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Appl Environ Microbiol, January 1998, p. 27-33, Vol. 64, No. 1
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Anaerobic Killing of Oral Streptococci by Reduced,
Transition Metal Cations
J. C.
Dunning,1
Y.
Ma,1 and
R. E.
Marquis1,2,*
Departments of Microbiology and
Immunology1 and
Dental
Research,2 University of
Rochester, Rochester, New York 14642-8672
Received 12 September 1997/Accepted 5 October 1997
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ABSTRACT |
Reduced, transition metal cations commonly enhance oxidative damage
to cells caused by hydroperoxides formed as a result of oxygen
metabolism or added externally. As expected, the cations Fe2+ and Cu+ enhanced killing of
Streptococcus mutans GS-5 by hydroperoxides. However,
unexpectedly, they also induced lethal damage under fully anaerobic
conditions in a glove box with no exposure to O2 or hydroperoxides from initial treatment with the cations. Sensitivities to anaerobic killing by Fe2+ varied among the organisms
tested. The oral streptococci Streptococcus gordonii ATCC
10558, Streptococcus rattus FA-1, and Streptococcus sanguis NCTC 10904 were approximately as sensitive as S. mutans GS-5. Enterococcus hirae ATCC 9790, Actinomyces viscosus OMZ105E, and Actinomyces
naeslundii WVU45 had intermediate sensitivity, while
Lactobacillus casei ATCC 4646 and Escherichia
coli B were insensitive. Killing of S. mutans GS-5 in
response to millimolar levels of added Fe2+ occurred over a
wide range of temperatures and pH. The organism was able to take up
ferrous iron, but ferric reductase activity could not be detected.
Chelators, uric acid, and thiocyanate were not effective inhibitors of
the lethal damage. Sulfhydryl compounds, ferricyanide, and ferrocyanide
were protective if added prior to Fe2+ exposure.
Fe2+, but not Fe3+, acted to reduce the acid
tolerance of glycolysis by intact cells of S. mutans. The
reduction in acid tolerance appeared to be related directly to
Fe2+ inhibition of F-ATPase, which could be assayed with
permeabilized cells, isolated membranes, or F1 enzyme
separated from membranes. Cu+ and Cu2+ also
inhibited F-ATPase and sensitized glycolysis by intact cells to acid.
All of these damaging actions occurred anaerobically and thus did not
appear to involve reactive oxygen species.
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INTRODUCTION |
Oxidative damage involving reduced,
transition metal cations is well-known in a variety of biological
systems. The generally accepted mechanism for damage involves so-called
Fenton reactions, in which the reduced cations react with an oxidant to
produce hydroxyl radical, OH·, the agent considered
generally to cause the major damage to nucleic acids, proteins, or
lipids. However, other views of the specifics of damage are developing,
including ones in which oxidants such as superoxide radical act to
leach iron from storage proteins. The leached iron then catalyzes
damage to DNA or other biopolymers (11). If the oxidant
produced by cells or added from the outside is
H2O2, then the products of the Fenton reaction
involving Fe2+ are Fe3+, hydroxyl anion, and
hydroxyl radical. If the oxidant is an organic peroxide, then the
products include Fe3+ and an organic radical. If the
oxidant is HOCl, then the products are Fe3+, hydroxyl
radical, and Cl
(5).
Multiple experimental systems to produce oxidative damage have been
developed. The simplest systems include a reduced, transition metal
cation, say, Fe2+ or Cu+, and oxygen.
More-elaborate systems may include NAD(P)H
oxidase-NAD(P)H-O2-Fe3+, xanthine-xanthine
oxidase-O2-Fe3+, or other combinations. In many
systems, metabolizing organisms may act as catalysts for their own
destruction by reducing O2 to produce intermediates
involved in Fenton reactions, such as H2O2,
superoxide radical (O2·), or hydroperoxyl
radical (HO2·). Many organisms also produce
ferric reductases (4, 23), so Fe3+ added to the
systems is reduced by the organisms to Fe2+, which is then
a source of reducing equivalents for Fenton reactions.
