Acinetobacter baumannii - Journal of Bacteriology - American

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JOURNAL OF BACTERIOLOGY, Dec. 1992, p. 7670-7679 Vol. 174, No. 23 0021-9193/92/237670-10$02.00/0 Copyright ) 1992, American Society for Microbiology Characterization of a High-Affinity Iron Transport System in Acinetobacter baumannii JOSE R. ECHENIQUE,1 HECTOR ARIENTI,1 MARCELO E. TOLMASKY,2t RON R. READ,3 ROBERTO J. STANELONI,2 JORGE H. CROSA,3 AND LUIS A. AC11S1t* Departamento de Bioquimica Clinica, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Cordoba,1 and Instituto de Investigaciones Bioquimicas "Fundacion Campomar, " Buenos Aires 2 Argentina, and Department of Microbiology and Immunology, Oregon Health Sciences University, Portland, Oregon 97201-3o983 Received 6 April 1992/Accepted 30 September 1992 Analysis of a clinical isolate of Acinetobacter baumannii showed that this bacterium was able to grow under iron-limiting conditions, using chemically defined growth media containing different iron chelators such as human transferrin, ethylenediaminedi-(o-hydroxyphenyl)acetic acid, nitrilotriacetic acid, and 2,2'-bipyridyl. This iron uptake-proficient phenotype was due to the synthesis and secretion of a catechol-type siderophore compound. Utilization bioassays using the SalmoneUla typhimurium iron uptake mutants enb-1 and enb-7 proved that this siderophore is different from enterobactin. This catechol siderophore was partially purified from culture supernatants by adsorption chromatography using an XAD-7 resin. The purified component exhibited a chromatographic behavior and a UV-visible light absorption spectrum different from those of 2,3-dihydroxybenzoic acid and other bacterial catechol siderophores. Furthermore, the siderophore activity of this extraceliular catechol was confirmed by its ability to stimulate energy-dependent uptake of 55Fe(M) as well as to promote the growth of A. baumannii bacterial cells under iron-deficient conditions imposed by 60 ,uM human transferrin. Polyacrylamide gel electrophoresis analysis showed the presence of iron-regulated proteins in both inner and outer membranes of this clinical isolate of A. baumannii. Some of these membrane proteins may be involved in the recognition and internalization of the iron-siderophore complexes. Acinetobacter baumannii, formerly known asAcinetobac- ter calcoaceticus subsp. anitratus (15), belongs to a bacterial species that is widely distributed throughout the environ- ment (6). This microorganism can be isolated from the skin (3, 43) and respiratory tract (59) of healthy ambulatory adults as well as from hospital personnel and equipment (36, 39, 46, 67). Lately, numerous outbreaks of nosocomial infections caused by A. baumannii have been reported (7-10, 16, 42, 47, 56, 57, 73), which are of particular concern because of the widespread and increasing antibiotic resistance of the isolated strains (17, 30, 32, 33, 40, 41, 48). Nevertheless, most infections have occurred in patients compromised by either antibiotic therapy, respiratory instrumentation and manipulations, dialysis, or surgery (18, 19, 39, 70). Thus, it was proposed that reduced host defenses and interaction with other bacterial species, rather than the expression of specific bacterial virulence factors (49), are the factors responsible for opportunistic infections caused by Acineto- bacter species. Acinetobacter species are able to survive under restricted nutrient conditions, such as those imposed by the host. Iron is one of the essential bacterial nutrients that is tightly controlled by the host, through its chelation by the high- affinity binding glycoproteins transferrin (TF) and lactoferrin (23). Thus, bacteria have developed efficient iron uptake mechanisms that allow them to scavenge iron from these host proteins, either by direct interaction with the iron- protein complexes (34) or by the synthesis and secretion of * Corresponding author. t Present address: Department of Microbiology and Immunology L220, School of Medicine, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97201-3098. high-affinity extracellular siderophore compounds (23). It was recently reported that clinical isolates of A. baumannii produce 2,3-dihydroxybenzoic acid (DHBA) when grown in a chemically defined medium and can utilize Fe-DHBA complexes to grow in the presence of the iron chelator 2,2'-bipyridyl (DIP) (66). In this report, we describe the isolation and preliminary characterization of an iron-regulated catechol compound capable of promoting the uptake of 55Fe(III) as well as the growth of A. baumannii in the presence of human TF. MATERIALS AND METHODS Bacterial strains and plasmids. The clinical strain A. bau- mannii 8399 was isolated during a nosocomial outbreak of respiratory tract infections (39). Vibrio anguillarum 531A (69) was used as source of anguibactin, a catechol-type siderophore different from enterobactin (1). The Escherichia coli LG1522 mutant, which is deficient in aerobactin synthe- sis but carries the receptor for this siderophore, was used as the indicator strain to detect aerobactin production (72). Salmonella typhimurium mutants enb-1, which can use only enterobactin, and enb-7, which can use enterobactin as well as DHBA (55) to grow under iron-deficient conditions, were used as indicator strains to detect the production of entero- bactin and DHBA, respectively. Plasmids pCP111 (20) and pCP410 (54) were used as enterobactin probes. Plasmid pABN5 (11) was used as the aerobactin probe. Growth conditions. Trypticase soy agar and broth, L agar and broth, M9 minimal medium (22), and Fe-CDM chemi- cally defined medium (66) were used to culture the bacterial strains. The iron chelators human TF, nitrilotriacetic acid (NTA), DIP, and ethylenediaminedi-(o-hydroxyphenyl)ace- tic acid (EDDA; Sigma Chemical Co., St. Louis, Mo.) were 7670 Downloaded from https://journals.asm.org/journal/jb on 16 December 2021 by 191.240.119.209.

