INFECTION AND IMMUNITY, May 2010, p. 1841–1849 Vol. 78, No. 50019-9567/10/$12.00 doi:10.1128/IAI.01258-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Unique Host Iron Utilization Mechanisms of Helicobacter pyloriRevealed with Iron-Deficient Chemically Defined Media�

Olga Senkovich, Shantelle Ceaser, David J. McGee, and Traci L. Testerman*Louisiana State University Health Sciences Center—Shreveport, Department of Microbiology & Immunology,

1501 Kings Highway, Shreveport, Louisiana 71130

Received 9 November 2009/Returned for modification 10 December 2009/Accepted 13 February 2010

Helicobacter pylori chronically infects the gastric mucosa, where it can be found free in mucus, attached tocells, and intracellularly. H. pylori requires iron for growth, but the sources of iron used in vivo are unclear. Inprevious studies, the inability to culture H. pylori without serum made it difficult to determine which host ironsources might be used by H. pylori. Using iron-deficient, chemically defined medium, we determined that H.pylori can bind and extract iron from hemoglobin, transferrin, and lactoferrin. H. pylori can use both bovineand human versions of both lactoferrin and transferrin, contrary to previous reports. Unlike other pathogens,H. pylori preferentially binds the iron-free forms of transferrin and lactoferrin, which limits its ability to extractiron from normal serum, which is not iron saturated. This novel strategy may have evolved to permit limitedgrowth in host tissue during persistent colonization while excessive injury or iron depletion is prevented.

Like nearly all pathogenic bacteria, Helicobacter pylori mustacquire iron to grow. Many pathogens produce siderophores,which are potent chelators that are capable of extracting ironfrom a variety of host proteins. Despite numerous efforts, no H.pylori-associated siderophores have been found (12). Without au-tologous siderophores or the ability to use siderophores producedby other species, pathogenic bacteria require specific receptors toutilize host iron-binding molecules as iron sources. The potentialiron sources in the host include heme, hemoglobin (Hb), trans-ferrin (Tf), lactoferrin (Lf), ferritin, hemosiderin, and iron-con-taining enzymes. Lactoferrin is present in gastric mucus, and thelevel of this molecule has been found to be increased in individ-uals with H. pylori-mediated gastritis (28). Bacteria normally ex-tract iron from Tf or Lf before importing the iron and releasingthe apoprotein (1). In the case of heme-containing proteins, theentire heme molecule is removed from the protein and importedinto the cytoplasm, where it can be degraded to release iron orused as a prosthetic group (15).

H. pylori iron acquisition is also of interest because infectionwith H. pylori is associated with iron-deficiency anemia (27). H.pylori could cause iron deficiency indirectly by suppressing gas-tric acid secretion, thereby reducing solubilization and uptakeof dietary iron. There is also evidence obtained with a mouseinfection model suggesting that H. pylori successfully competesfor dietary iron when the diet is iron poor (20). Furthermore,a recent study showed that clinical isolates of H. pylori frompatients with iron-deficiency anemia have higher rates of inor-ganic iron uptake than strains from patients with non-iron-deficiency anemia (39). At present, it is not known to whatextent H. pylori relies on iron in the host’s diet and how muchiron is obtained from host molecules originating from hostblood or tissue.

H. pylori is intrinsically resistant to numerous antibiotics andcan acquire resistance to other antibiotics, which makes treat-ment failure common. Cost and unacceptable drug side effectsare also barriers to eradication of H. pylori infections, whichmakes development of new treatment methods a high priority.Based on the reported inability of H. pylori to utilize bovine Lf,this protein is being tested as a treatment adjuvant (11, 34, 41).Specific drugs are also being developed to inhibit heme utili-zation and other mechanisms of iron uptake by pathogens (7,32). A clear understanding of a pathogen’s iron acquisitionstrategies is helpful in designing drugs with the purpose ofstarving the organism of iron.

Iron may be periodically abundant in the gastric lumen asdietary iron is solubilized by gastric acid. H. pylori may there-fore have a short window of opportunity to acquire iron beforeit is bound by mucus lactoferrin or taken up by epithelial cells.As H. pylori invades gastric crypts, free iron is expected to bescarce, and host iron-binding molecules may play an importantrole in maintaining colonization in this niche.

In vitro studies of H. pylori iron utilization have been ham-pered by the lack of a suitable serum-free growth medium.Nonetheless, several investigators have made progress towardidentifying host-derived iron sources utilized by H. pylori. Onestudy reported that H. pylori can use heme as an iron source ina blood agar-based assay system (38). Another study claimedthat H. pylori can use human Lf, but not bovine Lf, as an ironsource (18). There are conflicting reports concerning the abil-ity of H. pylori to obtain iron from Tf (18, 35).

H. pylori has a tonB gene, but it lacks recognizable homologs ofthe Lf-, Tf-, and hemoglobin-binding proteins of other organisms.In our studies, we have found that H. pylori preferentially bindsapo-Tf and apo-Lf rather than the iron-containing proteins. Thismode of iron utilization is expected to reduce the potential forsepsis or overgrowth of the organism in tissue.