Although all the systems described above are very much dependent on
O2 or hydroperoxide, there are a few reports in the
literature of damage by transition metal cations apparently in the
absence of O2 or hydroperoxide. For example, Sagripanti et
al. (20) found that Fe3+ or Cu2+
alone at concentrations of about 16 to 18 mM could cause inactivation of a variety of viruses, although their actions were greatly enhanced by addition of H2O2. Jain et al.
(10) found that O2 was not required for
degradation of isolated DNA by glutathione plus Cu2+,
possibly because of formation of glutathione radicals, which then
damaged the DNA.
In this paper, we present a primary description of damage, including
lethal damage, to oral streptococci induced by Fe2+ and
Cu+ involving mechanisms which appear to be independent of
oxygen because the damage can occur even under anaerobic conditions.
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MATERIALS AND METHODS |
Bacteria.
Streptococcus mutans GS-5,
Streptococcus gordonii ATCC 10558, Streptococcus
rattus FA-1, and Streptococcus sanguis NCTC10904 are
maintained routinely in our laboratory with weekly subculture on
tryptic soy agar (Difco Laboratories, Detroit, Mich.) plates and
long-term storage at 
70°C in 50% glycerol solution. The organisms were routinely grown in static culture at 37°C in tryptone-yeast extract medium (1). Other organisms used that are maintained routinely in the laboratory include Actinomyces viscosus
OMZ105E, Enterococcus hirae ATCC 9790, Escherichia
coli B, and Lactobacillus casei ATCC 4646. Actinomyces naeslundii WVU45 was obtained from E. Morou-Bermudez of the Department of Dental Research, University of
Rochester. It was maintained by anaerobic growth on tryptic soy agar
plates and was grown for experiments in static culture in
tryptone-yeast extract broth.
Assays of lethality.
Freshly harvested cells were suspended
routinely in 1% (wt/vol) Difco peptone solution to yield suspensions
with approximately 109 CFU/ml. A sample was taken for a
zero time count, and then the various chemicals to be tested were added
to the suspension. Generally, chloride or sulfate salts were used, but
the anionic counterion did not significantly affect the results. At
intervals, samples were taken and immediately diluted in 1% Difco
peptone broth. Samples (0.1 ml) from 1:10 dilution series in peptone
broth were spread over the surfaces of tryptic soy agar plates, and the
plates were incubated appropriately to allow colony formation and
subsequent counting. For a few experiments, cells were suspended in a
solution containing 50 mM KCl and 1 mM MgCl2. The extent of
killing of the cells was reduced by about 50% compared with that of
cells in peptone; nevertheless, killing of more than 99% of the
population could be achieved after exposure to 10 mM Fe2+.
For experiments in which all manipulations were carried out
anaerobically, a Coy (Grass Lake, Mich.) glove box chamber was used. It
was gassed with 5% CO2, 10% H2, and 85%
N2. In a series of experiments, it was found that the
lethal effect of Fe2+ for S. mutans GS-5 was the
same when the experiment was carried out totally anaerobically or when
static suspensions were exposed to the cation with subsequent growth of
plated samples aerobically. However, if the suspensions were stirred
aerobically after exposure to Fe2+, the cells were much
more resistant to the lethal action of Fe2+. In fact, in
the extreme, cells suspended in 1% Difco peptone which were treated
with 10 mM Fe2+ and vigorously aerated on a shaker at
37°C showed essentially no killing, while cells in the same
experiment incubated statically or anaerobically showed about a 4-log
reduction in viable count.
Fe3+ reductase activity.
Ferric reductase
activities were assayed with whole cells, permeabilized cells
(1), isolated membranes (22), and cytoplasmic extracts of lysed spheroplasts according to the procedures described by
Mazoy and Lemos (15).
Glycolysis and pH drop assays.
Cells for pH drop assays were
harvested by centrifugation, washed once with water, and resuspended in
dense suspension (2 mg [dry weight] of cells per ml) in a solution
containing 50 mM KCl and 1 mM MgCl2. The suspension was
titrated with KOH to about 7.2. Then glucose was added to yield an
initial concentration of 55.6 mM. The resulting fall in pH was
monitored with a glass electrode. Glucose utilization was assessed by
use of glucose oxidase kits from Sigma Chemical Co. (St. Louis, Mo.).
Assays of the phosphotransferase system were carried out as described previously (2).