Transcript of Acinetobacter baumannii - Journal of Bacteriology - American

Page 1: Acinetobacter baumannii - Journal of Bacteriology - American

JOURNAL OF BACTERIOLOGY, Dec. 1992, p. 7670-7679 Vol. 174, No. 230021-9193/92/237670-10$02.00/0Copyright ) 1992, American Society for Microbiology

Characterization of a High-Affinity Iron Transport System inAcinetobacter baumannii

JOSE R. ECHENIQUE,1 HECTOR ARIENTI,1 MARCELO E. TOLMASKY,2t RON R. READ,3ROBERTO J. STANELONI,2 JORGE H. CROSA,3 AND LUIS A. AC11S1t*

Departamento de Bioquimica Clinica, Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba,Cordoba,1 and Instituto de Investigaciones Bioquimicas "Fundacion Campomar, " Buenos Aires 2

Argentina, and Department ofMicrobiology and Immunology, Oregon HealthSciences University, Portland, Oregon 97201-3o983

Received 6 April 1992/Accepted 30 September 1992

Analysis of a clinical isolate ofAcinetobacter baumannii showed that this bacterium was able to grow underiron-limiting conditions, using chemically defined growth media containing different iron chelators such ashuman transferrin, ethylenediaminedi-(o-hydroxyphenyl)acetic acid, nitrilotriacetic acid, and 2,2'-bipyridyl.This iron uptake-proficient phenotype was due to the synthesis and secretion of a catechol-type siderophorecompound. Utilization bioassays using the SalmoneUla typhimurium iron uptake mutants enb-1 and enb-7proved that this siderophore is different from enterobactin. This catechol siderophore was partially purifiedfrom culture supernatants by adsorption chromatography using an XAD-7 resin. The purified componentexhibited a chromatographic behavior and a UV-visible light absorption spectrum different from those of2,3-dihydroxybenzoic acid and other bacterial catechol siderophores. Furthermore, the siderophore activity ofthis extraceliular catechol was confirmed by its ability to stimulate energy-dependent uptake of55Fe(M) as wellas to promote the growth ofA. baumannii bacterial cells under iron-deficient conditions imposed by 60 ,uMhuman transferrin. Polyacrylamide gel electrophoresis analysis showed the presence of iron-regulated proteinsin both inner and outer membranes of this clinical isolate ofA. baumannii. Some of these membrane proteinsmay be involved in the recognition and internalization of the iron-siderophore complexes.

Acinetobacter baumannii, formerly known asAcinetobac-ter calcoaceticus subsp. anitratus (15), belongs to a bacterialspecies that is widely distributed throughout the environ-ment (6). This microorganism can be isolated from the skin(3, 43) and respiratory tract (59) of healthy ambulatory adultsas well as from hospital personnel and equipment (36, 39, 46,67).

Lately, numerous outbreaks of nosocomial infectionscaused by A. baumannii have been reported (7-10, 16, 42,47, 56, 57, 73), which are of particular concern because ofthe widespread and increasing antibiotic resistance of theisolated strains (17, 30, 32, 33, 40, 41, 48). Nevertheless,most infections have occurred in patients compromised byeither antibiotic therapy, respiratory instrumentation andmanipulations, dialysis, or surgery (18, 19, 39, 70). Thus, itwas proposed that reduced host defenses and interactionwith other bacterial species, rather than the expression ofspecific bacterial virulence factors (49), are the factorsresponsible for opportunistic infections caused by Acineto-bacter species.Acinetobacter species are able to survive under restricted

nutrient conditions, such as those imposed by the host. Ironis one of the essential bacterial nutrients that is tightlycontrolled by the host, through its chelation by the high-affinity binding glycoproteins transferrin (TF) and lactoferrin(23). Thus, bacteria have developed efficient iron uptakemechanisms that allow them to scavenge iron from thesehost proteins, either by direct interaction with the iron-protein complexes (34) or by the synthesis and secretion of

* Corresponding author.t Present address: Department of Microbiology and Immunology

L220, School of Medicine, Oregon Health Sciences University, 3181S.W. Sam Jackson Park Road, Portland, OR 97201-3098.

high-affinity extracellular siderophore compounds (23). Itwas recently reported that clinical isolates ofA. baumanniiproduce 2,3-dihydroxybenzoic acid (DHBA) when grown ina chemically defined medium and can utilize Fe-DHBAcomplexes to grow in the presence of the iron chelator2,2'-bipyridyl (DIP) (66).

In this report, we describe the isolation and preliminarycharacterization of an iron-regulated catechol compoundcapable of promoting the uptake of 55Fe(III) as well as thegrowth ofA. baumannii in the presence of human TF.

MATERIALS AND METHODS

Bacterial strains and plasmids. The clinical strain A. bau-mannii 8399 was isolated during a nosocomial outbreak ofrespiratory tract infections (39). Vibrio anguillarum 531A(69) was used as source of anguibactin, a catechol-typesiderophore different from enterobactin (1). The Escherichiacoli LG1522 mutant, which is deficient in aerobactin synthe-sis but carries the receptor for this siderophore, was used asthe indicator strain to detect aerobactin production (72).Salmonella typhimurium mutants enb-1, which can use onlyenterobactin, and enb-7, which can use enterobactin as wellas DHBA (55) to grow under iron-deficient conditions, wereused as indicator strains to detect the production of entero-bactin and DHBA, respectively. Plasmids pCP111 (20) andpCP410 (54) were used as enterobactin probes. PlasmidpABN5 (11) was used as the aerobactin probe.Growth conditions. Trypticase soy agar and broth, L agar

and broth, M9 minimal medium (22), and Fe-CDM chemi-cally defined medium (66) were used to culture the bacterialstrains. The iron chelators human TF, nitrilotriacetic acid(NTA), DIP, and ethylenediaminedi-(o-hydroxyphenyl)ace-tic acid (EDDA; Sigma Chemical Co., St. Louis, Mo.) were