MATERIALS AND METHODS

Culture of organisms. H. pylori strains (Table 1) were routinely cultured onCampylobacter blood agar containing 10% defibrinated sheep blood and in

* Corresponding author. Mailing address: Louisiana State UniversityHealth Sciences Center—Shreveport, Department of Microbiology &Immunology, 1501 Kings Highway, Shreveport, LA 71130. Phone: (318)675-8143. Fax: (318) 675-5764. E-mail: tteste@lsuhsc.edu.

� Published ahead of print on 22 February 2010.

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Ham’s F-12 medium (Mediatech, Inc., Manassas, VA) supplemented with 1%fetal bovine serum. Prior to experiments, organisms were inoculated into Ham’sF-12 medium without serum and allowed to reach mid- to late-log phase in orderto minimize the effects of serum carryover.

Preparation of an iron-deficient medium (TT18). Glassware was soaked for 4to 7 days in 2% nitric acid to remove iron and then rinsed with ultrapure water.The formula for TT18, the chemically defined medium that we used, is based onthe formula for Ham’s F-12 medium. The final (1�) concentrations of thecomponents of TT18 are as follows. The final concentrations of amino acids were8.9 mg/liter alanine, 211 mg/liter arginine-HCl, 15 mg/liter asparagine � H2O, 13mg/liter aspartic acid, 35 mg/liter cysteine-HCl, 14.7 mg/liter glutamic acid, 146mg/liter glutamine, 7.5 mg/liter glycine, 21 mg/liter histidine-HCl monohydrate,4 mg/liter isoleucine, 13 mg/liter leucine, 36.5 mg/liter lysine-HCl, 4.5 mg/litermethionine, 5 mg/liter phenylalanine, 34.5 mg/liter proline, 10.5 mg/liter serine,12 mg/liter threonine, 2 mg/liter tryptophan, 7.8 mg/liter tyrosine 2Na dihydrate,and 11.7 mg/liter valine. The metal-salt solution used contained 44 �g/mlCaCl2 � 2H2O, 2.5 ng/ml CuSO4 � 5H2O, 22.4 �g/ml KCl, 57.2 �g/ml MgCl2(anhydrous), 7.6 mg/ml NaCl, 0.86 ng/ml ZnSO4 � 7H2O, and 1.2 ng/ml phenolred. The medium also contained a sodium phosphate-sodium bicarbonate solu-tion consisting of 1.176 mg/ml NaHCO3 and 0.142 mg/ml Na2HPO4, as well as4.77 �g/ml hypoxanthine, 0.11 �g/ml sodium pyruvate, and 0.3 �g/ml thiamine-HCl. The medium was prepared by mixing appropriate ratios of stock solutions(10� L-amino acids, 5� metal-salt solution, 10� sodium phosphate-sodiumbicarbonate solution, 1,000� hypoxanthine, 1,000� sodium pyruvate, and1,000� thiamine-HCl) with ultrapure water. All solutions were filter sterilized,and all solutions except the metal-salt solution were treated with Chelex resin(Chelex 100 resin; catalog no. 143-2832; Bio-Rad Laboratories, Hercules, CA) tofurther reduce iron contamination. Since Chelex is not highly specific for iron,the metal-salt solution could not be treated without adversely impacting the zincand magnesium concentrations. Deferoxamine mesylate (Sigma-Aldrich, Inc., St.Louis, MO) was added to the medium in some experiments, as indicated below.

Preparation of iron-saturated serum and serum components. Dialysis cham-bers containing a protein sample were placed in 0.1 M sodium citrate-bicarbon-ate buffer (pH 8.2) containing 70 mM ferric chloride as previously described (24).Samples were subsequently dialyzed against phosphate-buffered saline (PBS)containing Chelex resin to remove any unbound iron. Total iron content andtotal iron-binding capacity assays were carried out by the LSUHSC HospitalClinical Chemistry Laboratory before and after the iron saturation procedure.The unsaturated iron-binding capacity was assayed using a Ferrozine-based col-orimetric assay (Equal Diagnostics, Exton, PA), and total iron was assayed usinga FERENE-based method (Abbott Diagnostics, Abbott Park, IL). Proteins werestored at 4°C in the presence of Chelex resin, which absorbed any residual iron.Since Chelex has a lower binding affinity for iron than Lf, Tf, or heme, weassumed that protein preparations that had been determined to be iron saturatedremained iron saturated after storage in the presence of Chelex.

Measurement of growth by ATP assay or plate assay. Twenty-four well platescontaining 1-ml aliquots of TT18 were supplemented with the iron-bindingmolecules indicated below and inoculated with 10 �l of a logarithmic-phase H.pylori culture grown in serum-free F-12 medium (starting inoculum, approxi-mately 1 � 104 CFU/ml). Experiments were performed in duplicate, triplicate, orquadruplicate, as indicated below. The culture plates were incubated at 37°C inan atmosphere containing 5% O2 and 10% CO2 for 16 to 42 h. Prior to the assay,the culture plates were evaluated by phase-contrast microscopy using an invertedmicroscope to assess the bacterial morphology and density of positive controls. Ifthe positive-control cultures were not sufficiently dense, the plate was incubatedfor a few more hours. Assay plates were not analyzed if positive-control cultures