ATPase assays.
Permeabilization of cells, membrane
isolation, and extraction of F1-ATPase were carried out as
described previously (22), as were assessments of ATPase
activity in terms of phosphate release from ATP.
Permeability measurements.
The space, or dense-suspension,
technique was used to assess the permeability of S. mutans
cells to Fe2+ (13). Centrifuged cell pellets
were mixed with equal volumes of Fe2+ solution, incubated
at 25°C for 45 min, and recentrifuged. Uptake values and R
values were calculated from knowledge of the original and final
Fe2+ concentrations, the relative volumes of cells and
suspending medium, and the volume of interstitial fluid. The
interstitial space in cell pellets was assessed by use of
high-molecular-weight dextrans (Dextran T-2000; Pharmacia Corp.,
Piscataway, N.J.). Iron was assayed by the method of Horak et al.
(9), which involves formation of ferrozine-ferrous complexes
that absorb light of 562-nm wavelength.
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RESULTS |
Killing of S. mutans GS-5 by hydroperoxides and iron or
copper cations.
The data presented in Fig.
1 show that S. mutans GS-5
cells in dense suspension were killed by 0.1% hydrogen peroxide (32.6 mM) with a time for killing of 90% of the initial population
(D value) of about 25 min and that killing could be markedly
enhanced by addition of transition metal ions. The reduced cations
Fe2+ and Cu+ were more effective, at a 10 mM
level, than the oxidized cations Fe3+ and Cu2+.
In fact, with Fe2+ added (Fig. 1A), the initial
D value was reduced to less than 3 min, and with
Cu+ added (Fig. 1B), the value was about 3.8 min. This type
of enhancement of hydroperoxide killing is found commonly and is
thought to be due to the reduced, transition metal cations acting to
catalyze formation of hydroxyl radicals from hydroperoxides via Fenton reactions.
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FIG. 1.
Transition metal cation enhancement of hydroperoxide
killing of S. mutans GS-5. (A) Effects of 10 mM
Fe2+ ( ) or Fe3+ ( ) on killing by 0.1%
H2O2 ( ). (B) Effects of 10 mM
Cu+ ( ) or Cu2+ ( ) on killing by 0.1%
H2O2 ().
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Similar enhancement by Fe
2+ and Cu
+ of killing
of
S. mutans GS-5 cells by the organic hydroperoxide
tertiary butyl hydroperoxide
at a concentration of 7.2 mM was found
(data not shown).
In subsequent experiments, we found that Fe
2+ alone, but
not Fe
3+, in the absence of any hydroperoxide was lethal
for
S. mutans GS-5 cells with approximately the same
kinetics and to the same
extent as shown in Fig.
1A for
Fe
2+ combined with 0.1% H
2O
2.
These results were somewhat unexpected
but still were interpretable in
terms of knowledge that the organism
has active oxygen metabolism,
mainly involving NADH oxidases,
one of which produces
H
2O
2 (
8). Thus, the organism in
suspensions
in contact with air could have produced sufficient
H
2O
2 through
endogenous metabolism of
O
2 diffusing into the suspension to allow
for
Fe
2+-H
2O
2 killing. However, a
completely unexpected finding (Fig.
2A)
was that the cells were killed when the entire experiment
was carried
out in an anaerobic glove box at 37°C. At no time
from first exposure
to Fe
2+ to growth of colonies were the cells exposed to air
or oxygen.
The extent of killing varied somewhat from experiment to
experiment.
In 59 experiments carried out over a 9-month period, the
average
final log (
N/N0) was
4.6 with a
standard deviation of ±0.99,
where
N/N0 is the
ratio of the final plate count to the initial
plate count. The
important point is that nearly all of the cells
in populations exposed
to Fe
2+ or Cu
+ were killed, so that generally
fewer than 1 cell in 1,000 survived.
Under anaerobic conditions,
Fe
3+ was essentially ineffective for killing. Addition of a
reducing
agent, such as phenazine methosulfate, was not found to be
effective
for enhancing killing, although Evans et al. (
7)
had found
that phenazine methosulfate could enhance iron uptake by
S. mutans (
sobrinus) OMZ176, which is thought to
involve only the reduced
cation.