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added to the media to achieve iron-limiting conditions. TFwas prepared as a 1 mM stock solution in 100 mM Tris-HCl(pH 7.5)-0.15 M NaCl-50 mM NaHCO3. The MICs for theiron chelators were determined in either liquid or solid mediacontaining increasing concentrations of each iron-chelatingcompound. Iron-proficient conditions were obtained by add-ing 50 p.M FeCl3 in 0.5 M HCl. The inocula used for growthand "Fe(III) uptake studies as well as for siderophorepurification were prepared by using cells iron starved byculturing in M9 or Fe-CDM medium containing 50 p.MEDDA or DIP.

Cloacin sensitivity and TF binding assays. Cloacin was

prepared from Enterobacter cloacae DF13 as previouslydescribed (71). Purified cloacin was spotted onto L agar

plates containing a lawn of the bacterial strain to be testedand increasing concentrations of EDDA. Bacterial growthwas recorded after overnight incubation at 37°C. E. coliLG1315 (72) was used as a positive control.The ability of bacteria to bind TF directly was assayed by

the method of Schryvers and Morris (61), using a horserad-ish peroxidase (HRP) conjugate (Pierce, Rockford, Ill.). Aclinical isolate of Haemophilus influenzae was used as a

positive control.Siderophore production. The production of extracellular

compounds with siderophore activity was investigated byusing the method of Schwyn and Neilands (62). The pres-

ence of phenolic or hydroxamate extracellular compoundswas detected in culture supernatants with the Arnow phe-nolic acid assay (4) or the Csaky hydroxylamine/hydroxamicacid assay (27), respectively. Catechol siderophores were

extracted with ethyl acetate from iron-deficient culture su-

pernatants adjusted to pH 1.5 with HCl (58). The ethylacetate samples were dried and dissolved in either ethanol or

phosphate-buffered saline. Hydroxamate siderophores were

extracted with chloroform-phenol from culture supernatantsadjusted to pH 7.0 as described by Simpson and Oliver (64).

Siderophore bioassays. Siderophore activity was deter-mined in liquid or solid bioassays by testing the ability ofcell-free culture supernatants to promote growth of bacterialstrains under iron-limiting conditions (2). Aerobactin synthe-sis was determined as previously described (26), using E.coli LG1522 as the indicator strain. The presence of entero-bactin and DHBA was examined by plate assays, using theS. typhimurium enb-1 and enb-7 mutants, as previouslydescribed (44). The purified siderophores enterobactin,amonabactin, and pyochelin and pyoverdin were generouslyprovided by J. B. Neilands, B. R. Byers, and C. Cox,respectively.

Radioactive iron uptake. A protocol similar to that de-scribed for V. anguillarum (2) was used. Briefly,A. bauman-nii 8399 was grown at 37°C for 12 h in 30 ml of M9 containing50 p.M EDDA. The cells were washed and suspended in thesame volume of M9 medium containing only CasaminoAcids and 50 p.M EDDA. After incubation for 1 h, the cellswere collected, washed, resuspended in M9 medium withoutCasamino Acids and containing 100 pM NTA, and furtherincubated for 1 h at 37°C. Cell concentration was adjusted toan A6. of 0.4 with the same medium. The 15Fe(III)-sidero-phore complex was prepared by incubating the purifiedAcinetobacter siderophore with 55FeC13 (specific activity,62.10 mCi/mg; NEN Research Products, DuPont Co., Bos-ton, Mass.) at room temperature for 10 min in M9 salts. Thefinal concentration of 55FeC13 in the uptake reaction was 0.13p.Ci/ml. Since the structure and composition of the Acineto-bacter siderophore are not known, its concentration was

determined as DHBA equivalents by the Arnow test, using 1

mM DHBA as the standard solution. The final concentrationof the Acinetobacter siderophore in the uptake mixtures, asDHBA equivalents, was 12 p,M. The uptake assays wereinitiated by the addition of 55FeCl3 or 55Fe(III)-siderophoreto the cell suspensions. Samples of 1 ml were removed after5 and 10 min of incubation at 37°C and immediately filteredthrough 0.45-p,m-pore-size HA filters (Millipore Corp., SanFrancisco, Calif.) in a vacuum-drawn manifold. The filterswere washed with 5 ml of 0.1 M sodium citrate. The amountof 5"Fe(III) retained on the filters was measured by liquidscintillation counting.

Cells suspensions were preincubated for 30 min at 37°Cwith either 20 mM potassium cyanate (KCN) or 1 mMcarbonyl m-chlorophenylhydrazone (CCCP5) (Sigma) to ex-amine whether the A. baumannii 8399 5Fe(III) uptakesystem requires cell energy.