did not grow or if a significant percentage of the bacteria in the positive-controlcultures were coccoid, which indicated that the viability was reduced. The ATPcontents of adherent and planktonic populations were measured as describedpreviously (36), except that 25-�l samples instead of 100-�l samples were mixedwith 25 �l of CellTiter-Glo reagent (Promega) instead of 100 �l of CellTiter-Gloreagent. Although this assay gives reproducible results, levels of growth werecompared only within an experiment, not between experiments. All experimentswere carried out at least three times in order to ensure that the results werereproducible. All iron-binding molecules were tested to ensure that they did notinterfere with the ATP assay. Numbers of CFU were assessed by preparing serial10-fold dilutions in PBS. Aliquots (50 �l) of each dilution were spotted induplicate onto predried CBA plates and incubated at 37°C in an atmospherecontaining 10% CO2 and 5% O2 for 4 to 5 days. Type 2 two-tailed Student’s ttests were performed with growth data using Microsoft Excel software.

Flow cytometry analysis. Labeling of proteins with fluorescein isothiocyanate(FITC) was performed using an FITC labeling kit (Calbiochem catalog no.343210; EMD Biosciences, La Jolla, CA). Unbound FITC was removed bypassing proteins over a 5-ml desalting column (GE Healthcare), which waseluted with PBS containing 0.1% sodium azide. Removal of free FITC wasconfirmed by loss of a low-molecular-weight fluorescent band following polyacryl-amide gel electrophoresis of labeled protein. One-milliliter aliquots of logarith-mic-phase H. pylori cultures grown in Ham’s F-12 medium containing 1% fetalbovine serum (FBS) were pelleted with a microcentrifuge at 10,000 � g andresuspended in filtered phosphate-buffered saline (PBS) containing 500 �g/mlbovine serum albumin. FITC-labeled protein was added to tubes, and sampleswere incubated at 37°C in the dark for 1 h. Samples were centrifuged andresuspended in a solution containing 2% (vol/vol) ultrapure, methanol-free for-malin (catalog no. 04018; Polysciences, Warrington, PA) diluted in PBS. Forcompetition experiments, labeled and unlabeled proteins were added at the sametime. Flow cytometric determination of protein binding was performed with aBD LSRII (BD Biosciences, San Jose, CA) that was made available by theResearch Core Facility at the Louisiana State University Health Sciences Cen-ter—Shreveport (Shreveport, LA). The LSRII has a Coherent Sapphire laser for488-nm excitation, a JDS Uniphase HeNe laser for 633-nm excitation, and aCoherent VioFlame for 405-nm excitation. Data analysis was performed usingFACS Diva software (BD Biosciences) and FlowJo software (Tree Star, Inc.,Ashland, OR). A minimum of 20,000 events (bacteria) were collected for eachsample. Gating of P2 was set to exclude at least 97.5% of the unstained popu-lation, and the same gate setting was used for all comparisons in a singleexperiment. Histograms were prepared by plotting the percentage of maximalfluorescence versus fluorescence.

RESULTS

H. pylori can utilize hemoglobin as an iron source. Previ-ously, studies of acquisition of iron by H. pylori from host-derived sources were hindered by the lack of a serum- orblood-free growth medium. We developed an iron-deficientchemically defined medium, TT18, which contains less than 0.1�M iron (based on atomic absorption spectroscopy). This me-dium is similar to the previously reported TT8 medium (33),but it contains NaCl at the same concentration as Ham’s F-12medium and lacks iron. This medium supports minimal growthof H. pylori unless another source of iron is provided, whichpermits evaluation of iron utilization in the absence of serumor chelators. The absence of chelators is particularly importantfor studying iron acquisition from Tf and Lf because manycommonly used iron chelators, such as deferoxamine, haveaffinities that are several orders of magnitude higher than thatof transferrin (6), which makes them unsuitable for experi-ments with a relatively slow-growing organism such as H. pylori.

We first examined the ability of H. pylori to obtain iron fromhemoglobin. Since dehydrated hemoglobin powder may con-tain a small amount of degraded protein, free heme, and pos-sibly even free iron, we dialyzed a hemoglobin solution using amembrane with a 10-kDa cutoff against PBS supplementedwith Chelex resin. H. pylori growth was monitored by assaying

TABLE 1. H. pylori strains used in this study

Strain Source Reference

26695m Motile 26695 33UMAB41 26HpDJM17 Clinical isolate 2543504 ATCCSS1 21J166 R. Peek, duodenal ulcer isolatea 22J178 R. Peek, gastritisa

B134A R. Peek, gastric ulcera

J54 R. Peek, duodenal ulcera

a Kindly provided by R. Peek from his personal collection.

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the ATP content in 25-�l aliquots of a culture. In this assay,20,000 relative light units (RLU) corresponds to a culturedensity of 5 � 106 CFU/ml, while 100,000 RLU corresponds to3 � 107 CFU/ml. The lower limit of detection is around 1 �104 CFU/ml. As shown in Fig. 1, H. pylori exhibited dose-dependent growth in the presence of hemoglobin, and toxicitywas observed at concentrations greater than 3 �M. Since he-moglobin is a tetramer, 1 �M hemoglobin corresponds to 4�M heme iron. To further prove that the growth was due to theheme iron in hemoglobin and not due to free inorganic iron,we repeated the experiment using deferoxamine (DFO). DFOis a strong chelator which can extract iron from proteins suchas Tf but cannot extract iron from heme (10). Addition of DFOto TT18 further suppressed growth of H. pylori. Addition ofhemoglobin restored the ability to grow in the presence ofDFO (data not shown).

