Department of Microbiology and Hygiene,
Institute of Medical Microbiology and Hygiene,1
Institute of Microbiology,3 and
Institute of Organic Chemistry and
Biochemistry,4 University of Freiburg, D-79104
Freiburg, Germany, and Faculty of Medicine, Department of
Medical Microbiology, Vrije Universiteit, 1081 BT Amsterdam, The
Netherlands2
Homologs of the ferric uptake regulator Fur and the iron storage
protein ferritin play a central role in maintaining iron homeostasis in
bacteria. The gastric pathogen Helicobacter pylori contains
an iron-induced prokaryotic ferritin (Pfr) which has been shown to be
involved in protection against metal toxicity and a Fur homolog which
has not been functionally characterized in H. pylori.
Analysis of an isogenic fur-negative mutant revealed that
H. pylori Fur is required for metal-dependent regulation of
ferritin. Iron starvation, as well as medium supplementation with
nickel, zinc, copper, and manganese at nontoxic concentrations, repressed synthesis of ferritin in the wild-type strain but not in the
H. pylori fur mutant. Fur-mediated regulation of ferritin synthesis occurs at the mRNA level. With respect to the regulation of
ferritin expression, Fur behaves like a global metal-dependent repressor which is activated under iron-restricted conditions but also
responds to different metals. Downregulation of ferritin expression by
Fur might secure the availability of free iron in the cytoplasm,
especially if iron is scarce or titrated out by other metals.
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INTRODUCTION |
The gram-negative microaerophilic
bacterial pathogen Helicobacter pylori colonizes the
mucus layer of the human stomach (12, 20). Its hostile
ecological niche has necessitated the development of regulatory
mechanisms which allow the bacterium to survive unfavorable changes in
the environment. Adaptation to the conditions in the gastric mucosa
includes acquisition mechanisms that overcome a temporary lack of the
metals iron and nickel. Iron is essential for maintaining the basic
energy and redox metabolism, whereas nickel is an essential cofactor of
urease, an important virulence determinant of H. pylori
(21). However, as overacquisition of iron, nickel, and other
metals is deleterious, the control mechanisms regulating the
intracellular availability of these metals are of crucial importance.
Iron-responsive regulation in prokaryotes is usually mediated through
the ferric uptake regulator (Fur) protein. Fur homologs downregulate
the expression of genes involved in iron uptake when the cytoplasmatic
ferrous iron concentration increases, thus abolishing iron acquisition
(24). Fur homologs using metal ions as cofactors have been
identified in many bacterial species (7, 9, 13, 16, 19, 23, 30,
32, 33), and their regulatory functions range from regulation of
metal uptake to more specific processes, like oxidative-stress defense,
production of virulence factors, and acid resistance. That the Fur
protein also activates transcription in response to iron was recently
shown for the iron-induced superoxide dismutase SodB of
Escherichia coli (11, 13).
In addition to regulation of iron uptake, the internal iron
concentration within the cell can be modulated by the removal of free
ionic iron from the cytoplasm. This function is catalyzed by bacterial
ferritins, which constitute a specialized intracellular compartment for
the storage of iron in a nonreactive state and thus separate it from
the biochemical processes in the cytoplasm. Knowledge about the role of
Fur in the regulation of ferritin-mediated iron storage is very
limited, as it has only been studied in E. coli, where
ferritin expression was suggested to be positively regulated by the Fur
protein (1). Iron-responsive regulation has been observed in
H. pylori (35, 36), and genetic analysis revealed
that H. pylori possesses a Fur homolog (2, 6, 7, 14,
28). It was shown that in E. coli, the H. pylori Fur protein acts as an iron-dependent repressor of the
Fur-regulated E. coli fhuF and fiu promoters
(6, 14). The H. pylori ferritin protein Pfr is a
member of the nonheme ferritin subfamily, all of which store iron in
the inner space of a multimeric protein shell consisting of 24 identical subunits (4). The protein plays a substantial role
in the storage of iron and protects the bacteria from metal toxicity
(8, 10, 15). Ferritins thus catalyze a function which is the
exact opposite of that of iron uptake systems, which increase the
cytoplasmic iron concentration. In bacteria, the main functions of
ferritins have been iron storage and protection against metal toxicity
and oxidative stress (1, 8, 18, 29, 34).