Siderophore purification. The extracellular siderophore(s)secreted by A. baumannii 8399 was isolated from iron-deficient culture supernatants by adsorption chromatogra-phy onto XAD-7 resin (1). Cells were grown for 16 h at 37°Cin minimal medium. A 4-liter volume of culture supernatant,adjusted to neutral pH, was applied to an XAD-7 column (2.5by 17 cm). The column was washed with 2 volumes ofdeionized water, and the adsorbed material was eluted withpure methanol. Chromatographic fractions containing cate-chol and siderophore compounds were identified by usingthe Arnow and Chrome azurol S (CAS) reactions, respec-tively. The peak column fractions containing the iron-bind-ing activity were concentrated by evaporation at reducedpressure and kept at 4°C protected from the light.TLC and paper chromatography. Thin-layer chromatogra-

phy (TLC) was performed on 0.2-mm precoated Silica Gel 60F-254 TLC sheets (E. Merk, Elmsford, N.Y.), using one ofthe following solvent systems: chloroform-methanol (2:1),butanol-acetic acid-water (60:15:25), benzene-acetic acid-water (125:72:3), and butanol-pyridine-water (14:3:3). Paperchromatography was performed on 3MM chromatographypaper (Whatman Ltd., Maidstone, England), using one ofthe following solvent systems: chloroform-methanol (2:1),chloroform-methanol-water (10:20:3), and butanol-acetic ac-id-water (60:15:25). The chromatograms were examinedunder UV light, and iron-binding compounds were detectedby spraying either the CAS reagent or 0.1 M FeCl3 in 0.1 MHCl. Catechol compounds were detected by spraying thereagents of the Arnow test.Mass spectrometry. Fast atom bombardment spectra were

obtained on a Kratos MS5OTC mass spectrometer at theDepartment of Agricultural and Life Sciences, Oregon StateUniversity. Samples were analyzed in either 3-nitrobenzoicacid or dithioerythritol-dithiothreitol matrixes.DNA techniques. Total DNA from the A. baumannii 8399

was obtained by the method described by Meade et al. (45).Plasmid DNA was isolated from E. coli HB101 (13) by themethod of Birnboim and Doly (12) and further purified bycentrifugation in CsCl-ethidium bromide density gradients(60). Southern blot hybridizations were performed as de-scribed previously (68). DNA probes were obtained by theoligolabeling method described by Feinberg and Vogelstein(31), using [a-32P]dATP. Electrophoresis of DNA restrictionfragments was performed in horizontal slab gels as previ-ously described (25). Restriction enzymes were used asdescribed by the supplier (Bethesda Research Laboratories,Gaithersburg, Md.). DNA restriction fragments were iso-lated from agarose gels by using Geneclean (Bio 101, LaJolla, Calif.).

Electrophoretic analysis of membrane proteins. Overnight

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TABLE 1. Cell growth and siderophore production byA. baumannii 8399 under iron-deficient and

iron-proficient conditions

Compound Concn Cell Colorimetric reactionin medium (PM) growta CASb Arnowc Csakyd

TF 30 + + + NDeDIP 120 + + + NDEDDA 800 + + + -NTA 800 + + + NDFeCl3 50 + - ND

a Determined spectrophotometrically at 600 nm. Optical densities were>1.0 after overnight incubation.b A color change from blue to pink-orange was considered a positive

reaction.c See reference 4.d See reference 27.e ND, not determined.

bacterial cultures were washed by centrifugation, resus-pended in 10 mM Tris-HCl (pH 8.0) containing 0.3% NaCl,and sonically disrupted at 4°C. Total cell envelopes wereprepared as described previously (24), and the outer mem-brane fractions were obtained by differential solubilization ofthe cell envelopes, using 1.5% Sarkosyl. Total and outermembrane proteins (100 ,ug) were examined by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), using 12.5% separating gels (2). After electrophore-sis (30 mA for 7 h), the gels were stained with Coomassieblue and then destained with 5% acetic acid. Protein con-centrations were determined as described by Bradford (14).

RESULTSBacterial growth under iron-limiting conditions. The initial

experiments showed that the clinical isolate A. baumannii8399, obtained during a nosocomial outbreak of lower respi-ratory tract infections, was able to grow in M9 minimalmedium containing either 50 p,M EDDA or 10 ,uM TF,suggesting that this strain was iron uptake proficient. Deter-mination of the MICs revealed that this isolate can grow inthe presence of 30 ,uM TF as well as in M9 mediumcontaining up to 800 p,M EDDA or NTA or up to 120 ,uMDIP (Table 1). These results indicated that this bacterialstrain acquires iron from chelated medium, either throughdirect interaction with ferrated TF or through an efficientiron uptake system that may involve an extracellular sidero-phore.HRP-TF binding assays showed that iron-starved A. bau-

mannii 8399 cells did not bind the labeled human TF (Fig. 1).Conversely, the H. influenzae strain used as a positivecontrol bound TF under the same experimental conditions.Therefore, it seems that the direct interaction between A.baumannii cells and human HRP-labeled TF is not requiredto scavenge the iron from the metal-protein complexes.The production of extracellular siderophore(s) was then

investigated by using the CAS reagent as described bySchwyn and Neilands (62). The appearance of orange halosaround the bacterial colonies on CAS-agar plates revealedthe production and secretion of an iron chelator compound(data not shown). Furthermore, the addition of iron-limited,but not iron-rich, culture supernatants to the CAS reagentproduced a color change of the reagent from blue to orange(Table 1). These results strongly suggested thatA. bauman-nii 8399 excreted a siderophore compound into the iron-limited culture medium.

1

2

3

4FIG. 1. Binding of HRP-labeled human TF by H. influenzae (1)

and A. baumannii 8399 cells grown in M9 minimal medium contain-ing either 50 ,uM FeCl3 (2), 50 ,uM EDDA (3), or 100 F.M EDDA (4).