Free heme is toxic to H. pylori. All hemoglobin-utilizingbacteria studied to date extract the heme molecule from he-moglobin and transport it to the cytoplasm, where it can beused directly or degraded to release the iron. Therefore, or-ganisms that utilize hemoglobin are expected to utilize freeheme as well. It should be noted that when heme is free insolution, the iron is oxidized to Fe(III) and the molecule isproperly referred to as hemin. For simplicity, here we use theterm heme to refer to either the oxidized form or the reducedform. We attempted to culture H. pylori in the presence ofheme concentrations ranging from 1 �M to 50 �M. In eachcase, the bacteria became coccoid, often appeared to be de-graded, and lost ATP, indicating that there was toxicity (datanot shown). Inorganic iron is not toxic to H. pylori at this rangeof concentrations (33). As positive controls, we used Vibriocholerae and uropathogenic Escherichia coli strain CFT073.These organisms exhibited dose-dependent increases in growthwith heme concentrations ranging from 1 �M to 50 �M, andno toxicity was noted (data not shown). Thus far, every H.pylori strain tested (J166, J178, B134A, J54, 43504, UMAB4,and G27) has shown sensitivity to heme at concentrationsabove 1 �M. Consistent with the measured heme toxicity,predigestion of hemoglobin with trypsin made the hemoglobinsolution toxic at concentrations that ordinarily stimulate

growth (data not shown). Refrigerated hemoglobin solutionsalso become toxic after storage for more than a few weeks.

Exogenous heme should be no more toxic than inorganiciron unless it is internalized. Therefore, we reasoned that tox-icity could result from unregulated heme uptake and that hememight stimulate growth if it was provided at an appropriateconcentration. We prepared serial 2-fold dilutions of heme inTT18 and found that the toxicity was reduced at concentrationsbelow �60 nM. We detected growth close to or slightly greaterthan that of the TT18 control at heme concentrations rangingfrom 15 to 30 nM in several experiments. These experimentsclearly showed that there was a reduction in toxicity, but therewas great variability between replicates. In order to reduce thevariability and growth due to free iron, we added 1 �M DFOto all wells and used a larger inoculum. As shown in Fig. 2,there was significant stimulation of growth by heme at concen-trations above 15.6 nM. The RLU seen in the presence of DFOwithout added heme represent ATP from the inoculum alongwith limited growth using existing iron stores. The concentra-tion of heme that results in toxicity appears to vary somewhatbased on the inoculum size, and cultures started with a largerinoculum tolerated slightly higher heme concentrations.

H. pylori can acquire iron from human and bovine lactofer-rin and transferrin. Iron-saturated bovine and human Tf andLf were dialyzed against Chelex-containing PBS and werestored in the presence of Chelex resin to remove all traces ofunbound iron. These proteins were then added to TT18 at aconcentration of 9 �g/ml (�113 nM). Either human or bovineTf or Lf consistently stimulated growth of H. pylori strain26695m in iron-deficient TT18 (Fig. 3 and data not shown). Inorder to be certain that increased ATP levels represented realgrowth, we plated serial dilutions of cultures from one assay.The relative levels of growth were similar regardless of theassay method; 100,000 RLU corresponded to about 1 � 107

CFU/ml, and the lower limit of detection for the ATP assaycorresponded to a culture density near 1 � 104 CFU/ml. Asidefrom speed, two major advantages of the ATP assay are that itpermits enumeration of both planktonic and adherent bacteria

FIG. 1. H. pylori utilizes hemoglobin as an iron source. Hemoglobinwas added to TT18 at the concentrations indicated. Inocula containingthe same amount of H. pylori strain 26695m were added to triplicatewells. Growth was measured by analysis of ATP after overnight incu-bation. rlu, relative light units. The error bars indicate standard devi-ations. *, P � 0.01 compared with the control.

FIG. 2. Growth on heme iron in the presence of deferoxamine.Deferoxamine mesylate was added to TT18 at a final concentration of1 �M. Hemin chloride was added at a starting concentration of 62.5nM, and serial 2-fold dilutions were prepared to obtain the concen-trations shown. Inocula containing the same amount of strain 26695mwere added to triplicate wells. Growth was assessed by measuring theATP content after 30 h. rlu, relative light units. The error bars indicatestandard deviations. *, P � 0.05 compared with the culture containingno heme.

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and that it is not affected by bacterial aggregation (33, 36).There are conflicting reports about the ability of H. pylori touse Tf, and use of Lf is reportedly species specific (18, 35). Incontrast, we found that H. pylori grew with either Tf or Lf andthat growth was similar when either human or bovine proteinswere used. Strains J99 and B134A were also found to usebovine Tf and Lf (data not shown), leading us to believe thatthe lack of species specificity is common in H. pylori isolates.

