of the probe by ROS. ROS
formation in both the blastoconidial and the germinated growth form of
C. albicans is consistent with earlier reports that both
forms are sensitive to histatin 5 killing activity ( 20). In contrast to
histatin 5, PGLa, a highly fungicidal peptide that acts by forming
membrane-spanning pore complexes ( 21), did not induce ROS formation
(data not shown). Importantly, two conventional inhibitors of the
respiratory chain, sodium cyanide and sodium azide, did not induce ROS
formation in C. albicans (Fig. B). These data
indicated that neither cell death (i.e., PGLa killing) nor inhibition
of respiration (i.e., cyanide and azide), per se, leads to
the production of ROS and that the effect observed with histatin 5 was
specific.
ROS Formation in C. albicans Mitochondria. To investigate whether the histatin induced promotion of oxygen radical
formation in C. albicans blastoconidia occurs at the
mitochondrial level, similar experiments were carried out with
mitochondria in their isolated form. Histatin 5 added to a suspension
of isolated mitochondria in the presence of dihydroethidium caused a
concentration dependent increase in its fluorescence, consistent with
ROS formation. This effect was abolished in the presence of the oxygen
scavenger l-cysteine or the membrane-permeant
mimetic of superoxide dismutase
2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) indicating that
the increase in fluorescence was indeed because of ROS formation (Fig.
).
Correlation Between ROS Formation and C. albicans
Killing. The results above suggest that killing of C. albicans by
histatin is induced by ROS. To further investigate such a
histatin-induced cell suicide mechanism, we carried out additional
experiments to assess the interrelationship between cell killing and
the intracellular formation of ROS. Cells were loaded with
dihydroethidium and incubated for 1 h with various concentrations
of histatin 5, after which the fluorescence intensity and cell
viability of the same inoculum were determined. It was found that the
amount of ROS produced, expressed as a percentage of the maximum
fluorescence intensity observed after that incubation period, was
highly correlated with cell killing (Fig.
A). When experiments were
conducted in the presence of l-cysteine, neither
ROS formation nor cell killing occurred (Fig. B). These
data strongly support the hypothesis that ROS formation is the key
event in histatin-induced killing of C. albicans.
Earlier studies have shown that the interaction of histatin 5 with
C. albicans leads to cellular uptake and mitochondrial
targeting of this peptide, ultimately resulting in the loss of cell
viability. In the present investigation, the relationship between this
specific subcellular targeting and cell death was established. Evidence
is presented showing that the respiratory apparatus is the target for
histatin 5 and that the derangement of respiratory activity, leading to
the formation of oxygen radicals, is the cause of cell death. We
therefore propose a histatin-triggered, self-inflicted cell suicide
mechanism in which ROS formation is the key element of the histatin 5
killing mechanism (Fig. ). This model
involves the following steps: (i) uptake of histatin 5 by
means of a receptor-mediated or transmembrane-potential-driven
mechanism, (ii) targeting of histatin 5 to the mitochondria,
(iii) inhibition of mitochondrial respiration,
(iv) generation of ROS by means of out-of-sequence electron
transfer to molecular oxygen, and (v) cell death because of
the oxidation of biological macromolecules and the loss of cellular
integrity.
The results of the present investigation identified histatin 5 as an
inhibitor of respiration. Our respiratory measurements contrast with
previous publications by Edgerton and coworkers, who reported that
histatin 5 had virtually no effect on yeast cellular respiration ( 15,
22). Surprisingly, data generated by this group suggested that cells
exposed to histatin were dead and had released all of their ATP, but
continued to respire for 1.5 h. Although these observations remain
unexplained, the results reported here show that there is an almost
perfect correlation between inhibition of respiration and cell killing
when either logarithmic-phase or stationary-phase cells were used. It
was not clear at first sight, however, how blockage of the respiratory
chain alone would lead to cell death, because Candida, like
most yeasts, is fully capable of fermentation. For example, high
concentrations of respiratory inhibitors (azide, cyanide, antimycin, or
combinations of these agents) are not fungicidal ( 23). The observation
that histatin 5, but neither sodium azide nor sodium cyanide, generates
ROS is consistent with the fact that histatin 5 is fungicidal, whereas
these conventional respiratory inhibitors are not. Therefore, the
inhibition of respiration afforded by histatin 5 should be considered
different in principle from that caused by cyanide and azide.