Characterization of the A. baumannii siderophore. Thechemical nature of the siderophore was determined by usingthe Arnow (4) and Csaky (27) colorimetric reactions. Cate-chol compounds were detected in iron-deficient culturesupernatants with the Arnow test, while no reaction wasobserved with the Csaky test (Table 1), indicating that nohydroxamate compounds were present in the medium. Theuninoculated culture medium gave negative results with bothcolorimetric tests. To confirm these results, A. baumannii8399 iron-chelated culture supernatants were treated withethyl acetate or phenol-chloroform, to extract and concen-trate either catechol or hydroxamate siderophores, respec-tively. The ethyl acetate extracts were positive for cate-chols, while the phenol-chloroform extracts remainedhydroxamate negative. We also investigated at the biologicaland genetic levels the ability of A. baumannii 8399 toproduce aerobactin. Biologically, the presence of this hy-droxamate siderophore was ruled out since utilization bio-assays capable of detecting it were negative. Furthermore,the cloacin sensitivity test, which can detect the presence ofthe aerobactin receptor in bacterial membranes, showed thatA. baumannii 8399 was cloacin resistant, suggesting theabsence of this outer membrane receptor. Genetically, nohomology was detected between A. baumannii 8399 totalDNA and the aerobactin genes probe pABN5 by DNAhybridization under stringent conditions (data not shown).Thus, only the production of a catechol siderophore(s) couldbe detected in culture supernatants. This extracellular cate-chol(s) was detected in the culture medium of cells grown atbetween 30 and 42°C, with the highest levels after overnightculture at 37°C (Fig. 2a). Figure 2b shows thatA. baumannii8399 cells grew properly under iron-deficient conditions,although they exhibited an increased generation time and areduced growth yield compared with bacteria cultured iniron-proficient conditions (Fig. 2c). The secretion of cate-chol(s) paralleled the bacterial growth in iron-deficient M9minimal medium, reaching the maximum after 24 h ofincubation (Fig. 2b), whereas the amount of this com-pound(s) in iron-rich medium was below the detectablelevels of the Arnow test (Fig. 2c).

Siderophore utilization bioassays showed that iron-defi-cient culture supernatants of this bacterium were able toreverse the EDDA inhibitory effect and enhance the growthof the S. typhimurium enb-7 mutant (Table 2), an ironuptake-deficient mutant that can utilize DHBA as a precur-

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a

,lMM EDDA'

b

E

0

10~ 1.0

-~~~~~~~~~~U-

0.5

.1

.01 0.00 1 0 20 30

HOURS

c

0 10

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20 30

FIG. 2. (a) Synthesis and secretion of catechol compounds inculture supernatants ofA. baumannii 8399 grown at 32, 37, or 42°Cin M9 minimal medium containing different amounts of EDDA; (band c) growth (optical density at 600 nm [OD 600 nm]) and catecholsynthesis (optical density at 510 nm) by A. baumannui 8399 in thepresence of either 100 ,uM EDDA (b) or 50 FM FeCl3 (c). Cells werecultured 24 h in M9 minimal medium at 37C with constant shaking.Catechol compounds were detected by the method of Arnow (4).

TABLE 2. Siderophore utilization bioassays

S. tphimwium growth halos (mm)bMaterial tested0

enb-1 enb-7

FeCl3 14 15Enterobactin 12 13V. anguillarum 531A 0 10A. baumannu 8399 0 7

a Samples of sterile culture supernatants from either V. anguillanan 531Aor A. baumannii 8399 were added to filter disks and placed on top of solidmedium. FeCl3 and enterobactin were used as positive controls.

b M9 minimal medium agar plates containing 50 ,M EDDA were seededwith S. typhimwium enb-1 orenb-7 iron uptake mutant cells. Diameters of thegrowth halos were recorded after 16 h of incubation at 37C.

sor to produce enterobactin (55). A similar effect was de-tected in iron-deficient culture supernatants of V. anguil-larum 531A, a bacterial strain that also produces DHBAtogether with anguibactin, a catechol siderophore unrelatedto enterobactin (2, 69). Conversely, the V. anguillarum 531AandA. baumannii 8399 culture supernatants were not able topromote the growth of the S. typhimurium enb-1 mutant(Table 2), an iron uptake mutant that requires the addition ofenterobactin to grow under iron limitation (55). Thus, DHBAbut not enterobactin is secreted into the growth medium byA. baumannii 8399. This observation was further supportedby the fact that the enterobactin genes could not be detectedin the genome of this bacterium by DNA hybridizationanalysis (data not shown), using as labeled probes plasmidspCP111 and pCP410, which harbor the enterobactin genesfepC, fepB, and entF (pCP111) and entA, entC, !entG, entB,and entE (pCP410) (20, 54).

Isolation of theA. baumannii siderophore. The facts thatA.baumannii 8399 is able to grow in the presence of highconcentrations of TF and secretes an iron-regulated com-pound(s) reacting with the CAS reagent suggest that thisbacterium produces, besides DHBA, an extracellular sidero-phore that is not related to enterobactin. To prove thishypothesis, iron-deficient culture supernatants were sub-jected to adsorption chromatography on XAD-7 resin. Cul-ture supernatants adjusted to pH 7.0, to avoid the coadsorp-tion of low-molecular-weight organic acids (DHBA amongthem) (1), were applied to a chromatographic column. Apeak of catechol compound(s), with very low siderophoreactivity as determined with the CAS reagent, was elutedwhen the column was washed with 2 volumes of deionizedwater (Fig. 3). Elution of the XAD-7 column with puremethanol yielded a well-defined catechol peak that coelutedwith a very significant peak of siderophore activity (Fig. 3).The same elution profiles were obtained with use of eitherM9 or Fe-CDM minimal medium, although the latter gaveclearer results due to the utilization of arginine, aspartate,proline, and glutamic acid instead of Casamino Acids, thusavoiding the pigments that are normally present in thecommercial Casamino Acids preparations and that are alsoretained in the XAD-7 column (data not shown).