Interestingly, addition of apo-Tf or apo-Lf suppressed growthto levels below that in the control medium. Although these apo-proteins are expected to bind free iron in the medium, one wouldexpect that an organism capable of extracting iron from Tf or Lfwould still be able to grow. When bovine apo-Lf and holo-Lf weremixed at different ratios, we found that growth was substantiallyinhibited when the proteins were mixed so that the total ironsaturation was less than 75% (Fig. 4). A similar pattern wasobserved with bovine Tf (data not shown). These data suggestthat H. pylori binds the apoproteins more strongly than the holo-proteins or that the apoproteins have an inhibitory effect via adifferent mechanism. Lactoferrin is reported to have antibacterialproperties unrelated to iron chelation (14), but we observed noevidence of toxicity as determined by culture, ATP, or micros-copy. The growth of H. pylori was inhibited, but the bacteria werenot killed.

H. pylori cannot acquire iron from normal serum. Normalfetal bovine serum (FBS) contains Tf that is about 30% ironsaturated and therefore has the capacity to bind free iron inbacteriological growth media. In fact, serum has been used asa chelator by some investigators (37). FBS consistently inhibitsH. pylori growth in TT18 when it is added at a concentration of1% (data not shown). A greater serum concentration (30%)was required to inhibit growth in Ham’s F-12 medium, consis-tent with the �30-fold-higher iron concentration in F-12 me-dium (data not shown). When fetal bovine serum was ironsaturated and dialyzed against Chelex-containing buffer, itstimulated growth of H. pylori in TT18 (data not shown). If H.pylori does not differentiate between holo- and apo-Tf, thenone would expect that growth in serum that is partially iron

saturated would be slowed but not stopped. We thereforeperformed a time course experiment to follow growth of H.pylori in TT18 supplemented with 10% normal fetal bovineserum. The culture grew slightly during the first 24 h, reachinga peak ATP value of 7,700 RLU (�1.7 � 106 CFU/ml), itremained static for the next 24 h, and then the concentration ofbacteria declined (data not shown). H. pylori cultured with 10%FBS in iron-replete medium normally reaches a density of0.7 � 108 to 1 � 108 CFU/ml. The minimal growth observedduring the first 24 h could have been due to utilization ofexisting intracellular iron stores. Organisms in wells containingTT18 with serum retained the bacillary morphology during thefirst 48 h, even though they did not grow after 24 h. Themaintenance of bacillary morphology along with the ATP datasuggested that H. pylori is inhibited, but not killed, by theapo-Tf and apo-Lf in normal serum; however, these results donot explain why H. pylori is unable to utilize the holo-Tfpresent in serum.

H. pylori specifically binds transferrin, lactoferrin, and he-moglobin. Acquisition of iron from host proteins requires thepresence of specific bacterial receptors. It should therefore bepossible to detect binding of labeled host proteins to the bac-terial surface. H. pylori was incubated with Tf, Lf, or hemoglo-bin that had been labeled with fluorescein isothiocyanate(FITC). As a positive control, we used rabbit IgG specific forH. pylori, followed by FITC-labeled goat anti-rabbit IgG as asecondary antibody. As a negative control, we used FITC-labeled bovine serum albumin (BSA). We found that FITC-BSA binds minimally to H. pylori, and a very great increase influorescence was detected following incubation with H. pylori-specific antibodies and an FITC-labeled secondary antibody(data not shown). BSA was also included with the FITC-la-beled Tf, Lf, or Hb to reduce any nonspecific binding interac-tions that may have occurred. A significant increase in fluores-cence was seen following incubation of H. pylori with humanhemoglobin or Tf or Lf from either human or bovine sources(Fig. 5). Binding of FITC-labeled proteins (human LF, bovineLf, human Tf, bovine Tf, human Hb) was compared for strains26695m and SS1. The percentages of the bacterial populationswith bound protein were comparable for the two strains over arange of ligand concentrations for all proteins except hemo-globin; strain SS1 bound hemoglobin significantly better than26695m bound it (data not shown).

FIG. 4. Effect of lactoferrin iron saturation on H. pylori growth.Ferri-lactoferrin and apo-lactoferrin were added to TT18 at ratios thatresulted in the saturation levels indicated. The total lactoferrin con-centration was 9 �g/ml. Inocula containing the same amount of strain26695m were added to duplicate wells. Growth was assessed by mea-suring the ATP content. rlu, relative light units.

FIG. 3. Acquisition of iron from transferrin and lactoferrin. Iron-saturated human Tf (hTf), human Lf (hLf), bovine Tf (bTf), andbovine Lf (bLf) were added to TT18 at a concentration of 6 �g/ml. Asa positive control, 10 �M FeCl3 was added. Inocula containing thesame amount of H. pylori strain 26695m were added to quadruplicatewells. Growth was measured by analysis of ATP after overnight incu-bation. rlu, relative light units. The error bars indicate standard devi-ations. *, P � 0.01 compared with the TT18 control.