Although the structures and catalytic functions of bacterial ferritins
are well known, data on their regulation in bacteria are limited
(1, 4, 18). The accumulation of H. pylori Pfr in
response to an increased iron concentration and its downregulation in
response to iron starvation (8) indicated the presence of an
iron-responsive regulatory network mediating iron homeostasis through
modulation of cytoplasmic iron storage and release.
This study describes the role of H. pylori Fur in the
regulation of ferritin-mediated iron storage. The observed
Fur-dependent regulation of H. pylori Pfr was found to be
affected by iron, as well as by nickel, zinc, copper, and manganese.
| |
MATERIALS AND METHODS |
Bacterial strains and growth conditions.
H. pylori
strains NCTC11638 and G27 and mutant derivatives were routinely grown
on blood agar in a microaerobic atmosphere as described previously
(8). For determination of growth characteristics and
responses to metals, bacteria were cultivated in brucella broth
supplemented with 10% fetal calf serum (BBF; Gibco BRL). The total
contents of iron, manganese, copper, zinc, and nickel in BBF medium
were 20, 0.3, 0.5, 15, and 0.2 µM, respectively, as determined by
atomic absorption mass spectrometry (26). Specific metal-enriched conditions were established by supplementation of BBF
with chloride salts of iron (F2877; Sigma) manganese (M3634; Sigma),
zinc (Z4875; Sigma), copper (C6641; Sigma), or nickel (N5756; Sigma) at
100 and 500 µM. Iron starvation was achieved by addition of the iron
chelator desferrioxamine B (desferal) (D-9533; Sigma) at a
concentration of 20 µM. Control cultures were supplemented with
sodium chloride at 500 µM to exclude the effect of osmotic stress on
regulation. For growth experiments, bacteria were cultured in liquid
medium to an optical density at 600 nm of approximately 1 and
subsequently diluted 1:100 in medium supplemented with desferal and/or
different metals. All experiments were performed in triplicate and were
repeated at least three times.
DNA and RNA techniques.
The construction and basic
characterization of the ferritin-negative H. pylori mutant
strain G27-PFR1 (pfr::cat) are
described elsewhere (8). Cloning of DNA was performed in
E. coli according to standard protocols (5).
Construction of the plasmid pFUR3-CAT carrying the H. pylori
fur gene interrupted by a promoterless cat gene (Fig.
1A), is described elsewhere
(6). Analysis of DNA by Southern hybridization and by PCR
was performed as described earlier (6, 25).
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FIG. 1.
Transcriptional organization of the H. pylori
fur (A) and pfr (B) genes. The genes are numbered
according to the annotated genome sequence of H. pylori
strain 26695 (28). The insertion sites and orientations of
promoterless cat cassettes inserted in the fur
and pfr mutants of strains NCTC11638 and G27, respectively,
are marked by circles (cat). The transcriptional direction
of the cat gene is indicated by a short arrow.
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To achieve marker exchange mutagenesis of the fur gene in
H. pylori, plasmid pFUR3-CAT was transferred into H. pylori strain NCTC11638 by electroporation (17).
Mutants carrying the promoterless cat gene from plasmid
pFUR3-CAT inserted in the chromosomal fur gene were selected
by growth on BBF agar containing chloramphenicol at a concentration of
10 mg/liter.
Isolation of total RNA and Northern hybridization were performed
according to a standard protocol (5) as described earlier for the fecA2 gene (14). Briefly, a digoxigenin
(DIG)-labeled antisense RNA probe was produced by in vitro
transcription of the pfr gene on plasmid pPFR1
(6) with SP6 RNA polymerase using the DIG RNA labeling kit
(Roche Diagnostics). Hybridization and stringency washes were done at
68°C. Bound probe was detected with the DIG-labeling and detection
kit using the chemiluminescent substrate CPD-Star (Roche Diagnostics).