Analysis by TLC of the pooled peak fractions eluted withpure methanol revealed a single component by all visualiza-tion methods. This iron-binding catechol component mi-grated very poorly on the silica plates, showing R valuesdifferent from those of DHBA and enterobactin (Table 3).Similar results were obtained by paper chromatography,although two components with R1 of 0.06 and 0.44 weredetected by using the butanol-acetic acid-water solventsystem.

0

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84La

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7674 ECHENIQUE ET AL.

0.8

o.6fz

4

0o.4 -

dH20 1MErOH

0.2-

0.00 1 0 20 30 40 50

FRACTIONS

FIG. 3. XAD-7 column chromatography of A. baumannii 8399culture supernatant. Deionized water (dH20) and pure methanol(METOH) elution steps are indicated by the arrows. Elution ofcatechol(s) was monitored with the Arnow reaction (510 nm) (4).Siderophore activity of chromatographic fractions was determinedwith the CAS reagent (630 nm) (62). The decrease in absorbance ofthe CAS reagent is inversely related to the siderophore activitypresent in each sample. Therefore, the activity of each fraction isrepresented as the inverse of the decrease ofA630 with respect to theabsorbance of the blank reaction (minimal medium plus CASreagent). The siderophore activity could not be determined on all ofthe catechol-containing fractions eluted with pure methanol becauseof interference by this organic solvent.

UV-visible light (VIS) spectrum analysis also showed thatthe purified catechol is different from DHBA (Fig. 4A).Similar spectra were obtained whether the purified catecholwas dissolved in methanol or aqueous solution (Fig. 4A).The UV-VIS spectrum of this catechol compound was

further compared with the spectra described for other bac-terial catechol siderophores, using the experimental condi-tions described for analysis of the latter. The spectrum of theAcinetobacter catechol in aqueous solution at pH 7.5 shownin Fig. 4A is different from those reported for anguibactin,the siderophore isolated from V. anguillarum 775, whichexhibits absorption maxima at 214, 260, 315, and 410 nm (1),and pseudobactin 589A, an iron-chelating agent isolatedfrom Pseudomonas putida 589, which has a maximum ab-sorption peak at 403 nm (53). In addition, the spectra ofenterobactin and the Acinetobacter catechol were differentwhen both iron-chelating compounds were dissolved inmethanol (data not shown). Figure 4B shows that the spec-trum of theAcinetobacter catechol dissolved in HCl-ethanolis different from those described for agrobactin and vibrio-

bactin, the siderophores isolated from Agrobacterium tume-faciens (50) and Vibrio cholerae (35), respectively. In addi-tion, the isolated catechol did not show the broad peak at 316nm and the sharp peak at 252 nm described for agrobactinsolubilized in ethanol (50) (data not shown). The absorptionspectrum of the Acinetobacter catechol at pH 6.5 in 0.1 Mmorpholinopropanesulfonic acid (MOPS) exhibited an

absorbance maximum at 324 nm and a shoulder at 260 nm(Fig. 4B), values that are different from those reported forchrysobactin, a catechol-type siderophore isolated fromiron-deficient culture supernatants of the plant pathogenErwinia chrysanthemi (52). This UV-VIS spectral analysisalso showed that the electronic absorption spectra of amon-abactin, the catechol siderophore found in Aeromonas hy-drophila (5), and the Acinetobacter catechol are different(Fig. 4C). The siderophores pyochelin and pyoverdin, iso-lated from iron-deficient Pseudomonas culture supematants(21, 29), showed absorption spectra different from those ofthe Acinetobacter catechol when examined at pH 5.0 (Fig.4D). Figure 4D also shows that the Acinetobacter catecholdoes not exhibit at pH 5.0 the peak at 380 nm and theshoulder at 336 nm described for azotobactin D, a catecholsiderophore produced by Azotobacter vinelandii D (28).Mass spectral analysis of the XAD-7 column fraction

containing this catechol compound gave a prominent ion atmlz 391.3, corresponding to [MH]+ in the cationic detectionmode. The mass of this ion, which may represent theAcinetobacter catechol that possesses the siderophore activ-ity, is different from the values reported for the siderophoresagrobactin, amonabactin, anguibactin, azotobactin, chryso-bactin, enterobactin, pseudobactin, pyochelin, pyoverdin,and vibriobactin, which were used in the UV-VIS analysis.

In conclusion, we have shown the existence of a catecholcomponent in the culture supernatants ofA. baumannii 8399which possesses iron-binding properties and appears to bedifferent from DHBA and several other bacterial catechol-type siderophores.

Siderophore activity of the purified catechol. The sidero-phore activity of the purified component was tested by itsability to promote the growth of A. baumannii 8399 in thepresence of increasing concentrations of TF. Figure 5 showsthat the addition of this purified compound to M9 mediumcontaining 60 ,uM TF stimulated the bacterial growth by a

factor of 5, and no effect was observed in the presence of 90,uM TF. Conversely, when DHBA was added to M9 mediumin amounts equivalent to those of the purified catecholcompound, as measured by the Arnow test, insignificant

TABLE 3. Rf values of the catechol component(s) purified by XAD-7 column chromatography fromA. baumannu 8399 culture supernatants

RfSupport Solvent system A. baumnnii

8399 catechol DHBA Enterobactin

Silica Chloroform-methanol 0.00 0.16 0.56Butanol-acetic acid-water 0.02 0.62 0.10Benzene-acetic acid-water 0.04 0.41 0.73Butanol-pyridine-water 0.01 0.40 0.61

Paper Chloroform-methanol 0.00 0.37 NDaChloroform-methanol-water 0.00 0.52 NDButanol-acetic acid-water 0.06, 0.44 0.87 ND

a ND, not determined.