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To test the specificity of the binding interactions, we addeda 50-fold excess of unlabeled protein. In each case, fluores-cence decreased substantially in the presence of unlabeledprotein (data not shown). Next, we sought to determinewhether the same receptor recognized both Tf and Lf. WhenFITC-labeled human Lf (40 �g) was mixed with a 50-foldexcess of unlabeled human Tf, only a small decrease in fluo-rescence was observed (Fig. 6A). On the other hand, human Lfwas able to competitively reduce binding of FITC-labeled hu-man Tf (Fig. 6B). These data suggest that the Tf receptor alsobinds Lf. Since Tf is not able to significantly reduce binding ofLf, it is possible that there are two Lf receptors and only oneof them binds Tf or that the affinity for Lf is much higher thanthe affinity for Tf. When apo-Lf binding was measured over abroad concentration range, we observed binding greater thanapo-Tf binding, and there was evidence of bimodal binding, incontrast with the pattern observed with apo-Tf (Fig. 7). Al-though not conclusive, these data are consistent with the pat-tern expected when two Lf receptors are present and only oneof them is also able to bind Tf. This hypothesis is supported bythe appearance of flow cytometry histograms created using thesame samples. The bell curve of Tf binding is narrow over the

FIG. 5. Binding of FITC-labeled proteins to H. pylori strain SS1.Three micrograms of each protein was incubated with 1 ml of H. pylorisuspended in PBS for 1 h at 37°C. Samples were centrifuged andresuspended in PBS to remove unbound protein. The transferrin andlactoferrin were iron saturated. hTf, human Tf; hLf, human Lf; bTf,bovine Tf; bLf, bovine Lf; hHb, human Hb.

FIG. 6. Characteristics of transferrin and lactoferrin binding. H. pylori strain 26695m was incubated with FITC-labeled proteins at variousconcentrations (C and D) or in the presence or absence of excess unlabeled protein (A and B). The fluorescence of bacteria was measured by flowcytometry. (A) A 50-fold excess of transferrin does not prevent binding of lactoferrin. (B) A 50-fold excess of lactoferrin blocks binding bytransferrin. (C) Effect of apo-Tf concentration on fluorescence distribution. (D) Effect of apo-Lf concentration on fluorescence distribution. hTf,human Tf; hLf, human Lf; bLf, bovine Lf.

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entire concentration range (Fig. 6C), whereas the apo-Lf curveis narrow at the lower concentrations but broadens at higherconcentrations, suggesting that there are two receptors withdifferent Lf affinities (Fig. 6D).

Of greatest interest, we examined the specificity of H. pylorireceptors for apoproteins and holoproteins. Incubation ofstrain 26695m with 40 �g/ml iron-saturated bovine Lf resultedin a substantial increase in fluorescence. Addition of a 50-foldexcess of unlabeled bovine apo-Lf shifted fluorescence almostto the baseline level (Fig. 8A). In contrast, a 50-fold excess ofunlabeled bovine holo-Lf did not reduce the fluorescence ob-tained when H. pylori was incubated with FITC-labeled bovineapo-Tf (Fig. 8B). Similar results were obtained using humanapo- and holo-Tf (Fig. 8C and D). For comparison, we exam-ined binding of bovine Lf to Neisseria gonorrhoeae. The bindingpattern for this organism was the exact opposite of the patternfor H. pylori; apo-Lf was not able to compete with FITC-labeled holo-Lf, but unlabeled holo-Lf dramatically decreasedthe fluorescence of organisms incubated with FITC-labeledapo-Lf (Fig. 8E and F). Binding curves generated by compar-ing different concentrations of apo- and holo-Lf and differentconcentrations of apo- and holo-Tf also showed that H. pylorihad a clear preference for the apoproteins (data not shown).Thus, we concluded that H. pylori has a greater affinity forapo-Lf and apo-Tf, which is a pattern that differs substantiallyfrom the patterns for other pathogens.

Given the ability of apoproteins to inhibit binding of theiron-containing versions, we wondered whether apoproteinsare readily released from the receptors or remain bound, thusblocking subsequent binding of a holoprotein. In order toanswer this question, we incubated bacteria with 2 mg of un-labeled holo- or apo-Tf or -Lf in order to saturate receptors.After 1 h, the bacteria were pelleted and resuspended in freshbuffer containing 40 �g of FITC-labeled holo-Tf or holo-Lf.Bacteria were incubated for 1 h and formalin fixed prior toexamination by flow cytometry. The fluorescence was similarregardless of whether bacteria had been preincubated with noprotein, a holoprotein, or an apoprotein (data not shown).

Thus, there is no evidence that apoproteins are permanentlybound to the receptors.

Synthesis of receptors involved in iron uptake is typicallyiron regulated, and other workers have shown that H. pyloriproduces iron-repressible outer membrane proteins, althoughthe identities of the proteins were not determined (38). Inorder to explore iron regulation of Tf, Lf, and Hb receptors, wesplit a culture of strain 26695 into subcultures that were grownin iron-replete and iron-deficient media. After 18 h of incuba-tion, the abilities of the bacteria to bind FITC-labeled humanhemoglobin or human or bovine Tf or LF were evaluated byflow cytometry. In each case, the percentage of bacteria dis-playing fluorescence greater than the gating threshold washigher when bacteria were grown in iron-deficient medium(Fig. 9). These findings suggest that the receptors involved inacquisition of iron from host proteins are induced when theiron supply is limited.