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FIG. 2.
Anaerobic killing of S. mutans GS-5 by iron,
copper, and cobalt cations. Data are shown for control cells () (A
to C) and cells exposed to 5 mM Fe2+ () or
Fe3+ () (A), 5 mM Cu+ () or
Cu2+ () (B), and 5 mM Co2+ (+) (C).
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Cu
+, but not Cu
2+, proved to be nearly as
effective as Fe
2+ for anaerobic killing of
S. mutans GS-5 (Fig.
2B), but reduced cobalt
cations, at a level of 5 mM, were much less effective, although
some killing was evident (Fig.
2C). However, in most experiments,
killing was delayed and then
resulted in at most 90% reduction
in viable counts of populations.
Mg
2+ or Ca
2+ at a concentration of 20 mM had no
damaging effect on the bacteria.
The rates of killing by
Fe
2+ or Cu
+ alone under anaerobic conditions
were approximately the same
as those for killing by combinations of the
ions with 0.1% H
2O
2 or 7.2 mM tertiary butyl
hydroperoxide. In addition, the extent
of killing by the transition
metal ions alone was generally approximately
the same as that with
combinations of 0.1% H
2O
2 and Fe
2+
or Cu
+, as indicated by the data of Fig.
1 and
2.
Dose-response data for killing of
S. mutans GS-5 by
Fe
2+ are presented in Fig.
3.
Extensive killing occurred in response to
addition of Fe
2+
to a final millimolar concentration. The
D value for cells
exposed
to 2.0 mM Fe
2+ was about 15 min, and this value
fell to less than 5 min for
cells exposed to 10 mM Fe
2+.
The results of repeated experiments to determine the optimal
concentration of Fe
2+ or Cu
+ for killing of
S. mutans GS-5 showed that 5 to 10 mM levels were
close to
saturating for the lethal effect, and higher concentrations
were not
more effective, possibly because of precipitation. In
addition, when
cells were first treated with 10 mM Fe
2+ and incubated for
up to 45 min at 25°C before addition of a second
dose of
Fe
2+ to raise the total concentration to 20 mM, there was
no additional
killing detected, at least not within the errors of plate
counting.
Again, it appeared that 5 to 10 mM levels of Fe
2+
or Cu
+ were saturating in terms of the killing response
under the experimental
conditions.
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FIG. 3.
Anaerobic killing of cells of S. mutans GS-5
mediated by Fe2+ at 0 (), 0.25 (), 0.50 ( ), 1.0 (), and 2.0 () mM (A) or 0 (), 2.5 (×), 5.0 (+), and 10.0 () mM (B).
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Fe2+ killing of other bacteria.
Anaerobic killing
by Fe2+ could be demonstrated for the other oral
streptococci tested (S. gordonii ATCC 10558, S. rattus FA-1, and S. sanguis NCTC 10904) (Fig.
4A to C) but not for L. casei ATCC 4646 or E. coli B (data not shown because control and
treated suspensions all maintained essentially the same counts). Thus, anaerobic killing by Fe2+ did not seem to be universal
among bacteria but was common for oral streptococci. E. hirae ATCC 9790 was intermediate in its sensitivity to
Fe2+, and exposure to 10 mM Fe2+ resulted in
only about 90% killing of the organism over a period of an hour.
A. viscosus OMZ105E was somewhat more sensitive (Fig. 4D),
but again, the maximum killing was generally only about 99% of the
population. Catalase-negative A. naeslundii WVU45 was only slightly more sensitive than catalase-positive A. viscosus
(Fig. 4D).
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FIG. 4.
Iron-induced killing of various plaque bacteria. (A to
C) S. gordonii, S. rattus FA-1, and S. sanguis NCTC10904, respectively (, control cells; and ,
cells exposed to 10 mM Fe2+ or Fe3+,
respectively). (D) A. viscosus OMZ105E (, control cells;
and +, cells exposed to 10 or 20 mM Fe2+, respectively)
and A. naeslundii WVU45 (, cells exposed to 10 mM
Fe2+).
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Factors affecting ferrous-ion killing of S. mutans
GS-5.