Protein analysis.
Measurement of protein concentration,
electrophoretic separation of proteins by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein staining
with Coomassie brilliant blue, and immunoblot analysis of proteins with
the ferritin-specific antiserum AK198 were performed as described
earlier (6). Bound antibodies were detected with a protein
A-alkaline phosphatase conjugate.
N-terminal Edman degradation was performed on protein bands blotted to
polyvinylidene difluoride membranes in a protein-sequencing apparatus
(model 477A/120A; Perkin-Elmer Applied Biosystems) according to the
manufacturer's instructions.
To quantitate the Cat reporter protein produced in H. pylori
strain G27-PFR1 by enzyme-linked immunosorbent assay (ELISA), bacteria
grown in BBF media with different metal concentrations were harvested
by centrifugation. Lysis and determination of the amount of Cat protein
were performed using the Cat-ELISA system (Roche Diagnostics) according
to the manufacturer's recommendations. The amount of Cat was
calculated from a standard curve prepared with purified Cat protein
from E. coli and normalized to the amount of total protein.
| |
RESULTS |
Construction of a fur-negative mutant strain of
H. pylori.
To study the regulatory functions of the Fur
homolog in H. pylori, an isogenic
fur-negative mutant of strain NCTC11638 was constructed by
marker exchange mutagenesis with plasmid pFUR3-CAT (Fig. 1).
This resulted in an inactivation of fur by the insertion of
a promoterless chloramphenicol resistance cassette. Analysis by
Southern hybridization (Fig. 2) and by
PCR (data not shown) confirmed correct replacement of the original
wild-type fur gene by the interrupted version. The resulting
H. pylori mutant strain, NCTC11638-FUR, was motile and
positive for urease, catalase, and cytochrome oxidase.
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FIG. 2.
Analysis of the mutagenized fur gene by
Southern hybridization. HindIII- and
Sau3A-digested DNAs from the strain NCTC11638 (lanes 1) and
the fur mutant NCTC11638-FUR (lanes 2) were hybridized with
probes specific for fur and cat, respectively.
The arrows indicate hybridizing DNA fragments of the predicted sizes,
demonstrating correct replacement of the wild-type fur gene
with the interrupted version. The sizes of individual marker DNA
fragments are indicated on the left.
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Growth characteristics of the H. pylori NCTC11638
fur mutant.
To determine whether the fur
mutation resulted in growth deficiencies as observed in other bacterial
species (32), the fur mutant and the parent
strain were grown in liquid media with various iron and nickel
concentrations (Fig. 3). This analysis
showed that, irrespective of the metal concentration, growth of the
fur-negative mutant did not differ from that of the parent
strain (Fig. 3). Under all growth conditions and at all sampling times
in this study (see below), the growth of the mutant was identical to
that of the wild-type strain (data not shown).
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FIG. 3.
Growth characteristics of the H. pylori fur
mutant. The H. pylori strain NCTC11638 (solid bars) and the
fur mutant (shaded bars) were grown in BBF medium with iron
and nickel at various concentrations. For high-iron, low-iron, and
high-nickel conditions, the BBF medium was supplemented with 1 mM
FeCl3, 20 µM desferal, or 1 mM nickel, respectively.
Bacterial growth was determined after 48 h by measuring the
optical density (OD) at 600 nm. The values represent the means of three
independent determinations. The standard deviation is indicated above
each bar.
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Ferritin expression is derepressed in the H. pylori fur
mutant.
To identify iron-regulated genes controlled by Fur, the
parent H. pylori strain and the fur mutant were
grown in iron-restricted and iron-sufficient BBF media, and their total
protein profiles were compared (Fig. 4A).
In the parent strain, a 19-kDa protein was expressed under
iron-sufficient conditions but repressed under iron-restricted
conditions (Fig. 4A). This 19-kDa protein was highly expressed in the
fur-negative mutant under both iron-restricted and
iron-sufficient conditions (Fig. 4A). The iron-regulated 19-kDa protein
was identified as the ferritin protein Pfr by using the ferritin-specific antiserum AK198 (8) (Fig. 4B), and this
was confirmed by N-terminal sequence analysis.