DISCUSSION

H. pylori has hemolytic activity and has been found to adhereto red blood cells in capillaries in the lamina propria (3, 23).Although H. pylori has previously been reported to use hemo-globin for growth (12, 38), the methods used previously did notpermit the workers to prove that growth was not due to freeheme present as a contaminant or due to other heme-contain-ing complexes, such as heme-hemopexin or hemoglobin-hap-toglobin. By growing H. pylori in serum-free medium supple-mented with different concentrations of dialyzed hemoglobin,we showed that growth is not a result of free heme and thathaptoglobin is not required. We have not determined yetwhether heme can be extracted from hemopexin or from he-moglobin bound to haptoglobin. A recent report showed thatFrpB2 in strain J99, which is homologous to HP0915 in strain26695, allows E. coli BL21/pLysS to grow on hemoglobin (16).We plan to pursue this finding in future studies.

Since all hemoglobin-utilizing bacteria studied to date are alsoable to grow using free heme, the extreme toxicity of heme for H.pylori came as a surprise. Excess heme was not toxic to V. choleraeor uropathogenic E. coli when these organisms were grown in thesame medium. These data led us to suspect that uptake of hemefrom hemoglobin in H. pylori is tightly regulated but that freeheme can enter via a transporter that is not iron regulated, lead-ing to iron toxicity. We believe that the toxicity observed at higherhemoglobin concentrations is the result of hemoglobin degrada-tion and concomitant release of heme. Indeed, the hemoglobinconcentration required to cause toxicity is different for differentbatches of dissolved hemoglobin and increases as solutions age.Attempts to isolate heme-resistant mutants from a transposonlibrary have been unsuccessful so far, suggesting that the hemeuptake mechanism used by H. pylori could have another essentialrole or that multiple transport pathways are involved. Althoughsensitivity to free heme seems to be a disadvantage for a heme-utilizing organism, it is unlikely that H. pylori encounters toxic freeheme concentrations in vivo. Heme that is released by hemoglo-bin can be rapidly bound by plasma albumin or hemopexin, andthe level of the heme released by epithelial cells is not likely to betoxic.

A number of pathogens can use Tf or Lf as an iron source.Both of these proteins are found in blood, although the concen-

FIG. 7. Percentages of bacteria displaying levels of fluorescencegreater than the background level following incubation with differentamounts of protein. Bacteria were incubated with FITC-labeled apo-transferrin or apo-lactoferrin at concentrations ranging from 1 to 150�g/ml. Gating was set to exclude 99.5% of the unstained population.hTf, human Tf; bLf, bovine Lf.

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trations of Lf are higher in mucus and other bodily secretions(30). Lactoferrin is secreted into gastric mucus, and this proteinhas been suggested to be important for H. pylori growth in vivo(18). Organisms lacking siderophores must have specific Tf or Lfreceptors in order to extract iron from these proteins. For exam-ple, several pathogenic Neisseria species have specific receptors

for Tf and Lf (8), and Haemophilus influenzae can use Tf but notLf (17). V. cholerae has been shown to bind Lf and, to a lesserextent, Tf (2). To date, there has not been a report of a receptorcapable of binding both Lf and Tf, in spite of the significant aminoacid homology (60% identity and 72% similarity) of these twoproteins. Therefore, it was surprising that Lf could compete with

FIG. 8. Competition between ferri-Tf or ferri-Lf and apo-Tf or apo-Lf. H. pylori strain 26695m (A to D) or N. gonorrhoeae (E and F) wasincubated with FITC-labeled protein in the presence or absence of excess unlabeled protein. The fluorescence of bacteria was measured by flowcytometry. (A) FITC–ferri-Lf with or without a 50-fold excess of unlabeled apo-Lf. (B) FITC–apo-Lf with or without a 50-fold excess of unlabeledferri-Lf. (C) FITC–ferri-Tf with or without a 50-fold excess of unlabeled apo-Tf. (D) FITC–apo-Tf with or without a 50-fold excess of unlabeledferri-Tf. (E) N. gonorrhoeae incubated with FITC–ferri-Lf with or without a 50-fold excess of unlabeled apo-Lf. (F) N. gonorrhoeae incubated withFITC–apo-Lf with or without a 50-fold excess of unlabeled ferri-Lf. hTf, human Tf; bLf, bovine Lf.

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Tf for binding to H. pylori. Our data are consistent with thepresence of more than one H. pylori Lf receptor.