As shown by the data presented in Table
1, optimal pH values for killing of
S. mutans GS-5 by 10 mM Fe2+ were between 7 and
8. However, the range of pH over which killing occurred was broad, from
about 4 to 9.
The data presented in Table
2 show that
killing occurred over a broad range of temperatures, from 4 to 48°C.
Killing was
detected even when the exposure to 10 mM Fe
2+
was at 4°C. Thus, it appeared that metabolism by the cells during
initial exposure to Fe
2+ was not required for killing.
However, since this assay of damage
involves growth of the cells at
37°C to form colonies, Fe
2+ may be bound at a low
temperature and then cause damage only
when the cells are warmed. The
temperature range for killing is
broader than the growth range for the
organism. The minimal and
maximal growth temperatures are about 30 and
48°C (
12).
Attempts to reverse killing by treating cells initially exposed to
Fe
2+ at 4°C (or at 37°C) with chelators were not very
successful,
even with use of chelators such as 10 to 80 mM
deferoxamine, dipyridine,
or EDTA. Also, extensive washing of the cells
with water or peptone
solution did not restore viability. Conversely,
the use of various
chelators also did not enhance killing, probably
because the Fe
2+ form of iron was sufficiently water
soluble under the experimental
conditions, while the less soluble
Fe
3+ was not effective even in a chelate complex for this
organism
which appears not to have highly active ferric reductases.
Sulfhydryl
compounds such as dithiothreitol, cysteine, and methionine
were
only partially protective. For example, in typical experiments,
at
a concentration as high as 100 mM, methionine increased the
final
average (
n = 2) log (
N/N0) from
approximately
5.1 to
4.2
(increase in percent survivors from 0.0008 to 0.0063%). Dithiothreitol
was more protective, and the average
percent survivors was increased
by 10 mM dithiothreitol to
approximately 1%. Histidine or tryptophan
had almost no protective
effect. Ferricyanide and ferrocyanide
were highly protective against
killing and at concentrations as
low as 1 mM could totally protect
cells of
S. mutans GS-5 against
killing by 10 mM
Fe
2+. However, ferricyanide and ferrocyanide were not
effective in
reversing the effects of prior exposure to
Fe
2+. The major antioxidant in saliva is uric acid
(
17), but even
at 10 mM, it did not protect against
Fe
2+ killing. Moreover, thiocyanate, which is also in
saliva, had
no protective effect, even at a concentration of 10 mM.
Aeration of cells of
S. mutans GS-5 in flasks on a shaker
incubator during exposure to Fe
2+ protected them from
killing, presumably because of oxidation
of Fe
2+ to
Fe
3+ (
6). Cells grown in shaker culture, rather
than static culture,
were not found to have enhanced resistance.
Moreover, growth of
the bacteria in cultures to which
H
2O
2 was added repeatedly during
the growth
phase to yield a concentration of 0.005 or 0.010% did
not increase
resistance to anaerobic Fe
2+. Cells from the
mid-exponential phase of growth in static or
shaken cultures appeared
to be slightly more sensitive to anaerobic
Fe
2+ killing
than cells from late-exponential-phase or early-stationary-phase
cultures, but the difference was marginal.
Permeability of cells of S. mutans GS-5 to
Fe2+.
As shown by the data presented in Table
3, cells of S. mutans GS-5
took up Fe2+ present in suspensions at initial
concentrations in the lethal range. The pattern of uptake suggests that
binding is an important component of uptake at lower Fe2+
levels (R values of >1). However, at higher
concentrations (10 mM in this case), it seems that Fe2+
uptake involves mainly nonconcentrative movement into the cell water
(R = 0.86). Overall, the data suggest that cells of
S. mutans GS-5 are permeable to Fe2+.
Ferric reductase activity.
Attempts to detect ferric reductase
activity in S. mutans GS-5 with intact cells, permeabilized
cells, membranes, or cytoplasmic extracts indicated that activity was
minimal, basically at the limit of detection. The major reductant
tested was NADH in the presence of Mg2+ and flavin adenine
dinucleotide, but other reductants, such as NADPH, also were
ineffective.
Inhibition of glycolysis by Fe2+ and
Cu+.