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FIG. 4.
Influence of Fur on ferritin synthesis in response to
iron starvation. (A) Bacteria of strain NCTC11638 (wild type [wt])
and the isogenic mutant (fur) were grown for 48 h under
iron-rich conditions (BBF liquid medium [High]) and under
iron-restricted conditions generated with desferal at a concentration
of 20 µM (Low). Proteins were analyzed by SDS-PAGE (15% gel) and
stained with Coomassie blue. The ferritin protein is marked by the
arrow on the left. The sizes of individual marker proteins are
indicated on the right. (B) Western blot analysis of ferritin
production in strain NCTC11638 (wt) and the isogenic mutant
(fur) grown under iron-rich and iron-restricted conditions
as described for panel A with the ferritin-specific antiserum AK198.
The Pfr protein is marked by the arrow on the right.
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The involvement of Fur in the iron-dependent regulation of ferritin
levels was further confirmed by Western blotting (Fig. 4B). The
intensity of the ferritin protein band detected by the specific
antiserum clearly decreased under conditions of iron starvation in the
parent strain but not in the fur mutant.
Regulation of ferritin synthesis occurs at the mRNA level.
Analysis of ferritin mRNA by Northern hybridization indicated that the
pfr gene is transcribed as a monocistronic unit (Fig. 5). Synthesis of pfr mRNA was
completely repressed under conditions of iron starvation in the
wild-type strain but not in the fur mutant (Fig. 5). This
indicates that low iron levels lead to a Fur-mediated repression of
pfr mRNA synthesis.
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FIG. 5.
Influence of the fur mutation on
transcription of the pfr gene. Total RNAs (10 µg) isolated
from H. pylori strains NCTC11638 wild type (wt) and
NCTC11638-FUR (fur) grown in BBF medium (high) and in BBF
medium with the iron chelator desferal (20 µM) (low) were hybridized
with DIG-labeled pfr antisense mRNA (upper gel). The bands
corresponding to pfr mRNA are indicated on the right. The
lower gel shows a methylene blue stain of transferred RNA prior to
hybridization. The positions of 16S and 23S rRNAs are indicated on the
right.
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Regulation of ferritin synthesis in response to metals.
To
test whether H. pylori Fur regulates pfr
transcription in response to other metals, the H. pylori
parent strain and the fur mutant were grown in the presence
of nickel, copper, manganese, or zinc at 100 or 500 µM, and Pfr
expression was assessed by SDS-PAGE (Fig.
6). Ferritin expression was repressed by
all metals in the wild-type strain but not in the fur mutant
(Fig. 6). Regulation of ferritin synthesis was confirmed by Western
blotting (not shown). The complete derepression of ferritin production
in the fur-negative mutant strain in the presence of the
tested metals indicated that Fur is involved in downregulation of
ferritin synthesis mediated by other metals (Fig. 6). Supplementation
of the medium with sodium chloride at the highest nickel concentration
had no influence on production of the 19-kDa protein, excluding the
influence of osmotic stress on regulation (not shown).
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FIG. 6.
Role of Fur in repression of ferritin synthesis by
different metals. Strain NCTC11638 (wild type [wt]) and the
fur mutant (fur) were grown in BBF liquid medium
in the presence of zinc, copper, manganese, and nickel at
concentrations of 100 and 500 µM. Proteins were analyzed by SDS-PAGE
(15% gel) and stained with Coomassie blue. The ferritin protein (Pfr)
is indicated by the arrows on the right.
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The inhibitory effects of nickel and of iron starvation on Pfr
expression were quantitated using the promoterless cat
cassette (Fig. 1) inserted in the pfr gene in the H. pylori strain G27-PFR1 (Fig. 7).
Changes in pfr expression in response to iron starvation and
to nickel were measured as changes in the levels of the Cat protein
(Fig. 7). Iron starvation and nickel supplementation at concentrations
of 0.1 and 1 mM reduced the Cat concentrations to 20, 50, and 9% of
the levels detected under normal growth conditions, respectively (Fig.