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020033o 0 0.

Wavelength (nm) Wavelength (nm)

1.2 11.2

0.4 < 0.6

FIG. 4. WV-VIS absorption spectra of the purifiedA. baumannii 8399 catechol, DHBA, and some bacterial catechol sideropho'res. (A4)Spectra ofDHBA( )a'ndAcinetobacter catechol dissolved in either 0.05 M Tris-HCI-0.2M Na2SO4 (pH 7.5) ( )or methanol (O )v(B)spectra of the Acinetobacter catechol dissolved in either 10% 0.1 N HCI-90% ethanol (--)or 0.1 M MOPS (pH 6.5)( ;(C) spectra of amonabactin (---)and theAcinetobacter catechol ( )dissolved in 75%o ethanol; (D) spectra of the Acinetobacter

catechol ( ,pyoverdin (-),and pyochelin (0-0) at pH 5.0. The spectra were recorded on a B'eckman' DU-40 spectrophotometer.

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7676 ECHENIQUE ET AL.

E

0

0

30 60 90

TRANSFERRIN (i.M)

FIG. 5. Siderophore activity of the purified A. baumannii 8399catechol. Bacterial growth was assayed in the presence of differentconcentrations of TF either in plain M9 medium (-) or in thepresence ofA. baumannii 8399 catechol (1) or DHBA (U1). OD 600nm, optical density at 600 nm.

increases in bacterial growth were detected at both 60 and 90,M TF.The involvement of this catechol in iron uptake was

confirmed by its ability to promote iron accumulation iniron-starved A. baumannii 8399 cells. Figure 6 shows thatthe uptake of 55Fe(III) was significantly enhanced by theaddition of this compound to the uptake mixtures. The rateof this siderophore-mediated transport of 55Fe(III) is fast,with the maximum intracellular accumulation occurringwithin 5 min and remaining constant even after 20 min ofincubation under the experimental conditions described inMaterials and Methods. The energy dependence of thisuptake process was determined by using the inhibitorsCCCP and KCN. The addition of 1 mM CCCP to the uptakemixtures inhibited the uptake of 55Fe(III) in the presence ofthe purified catechol, while no effect was detected when 20mM KCN was added, indicating that the uptake of 55Fe(III)in A. baumannii is coupled to proton movement across themembranes and not to the respiratory chain (63).These results strongly suggest that the catechol compound

isolated from A. baumannii 8399 culture supernatants is

0

20 -

10 -

0-

5 10

MINUTES

FIG. 6. "Fe(III) uptake by nongrowingA. baumannii 8399 cellsunder iron-limiting conditions. The intracellular accumulation of"FeCl3 was determined after the cells were incubated at 37°C for 5and 10 min in the presence of either carrier-free 55FeC13 (-) or55Fe(III)-A. baumannii 8399 siderophore (5,, , / ). The energydependence of the uptake process was examined by adding either 20mM KCN (U) or 1 mM CCCP ( , ).

134

120

1 2 M 3 4

87-82

77\76

7068

FIG. 7. SDS-PAGE of total membranes (lanes 1 and 2) and outermembrane proteins (lanes 3 and 4) fromA. baumannii 8399 grown inM9 minimal medium containing either 50 p,M FeCl3 (lanes 2 and 4)or 100 p,M EDDA (lanes 1 and 3). Molecular weight marker proteins(lane M) were phosphorylase b (94,000), bovine serum albumin(67,000), ovalbumin (43,000), trypsin inhibitor (20,100), and a-lac-talbumin (14,200).

indeed an extracellular siderophore, capable of scavengingiron from high-affinity iron chelators such as human TF andbringing it into the bacterial cells across the membranes in anenergy-dependent mode.

Effect of iron on the expression of A. baumannii 8399membrane proteins. The effect of iron on the synthesis ofmembrane proteins was investigated by SDS-PAGE. Severalmembrane proteins are induced under iron-limiting condi-tions (Fig. 7, lanes 1 and 2). Treatment of the total mem-branes with 1.5% Sarkosyl revealed that most of theseiron-regulated proteins are located in the outer membrane ofstrain 8399 (lanes 1 and 3). However, two of them, of 120 and134 kDa, appear to be localized in the inner membrane, sincethey are present in the total membrane preparation (lane 1)but absent in the detergent-treated sample (lane 3). Con-versely, the latter sample contains at least seven iron-regulated proteins, with molecular masses ranging from 68 to87 kDa (lane 3). The fact that these inner and outer mem-brane proteins are expressed only under iron limitationsuggests that some of them may be involved in the recogni-tion and transport process for the iron-siderophore com-plexes.

DISCUSSION

A. baumannii is an opportunistic human pathogen thatpresents a potential risk of severe infections for patients whoare compromised or suffer from polymicrobial infections.Thus, the simple presence of this bacterium is not sufficientfor establishment of an infection, which is thus related to theclinical status of the colonized host. Furthermore, the natureof the factors affecting the virulence of this bacterium is notfully understood.