Fe2+, but not Fe3+, was
found to reduce the acid tolerance of S. mutans GS-5 cells
in standard pH drop assays (Fig. 5A to
C). In the suspensions used for these experiments, the extent of pH drop reflects the acid tolerance of the organism. Glycolytic acid tolerance in oral streptococci depends on the capacities of F-ATPases to move protons out of the cytoplasm, and the final pH values in
glycolyzing suspensions with excess glucose indicate minimal environmental values at which the F-ATPases can operate to maintain a
sufficient 
pH across the cell membrane to allow glycolysis (22). Acidification only reversibly inhibited glycolysis in control suspensions (3). Examples of pH drop curves are
presented in Fig. 5A. The plots in Fig. 5B and C show final pH values
as a function of the concentrations of Fe2+ or
Fe3+ added to the suspensions. The data indicate almost no
effect of Fe3+ on acid tolerance but major reductions in
acid tolerance due to Fe2+. Fe2+ was somewhat
more potent when the experiments were carried out with suspensions in
the anaerobic glove box (Fig. 5C) rather than in air (Fig. 5B).
Moreover, cells treated with Fe2+ were irreversibly
impaired, and extensive washing did not restore glycolytic capacity,
even though cells from control suspensions readily produced acid from
glucose after being washed and resuspended in KCl-MgCl2
solution.
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FIG. 5.
Reduced pH drop in suspensions of S. mutans
GS-5 cells in response to added Fe2+ under aerobic or
anaerobic conditions. Data are shown for control cells incubated
aerobically () or anaerobically (), cells exposed to 10 mM
Fe3+ incubated aerobically () or anaerobically (),
and cells exposed to 10 mM Fe2+ incubated aerobically ()
or anaerobically () (A); for cells exposed aerobically to
Fe3+ () or Fe2+ () (B); for cells exposed
anaerobically to Fe3+ () or Fe2+ () (C);
and for cells exposed anaerobically to Cu2+ () or
Cu+ () (D).
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Copper acted also to decrease acid tolerance (Fig.
5D), but even for
anaerobic suspensions, Cu
2+ was nearly as potent as
Cu
+. Again, the inhibition was not reversible.
Results of experiments to assess inhibition of glycolysis by
Fe
2+ at pH 6.5 in terms of changes in the rate of
disappearance of
glucose from suspensions assayed by means of the
glucose oxidase
reaction indicated an average control value for glucose
utilization
of 0.82 mmol/h/g (dry weight) of cells, compared with
values of
0.89 and 0.42 for cells exposed to 20 mM Fe
3+ or
Fe
2+, respectively.
The results of assays of the phosphotransferase system with
permeabilized cells (
2) indicated essentially no inhibition
over a range of Fe
2+ concentrations from 0 to 10 mM.
Effects on F-ATPase.
Fe2+ was found to be an
effective inhibitor of the F-ATPase of S. mutans GS-5 in
permeabilized cells. For example, activities (means ± standard
deviations) were reduced from 59.4 ± 8.20 (n = 4)
µmol of Pi released/min/g (dry weight) of cells to values of 28.0 ± 6.58 (n = 4), 19.95 ± 6.27 (n = 4), and 5.53 ± 1.04 (n = 3)
in the presence of 2.5, 5.0, or 10.0 mM added Fe2+,
respectively. Fe3+ served mainly to slightly stimulate
ATPase activity (data not shown). Fe2+ inhibition of
F-ATPase activity appeared to be irreversible in that extensive washing
of permeabilized cells with solutions containing 10 mM Mg2+
or treatment with chelators did not restore activity. Fe2+
also inhibited F-ATPase activities of isolated membranes and F1 complexes dissociated from the membrane. For example,
the activity of isolated membranes (mean ± standard deviation)
was reduced from 0.469 ± 0.032 (n = 3) µmol of
Pi released/min/mg of membrane protein to 0.031 ± 0.008 (n = 3) in the presence of 5 mM added Fe2+. The activity of F1 complexes (mean ± standard deviation) was reduced from 2.43 ± 0.22 (n = 4) µmol of Pi released/min/mg of protein to 0.24 ± 0.07 (n = 2) in the presence of
5 mM added Fe2+.
Cu
+ also was inhibitory for the F-ATPase, with a 50%
inhibitory concentration about equal to that of Fe
2+.