7).
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FIG. 7.
Expression of the pfr gene in response to
iron starvation and nickel supplementation determined with a
pfr::cat fusion. The isogenic
pfr mutant strain, in which the promoterless cat
gene is fused to the pfr gene, was grown in the presence of
nickel at increasing concentrations and under conditions of iron
starvation generated with the iron chelator desferal (20 µM). The
production of the Cat protein resembling transcriptional activity of
the pfr gene was monitored by Cat-specific ELISA. The amount
of Cat produced under iron-replete conditions in BBF medium was set at
100%. The values represent the means of three independent
determinations. The error bars indicate standard deviations.
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DISCUSSION |
The H. pylori ferritin Pfr is a major component of iron
storage, as well as iron distribution, in the cell (8, 10,
15). Under iron-rich conditions, which in the natural environment
of H. pylori could be caused by free iron and heme compounds
from nutrition or inflammation, ferritin protects the cell from iron toxicity. The same could be true for other metals. On the other hand,
in response to iron starvation caused by the absence of nutrients or by
the iron-binding functions of mucosal lactoferrin (22),
repression of ferritin synthesis secures the availability of free iron
in the cytoplasmatic space for incorporation into essential
iron-cofactored electron transport proteins (Fig.
8). For regulation of the free ferrous
iron concentration in the cytoplasm, ferritins catalyze a reaction
whose function is the exact opposite of that of iron uptake systems
(Fig. 8). In contrast to the latter, ferritins lower the cytoplasmic
concentration of ferrous iron by binding it and transporting it into
the inner shell of the ferritin holoprotein. This function also lowers
metal toxicity in other bacterial species, as shown for the E. coli ferritin FtnA (1, 29).
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FIG. 8.
Schematic representation of iron metabolism in H. pylori under iron-rich, iron-restricted, and metal-rich
conditions. The putative regulatory roles of Fur are indicated. Iron
flux in the cytoplasm is indicated by the arrows. Free iron is
indicated by the solid circles. The possible competitive inhibitory
interactions between free iron and other metals (shaded circles) are
indicated by a double arrow. The protein shell of ferritin is shown by
the open circle. Repression of ferritin synthesis under conditions of
iron starvation or metal overload, as well as release of ferritin-bound
iron, is indicated by the dashed circle. Thick and thin arrows at the
membrane indicate high and low transport, respectively. Arrows with two
bars indicate inhibition of ferritin synthesis.
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The concentration of free ferrous iron in the bacterial cytoplasm is
mainly controlled by repression of iron uptake mediated by Fur
homologs, by enhanced incorporation of iron into iron-cofactored proteins, and by the iron-binding properties of ferritins (Fig. 8).
Therefore, combining regulatory functions for iron uptake and iron
storage would be an advantage in maintaining iron homeostasis in the
cell. The regulatory functions of Fur homologs in balancing the
cytoplasmic metal concentration have been most extensively investigated
for the regulation of metal uptake, whereas almost nothing is known
about their role in the regulation of storage of iron and other metals
(13).
Our previous studies indicated that H. pylori Fur is
involved in regulation of iron metabolism and acts as a repressor of iron uptake systems in response to elevated levels of iron in the
environment (6, 7, 31). In the induction of iron uptake systems, H. pylori Fur behaves similarly to Fur homologs of
other bacteria (6, 14, 31), which are usually involved in
differential gene regulation in response to changes in the
environmental concentrations of metals (7, 9, 13, 16, 19, 23, 30,
32, 33). Evidence for binding of H. pylori Fur to the
promoter regions of genes involved in iron uptake is provided by the
presence of putative Fur binding sequences in some promoters of
H. pylori genes predicted to be involved in iron uptake
(28) and by isolation of putative H. pylori
Fur-regulated promoters using a modified Fur titration assay
(14). The comparison of Pfr expression in the wild-type and
fur mutants performed in this study confirmed that H. pylori Fur is involved in metal-dependent regulation and, especially in combining modulation of both uptake and storage of iron,
might have a wider range of functions in maintaining metal homeostasis
than is known so far.