Analysis of experimental infections in mice (49) showedthat the slime ofA. baumannii is a virulence factor that actsby impairing the functions and cell structure of mice phago-cytes. The concurrent intraperitoneal injection of slime-producing Acinetobacter strains or isolated slime enhancesthe virulence of E. coli, Serratia marcescens, and P. aerug-inosa strains with respect to single bacterial infections.However, this study showed that the virulence of A. bau-

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mannii is very low, and no differences in 50% lethal dosewere found between the slime-producing and non-slime-producing strains.More recently, Smith et al. (66) reported that a clinical

isolate of A. baumannii was able to grow in a chemicallydefined medium containing DIP. Chemical and chromato-graphic analysis of ethyl acetate extracts of culture superna-tants from that isolate revealed only the presence of DHBA,while bioassays showed that the organism could utilizeFe-DHBA complexes to grow under iron-restricted condi-tions. These findings suggested to the authors that A. bau-mannii expresses an iron uptake system involving DHBA asa putative extracellular siderophore. Other reports have alsoindicated that DHBA may act as a siderophore (37, 38, 51),although its activity appears to be somewhat disputableunder the iron-restricted conditions described in the humanhost. Consequently, we initiated a detailed analysis of theiron proficiency phenotype of the A. baumannii 8399 strainisolated during a nosocomial outbreak of lower tract respi-ratory infections. The free iron concentration of the culturemedium affected the growth of bacteria as well as thesynthesis of membrane proteins and extracellular products.However, this strain grew well under the iron-limiting con-ditions imposed by different iron chelators added in highconcentrations to chemically defined media. This iron scav-enging activity appears not to require a direct contactbetween the bacterial cells and the iron-chelating humanglycoprotein, as is the case with H. influenzae, Neisseriameningitidis, and N. gonorrhoeae (34).

Colorimetric and siderophore utilization tests, supportedby DNA hybridization experiments, revealed that the ironuptake proficiency of A. baumannii 8399 is related to thesynthesis and excretion of an iron-regulated catechol sidero-phore and not to a hydroxamate iron-chelating agent. Thesiderophore utilization bioassays, the chromatographic be-havior, and the UV-VIS and mass spectral properties con-firmed these results. In addition, they indicated that theisolated catechol siderophore is different from agrobactin,amonabactin, anguibactin, azotobactin, chrysobactin, enter-obactin, pseudobactin, pyochelin, pyoverdin, and vibriobac-tin. However, the possibility that the Acinetobacter sidero-phore is either similar or identical to other catecholsiderophores not included in our study will be clarified afterthe elucidation of its chemical structure.The actual siderophore activity of the purifiedA. bauman-

nii 8399 catechol was confirmed by utilization bioassays,since the addition of this compound to minimal mediumcontaining 60 ,uM TF facilitated significantly the growth ofA. baumannii 8399. Such growth facilitation was not ob-served when an equivalent amount of DHBA was added tothe chemically defined culture medium containing the sameTF concentration. These results also indicate that this cate-chol is a high-affinity siderophore, since it is able to scavengeiron from strong chelating molecules such as human TF. Therole of this extracellular component in the iron uptakeprocess was demonstrated by its ability to promote theintracellular accumulation of 55Fe(III). The fact that thisuptake process is sensitive to CCCP and not to KCNindicates that it requires energy coupled to a transmembraneproton gradient rather than to the electron transport chain(63).Although it was shown that A. baumannii secretes and

utilizes DHBA as a siderophore (66), the fact that DHBAwas added in that study as a preformed iron complex shouldbe taken into account. The experimental approach describedin that publication does not demonstrate that DHBA can

acquire iron from TF when the cells are cultured in thepresence of this protein. Indeed, our results indicate thatDHBA cannot promote the growth ofA. baumannii 8399 inminimal medium containing 60 ,uM TF, supporting thegeneral concept that DHBA is a weak iron chelator that canprovide iron only under mild restricted conditions. It hasbeen reported that DHBA can stimulate E. coli growth onnutrient plates containing DIP or iron-deficient M9 minimalmedium only if it is used as a precursor for the biosynthesisof enterobactin (37, 38). In the case ofA. vinelandii, DHBAwas shown to act as an effective iron-solubilizing agent, butwhen free iron becomes less abundant, this bacterium com-bines the production of DHBA with production of thehigher-affinity siderophores azotobactin and azotochelin(51).Thus, it is possible that A. baumannii 8399 can utilize

DHBA as a siderophore to grow in culture media containingmoderately low levels of free iron, but it must produce asiderophore with higher affinity for iron and its cognatetransport system to grow under more stringent iron-deficientconditions, such as those found in the human host.Our preliminary research in this work for such a high-

affinity transport system led to the identification of two innermembrane proteins and of at least seven outer membraneproteins that are expressed only when A. baumannii 8399 isgrown under iron-limiting conditions. Some of these outermembrane proteins may correspond to those already identi-fied by Coomassie blue staining (66) or immunoblotting withserum from septicemic patients (65), although no iron-regulated inner membrane proteins have previously beendetected in Acinetobacter species. Furthermore, some ofthese membrane proteins may play an important role ascomponents of the cell membrane receptors that recognizeand internalize the Fe-siderophore complexes, as was de-scribed for other bacterial iron uptake systems (23).The expression of this siderophore-mediated iron uptake

system could be another important factor in the pathogenesisof Acinetobacter infections, since such systems were dem-onstrated to be important virulence factors in the establish-ment of bacterial infections (23).

ACKNOWLEDGMENTSThis work was supported by grants from Consejo Nacional de

Investigaciones Cientificas y Tecnicas (CONICET) of Argentina toL.A.A., M.E.T., and R.J.S. and from Consejo de InvestigacionesCientificas y Tecnologicas de la Provincia de Cordoba (Argentina) toL.A.A. and by Public Health Service grant AI-19018 from theNational Institutes of Health to J.H.C. R.R.R. was supported by aMedical Research Council of Canada fellowship. M.E.T., R.J.S.,and L.A.A. are Career Investigator members of CONICET.We are thankful to L. M. Crosa for valuable help in performing

the aerobactin utilization bioassays.

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