Interestingly, the cationic pore-forming antifungal peptide PGLa did
not induce ROS, further supporting that different mechanisms of action
underlie the fungicidal activities of PGLa and histatin 5 ( 9). The interference of histatin 5 with the respiratory chain and the
formation of ROS indicate the out-of-sequence electron transfer from
some carrier in the respiratory chain to molecular oxygen. The specific
site of inhibition of histatin 5 has yet to be identified. In general,
it has been shown that mitochondrial ROS is formed both at the
NADH:ubiquinone reductase level (complex I) and the
ubiquinol:cytochrome c reductase level (complex III) ( 24,
25). Under normal physiological conditions, the highly reactive CoQ
semiquinone radical (CoQ ![[center dot]](/corehtml/pmc/pmcents/middot.gif) −) is rapidly
neutralized when a second electron is transferred to cytochromes
b566 and
b562, thereby regenerating the
oxidized form of CoQ (the Q cycle). In mammalian cells, inhibitors that
bind to cytochrome b562 and prevent
cytochrome b reoxidation, such as antimycin A, lead to the
accumulation of the unstable CoQ −
radical, and subsequent random collisions of this radical with
molecular oxygen will form various ROS ( 24, 25). It is feasible that
histatin 5 generates ROS by means of a similar mechanism. A highly
conserved target such as coenzyme Q, however, would not explain the
apparent specificity of histatins 5 for yeast-cell killing and absence
of mammalian-cell killing. It is possible that such specificity is
accomplished at a different level, e.g., by species-specific cell
surface receptors or specific peptide translocation mechanisms.
Additional studies are required to elucidate the basis for this
histatin 5 specificity. We previously demonstrated that addition of histatin 5 to C.
albicans cells leads to the release of rhodamine 123 from
mitochondria, indicating the dissipation of the mitochondrial
transmembrane potential, ΔΨ mito ( 14). Here we
present evidence that histatin 5 has no direct membrane-permeablizing
effect (no uncoupling effect) on yeast mitochondria in their isolated
form (Fig. and ref. 9). It is possible that the dissipation of
ΔΨ mito in cells is a result of the inhibition
of electron transport. Alternatively, the dissipation of
ΔΨ mito is due to mitochondrial membrane
permeabilization, but occurs as a secondary event after histatin 5
exposure. Such membrane-permeabilizing effects are consistent with the
fact that ROS are formed because it is well recognized that a local
accumulation of such radicals will rapidly lead to disintegration of
biological membranes. With respect to the above-mentioned membrane
effects, it should be noted that the primary site of action of ROS will
be in close proximity to their site of generation, the mitochondrion,
because of the high reactivity of ROS ( 26). Indeed, the cell
cytoplasmic membrane, distant from the site of oxygen radical
generation, is only moderately affected shortly after exposure of cells
to histatin 5 ( 9– 11). These minor cytoplasmic membrane permeability
changes are reflected by the influx of the cell necrosis marker
propidium iodide ( 14) and the efflux of intracellular ATP ( 15, 22). The
close correlation between the release of these intracellular
constituents and cell death ( 22) is an indication that the efflux of
such molecules is not compatible with cell viability. It has become evident that histatin 5 causes the release of not only
ATP but of a variety of nucleotides (C. Gyurko, E.J.H., R.F.T., and
F.G.O., unpublished data). It has been suggested that one of these
nucleotides, ATP, is not only released from the cell, but actually
serves as an effector molecule by binding to purinergic receptors, and
that this event in some way is responsible for causing cell death ( 15).
Although the release of such nucleotides, including ATP, is likely part
of a cascade of events that occur during the killing process, the
purinergic receptor model for histatin killing does not adequately
explain the requirement for cellular respiration in the sensitivity of
the cells to histatin 5. The ROS model for histatin killing as outlined
in Fig. is consistent with the protective effects of anaerobiosis and
“petite” mutations of C. albicans ( 16, 18), and has
become attractive because the present weight of evidence from studies
by us ( 9, 13, 14, 16) and others ( 12, 15, 22) are consistent with this
model. It is of great interest that the restricted conditions under which
histatin 5 is active are not unique to this peptide. A number of
reports have appeared showing that the activity of a number of toxins
from either plant, bacterial, or human origin, are dependent on active
participation by the target cell. For example, studies on human
defensins (HNP1, -2, and -3)-mediated cytotoxicity showed the
requirement for peptide internalization and for target cell metabolic
processes ( 27). Other examples are the candidacidal activity of HNP-1,
which is negatively affected in the presence of respiratory inhibitors
( 28), and the activity of killer toxin from Pichia kluyveri
against Saccharomyces cerevisiae cells that is highly
dependent on the physiological state of cells ( 29) in a very similar
fashion as that described for histatin 5. It is therefore feasible that
the present discovery that ROS formation is a quintessential step in
the killing process provoked by histatin 5 is not restricted to
histatin 5 activity, but may represent a common mechanism of
target-cell killing afforded by agents that are dependent on active
cell metabolism. We acknowledge Dr. H. A. Lucero for assistance in
spectrofluorimetric analyses. This study was supported by National
Institutes of Health/National Institute of Dental and Craniofacial
Research Grants DE05672 and DE07652. | | ROS | reactive oxygen species | | | TEMPO | 2,2,6,6-tetramethylpiperidine-N-oxyl | | | CCCP | carbonyl
cyanide m-chlorophenylhydrazone |
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