We showed that the utilization of Tf and Lf by H. pylori isnot species specific, contrary to a previous report (18). Thepreviously reported inability of H. pylori to use bovine Lf led toa number of clinical trials in which Lf was used as a treatmentadjuvant with the hope of starving H. pylori for iron. Ourfinding that H. pylori is able to utilize bovine holo-Lf mayexplain some of the disappointing results of clinical trials em-ploying Lf as a treatment adjuvant (40, 41). A study comparingtreatment of H. pylori-infected mice with bovine Lf and treat-ment of H. pylori-infected mice with human Lf concluded thatneither protein reduced the bacterial load, and both groups ofmice had higher levels of inflammation (based on myeloper-oxidase levels) than controls (19). Our results suggest thatbovine Lf inhibits H. pylori growth only if it is ingested in theabsence of dietary iron that could be bound by the Lf. Withsufficient iron intake, bovine Lf could augment rather thaninhibit H. pylori growth, and ingested dairy products could alsoserve as an iron source. Although the Lf in dairy products isprobably not completely iron saturated, it could absorb ironreleased from other foods as iron becomes solubilized by gas-tric acid. Transferrin from meat could also be a utilizable ironsource under the right conditions. Several other pathogenshave been shown to bind Tf from the host species but not Tffrom nonhost species, suggesting that iron acquisition mecha-nisms can contribute to host tropism (4, 29). In contrast, wehave found no evidence that iron acquisition contributes tohost tropism in H. pylori.

It has been reported that Lf has bactericidal activity forsome microorganisms in addition to its ability to sequester iron(9, 13), but we have found no evidence that apo-Lf kills ordamages H. pylori. The bacteria maintain the bacillary shapeand do not die following exposure to apo-Lf; their growth ismerely suppressed by iron limitation.

H. pylori also appears to be unique in its preference forbinding apo-Tf and apo-Lf. Several other pathogens, such asNeisseria meningitidis and Moraxella catarrhalis, specifically

bind holo-Tf (5, 31). We are not aware of any pathogen thatpreferentially binds apoproteins. This pattern of host iron uti-lization could explain why examination of predicted H. pyloriprotein sequences has not revealed homology with known Tf-or Lf-binding proteins from other organisms. Since otherpathogens that have been studied either require specific ferri-proteins or show no preference, it is reasonable to speculatethat receptors of these organisms recognize different aminoacid residues of Tf and Lf.

Much has been made of the ability of H. pylori to causesevere diseases, such as ulcers and gastric cancers, but it isperhaps more remarkable that in the vast majority of cases H.pylori is able to coexist with its host for decades while causingonly asymptomatic inflammation. Many otherwise innocuousorganisms, such as Staphylococcus epidermidis and Serratiamarcescens, can cause severe invasive disease in immunocom-promised individuals, yet H. pylori sepsis has never been re-ported. It is therefore possible that H. pylori has evolved mech-anisms to avoid excessive tissue destruction in most cases.Decreased pathogenic potential is a distinct advantage for anorganism that colonizes its host for life. Our data suggest thatH. pylori limits its pathogenicity by binding apo-Tf and apo-Lfmore strongly than the iron-containing forms, thus preventinggrowth in the bloodstream. This characteristic may also mod-erate competition with the host for iron. Although the pre-ponderance of evidence suggests that there is an associationbetween H. pylori and iron-deficiency anemia (27), moreaggressive bacterial iron acquisition strategies might makeanemia a more dominant symptom of H. pylori infection.

The model shown in Fig. 10 suggests that H. pylori couldeasily acquire iron in the upper regions of the mucous layer butthat growth in gastric crypts would be exceedingly difficult dueto the prevalence of apo-LF. Since H. pylori is frequently at-tached to the epithelial surface and is present in crypts, it islikely able to acquire iron in this milieu. We have preliminarydata suggesting that host epithelial cells do indeed improve theability of H. pylori to grow in iron-limited media containingapo-Tf. This will be the topic of future investigations.

In summary, our studies demonstrate that H. pylori uses ironacquisition strategies analogous to skimming cream from the toprather than stealing the whole milk can. Preferential binding ofapoproteins ensures that the host does not suffer excessive iron

FIG. 9. Effect of iron restriction on binding of H. pylori to host iron-containing proteins. Binding of FITC-labeled Lf, Tf, and Hb (40 �g/ml) towild-type strain 26695m was measured by flow cytometry following growthin iron-replete medium (F-12 with 1% FBS) or iron-restricted medium(TT18 with 1 �M DFO) for 18 h. The data are the percentages of thefluorescent population (P2) in the total population (50,000 events). Fe�,iron-replete medium; Fe�, iron-restricted medium; hTf, human Tf; hLf,human Lf; bTf, bovine Tf; bLf, bovine Lf; hHb, human Hb.

FIG. 10. Free iron in the gastric mucosa. Lf, lactoferrin; Tf, trans-ferrin; Hb, hemoglobin. Blue hexagons indicate iron-free Lf; red hexagonsindicate iron-saturated Lf; curved blue bars indicate H. pylori cells.

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deprivation and prevents disseminated infection. Although the invivo consequences of heme toxicity are unclear, heme toxicitycould serve as a disincentive for acquisition of destructive extra-cellular proteases. H. pylori has coevolved with humans for mil-lennia, and its iron acquisition strategies are further evidencesuggesting that there has been an evolutionary path toward com-mensalism rather than toward pathogenesis; however, it is stillunclear whether certain H. pylori strains are truly benign.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grant K22AI063307-01 from the National Institutes of Health and by a Bridgeaward from LSU Health Sciences Center.

We thank Dalton Gossett for performing atomic absorption spec-troscopy and Robert Perry for advice concerning iron removal. We aregrateful to Shannon Mumphrey, Rob Chervenak, and Deborah Cher-venak for their assistance with flow cytometry.

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Editor: S. M. Payne

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