However, Cu
2+ had approximately the same inhibitory potency
as Cu
+. Thus, the effect on F-ATPases appeared to be
related to reduction
of acid tolerance of glycolysis by intact cells
but not to lethality.
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DISCUSSION |
We present here a description of what appears to be a phenomenon
not previously described, anaerobic killing of bacterial cells induced
by the reduced, transition metal cations Fe2+ and
Cu+. There is at least some precedent in the literature in
the report of Sagripanti et al. (20) that viruses can be
inactivated by iron or copper cations in the absence of hydroperoxides.
The phenomenon we describe has unique characteristics, especially the
effectiveness of only the reduced cations for damage. The detailed
nature of the damage caused by Fe2+ and Cu+
remains to be determined. Presumably, damage involves formation of
organic radicals, such as glutathione radicals, with subsequent electron transfer to or from various biopolymers. In our initial search
for enzyme targets, we found also that Fe2+ and
Cu+ could irreversibly inhibit the F-ATPase of S. mutans GS-5 and, as a consequence, reduce the capacity of the
organism to carry out glycolysis in acid environments. Previously,
Senior et al. (21) had found that Fe2+ was bound
by beef heart F1 enzyme with high avidity, and there was
some 40% loss of activity when 4 mol of Fe was bound per mol of
F1 at about 20 µM Fe. Inhibition of F-ATPase could be
partly responsible for lethal damage. However, it seems that other
enzymes or structures must be the prime targets for lethality, since
Cu+ and Cu2+ both inhibited F-ATPase, while
only Cu+ was effective for anaerobic killing.
The type of killing described here seems to be limited in terms of
types of susceptible bacteria. The oral streptococci were generally
susceptible, but lactic acid bacteria are not all susceptible, as shown
by the high level of resistance of L. casei and the moderate susceptibility of E. hirae. Both catalase-positive and
catalase-negative oral actinomycetes were susceptible but not as
sensitive as the oral streptococci. E. coli was insensitive.
The role of iron in the physiology of oral streptococci has remained
somewhat unclear. The organisms do produce a superoxide dismutase that
is active with iron as metal. However, the enzyme is active also with
Mn, and at least for S. sobrinus OMZ176, iron is not
required for anaerobic growth (14). It can substitute partly
for the Mn required for aerobic growth. Evans et al. (7)
concluded that only the ferrous form is transported across the cell
membrane by S. sobrinus OMZ176, and the organism did not
appear to produce iron siderophores. Iron was taken up mainly under
anaerobic conditions. Overall, it appears that oral streptococci seem
not well adapted to manage iron. However, they are not unique in this
regard, and other lactic acid bacteria do not produce hemes and do not
have major needs for iron.
It appears from our work and that of others that iron may be useful as
a plaque control agent. In fact, there is already evidence in the
literature that iron and copper are anticaries agents. Previously,
Oppermann and Rölla (18) found that FeCl3
can reduce acid production by plaque in vivo. Our findings indicate
that Fe3+ is actually slightly stimulatory for glycolysis
at near-neutral pH. However, it is probable that polymicrobic plaque in
vivo may bring about reduction of Fe3+ to Fe2+,
which may then be inhibitory. Iron in the diet has been correlated with
a low incidence of caries, and recently, Rosalen et al. (19) and Miguel et al. (16) have shown that iron cocrystalized
with sugar can reduce caries in rats. Copper had a similar effect, and
the effects of Cu or Fe were found to be separate from any anticaries
effects of fluoride. The work described here provides a basis for
consideration of antimicrobial actions in attempts to interpret the
known anticaries effects of Fe and Cu.
| |
ACKNOWLEDGMENTS |
This study was supported by grants R01DE06127 and P01DE11549 from
the U.S. National Institute of Dental Research.
We thank Anne Clancy for help with the experiments involving
hydroperoxide killing.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Box 672, University of Rochester Medical Center, Rochester, NY 14642-8672. Phone: (716) 275-1674. Fax: (716) 473-9573. E-mail:
mutansst{at}aol.com.
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Appl Environ Microbiol, January 1998, p. 27-33, Vol. 64, No. 1
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Top
Abstract
Introduction