The regulatory role of Fur under iron-restricted conditions has not yet
been studied, nor has its role in positive gene regulation (33). A possible influence of iron starvation on
metal-dependent transcriptional repressor proteins was recently
suggested for the zinc uptake regulator Zur of E. coli, for
which activation by iron starvation was reported (23). In
addition, the E. coli Fur protein has been demonstrated to
contain two zinc atoms, and binding specificity is altered when these
zinc atoms are removed (3).
The finding that H. pylori Fur is necessary for repression
of ferritin synthesis in response to iron starvation suggests that Fur
is active in the absence of iron and that it secures, via repression of
ferritin, the availability of free iron in the cytoplasm under
conditions where environmental iron is scarce. The fact that
Fur-mediated regulation of Pfr expression occurs at the mRNA level
provides evidence for DNA-binding activity of Fur in the absence of
iron, but other regulatory mechanisms acting on mRNA stability, like
those present in eukaryotes, cannot be excluded (27). The
transcriptional regulation of ferritin synthesis and the repression in
response to iron starvation and to nickel was well reflected by using
the promoterless Cat gene as a reporter (Fig. 7), indicating that Cat
is well suited for reporter gene analysis in H. pylori.
The increase in ferritin production observed in the fur
mutant under conditions of iron starvation suggests that other
regulators might be involved in regulation of iron storage. A direct
interaction of Fur with ferritin transcription is supported by the
metal-dependent function observed. If iron is scarce, the metal/iron
ratio in the cell could increase, and it can be suggested that under
these conditions other metals become dominant over iron and activate Fur, resulting in repression of ferritin synthesis (Fig. 6 and 8). In
this context, it can also be speculated that other metals compete with
iron for incorporation in iron proteins and thus mimic iron starvation
in the cytoplasm. Competition of nickel, manganese, and copper with
iron is only possible at high concentrations of these metals, because
their concentrations in BBF medium are about 50 to 100 times lower than
the iron concentration (see Materials and Methods). Of the metals
investigated, only zinc is present at a concentration which is similar
to the concentration of iron. In this context, the Fur-dependent
repression of ferritin synthesis by nickel, copper, manganese, and zinc
suggests that H. pylori Fur acts like a more global
metal-dependent regulator. Multiple functions of Fur are not
unexpected, because Fur homologs of other bacteria are involved in
responses to iron and zinc and to peroxide stress, respectively
(3, 9, 16). Because H. pylori possesses only a
small set of regulatory proteins, and only one Fur homolog (2,
28), it would be reasonable if H. pylori Fur had
multiple functions which would compensate for the absence of other
regulators. The functions of H. pylori Fur in the regulation
of iron storage seem to differ considerably from those of E. coli Fur, which regulates the ferritin homolog FtnA in a way
opposite to that of H. pylori Pfr, as an E. coli
fur mutant produces less FtnA than the parent strain.
In summary, H. pylori Fur acts like a metal-dependent
regulator of Pfr-mediated iron storage. The question of whether
regulation of ferritin synthesis is mediated directly by Fur or
includes other proteins, as in eukaryotes (27), is the topic
of ongoing studies focused on the functional DNA-binding properties of
the Fur protein itself and on the investigation of member genes of the
H. pylori Fur regulon.
We thank Klaus Hantke (Tübingen, Germany) for advice on
experiments. Tanja Vey provided excellent technical assistance. The ferritin-specific antiserum AK198 was kindly provided by Stefan Odenbreit and Rainer Haas (University of Munich, Munich, Germany).
This work was supported by grants from the Deutsche
Forschungsgemeinschaft (Ki201/8-2) to M.K. and from the
Universitair Stimulerings Fonds of the Vrije Universiteit
Amsterdam and the Nederlandse Organisatie voor Wetenschappelijk
Onderzoek (901-14-206) to A.H.M.V.V.
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Abstract
Introduction
