Role of Acinetobactin-Mediated Iron Acquisition Functions in theInteraction of Acinetobacter baumannii Strain ATCC 19606T withHuman Lung Epithelial Cells, Galleria mellonella Caterpillars,and Mice

Jennifer A. Gaddy,a Brock A. Arivett,a Michael J. McConnell,b Rafael López-Rojas,b Jerónimo Pachón,b and Luis A. Actisa

Department of Microbiology, Miami University, Oxford, Ohio, USA,a and Unit of Infectious Disease, Microbiology, and Preventive Medicine and Institute of Biomedicine ofSeville (IBiS), University Hospital Virgen del Rocío/CSIC/University of Seville, Seville, Spainb

Acinetobacter baumannii, which causes serious infections in immunocompromised patients, expresses high-affinity iron acqui-sition functions needed for growth under iron-limiting laboratory conditions. In this study, we determined that the initial inter-action of the ATCC 19606T type strain with A549 human alveolar epithelial cells is independent of the production of BasD andBauA, proteins needed for acinetobactin biosynthesis and transport, respectively. In contrast, these proteins are required for thisstrain to persist within epithelial cells and cause their apoptotic death. Infection assays using Galleria mellonella larvae showedthat impairment of acinetobactin biosynthesis and transport functions significantly reduces the ability of ATCC 19606T cells topersist and kill this host, a defect that was corrected by adding inorganic iron to the inocula. The results obtained with these exvivo and in vivo approaches were validated using a mouse sepsis model, which showed that expression of the acinetobactin-mediated iron acquisition system is critical for ATCC 19606T to establish an infection and kill this vertebrate host. These obser-vations demonstrate that the virulence of the ATCC 19606T strain depends on the expression of a fully active acinetobactin-mediated system. Interestingly, the three models also showed that impairment of BasD production results in an intermediatevirulence phenotype compared to those of the parental strain and the BauA mutant. This observation suggests that acinetobactinintermediates or precursors play a virulence role, although their contribution to iron acquisition is less relevant than that of ma-ture acinetobactin.

Acinetobacter baumannii is a Gram-negative opportunistic bac-terial pathogen that has emerged as a serious threat to human

health, particularly in hospitalized and/or immunocompromisedpatients (18, 36). Pneumonia has been the main manifestation ofnosocomial infections caused by this pathogen that results in asignificant impact on the mortality rate of patients (44). However,this microorganism has also been identified as the etiologicalagent responsible for a wide range of other infections, includingsepticemia, meningitis, and more recently, severe and deadly casesof necrotizing fasciitis (4, 9, 14, 36). In addition, the emergence ofmultiple-drug-resistant strains responsible for infections in sus-ceptible populations, such as wounded military personnel return-ing from the Middle East, has also presented a significant problemto clinicians (1, 7). Many clinical isolates of A. baumannii exhibita multiple- or pan-drug-resistant phenotype, limiting treatmentoptions and creating a burden upon health care facilities (22, 23).Thus, the importance of identifying novel targets for the develop-ment of alternative antimicrobial therapies for these infections is apriority. However, the role A. baumannii virulence factors play inthe pathogenesis of human infections remains largely obscure;therefore, the feasibility of these factors as therapeutic targets isuncertain. Recent work showed that A. baumannii interacts withhuman respiratory epithelial cells, an appropriate model consid-ering the serious respiratory infections it causes in hospitalizedpatients, through cellular processes that involve the expression ofbacterial cell surface components, such as the major outer mem-brane protein A (10, 11, 26). This surface-exposed protein plays aclear role in the abilities of A. baumannii to attach to, invade, andcause the apoptotic death of A549 human alveolar epithelial cell

monolayers. A. baumannii clinical isolates, including the ATCC19606T type strain, also cause deadly acute sepsis, pneumonia, andsoft tissue infections when tested in murine experimental infec-tions (30, 38) as well as the death of infected larvae of the greaterwax moth Galleria mellonella (35). Taken together, these studiesand observations indicate that A. baumannii is able to persistwithin an infected host by acquiring essential nutrients.

The host environment presents many nutritional possibilitiesas well as challenges that a bacterial pathogen must overcome. Oneof these challenges is the acquisition of iron, a micronutrient thatis essential for almost all living cells (15). Although abundant inthe human host, most of this metal is sequestered by high-affinityiron-binding chelators, such as hemoglobin, lactoferrin, andtransferrin, with the purpose of avoiding cytotoxic effects due tothe presence of free iron and controlling microbial infectionsthrough nonspecific mechanisms (6, 37, 47). To overcome thischallenge, bacteria express a wide variety of high-affinity iron ac-quisition systems to scavenge iron from the host (16, 45). Onesuch iron acquisition system, mediated by the biosynthesis and

Received 5 December 2011 Accepted 12 December 2011

Published ahead of print 9 January 2012

Editor: A. J. Bäumler

Address correspondence to Luis A. Actis, actisla@muohio.edu.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

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utilization of the siderophore acinetobactin, is produced by theA. baumannii ATCC 19606T type strain (20, 31). Our previousmolecular genetic and functional analyses showed that theacinetobactin-mediated iron acquisition system is the onlyfully active high-affinity iron acquisition system produced bythis strain when cultured in iron-chelated bacteriological me-dia (20), although recent genomic analyses showed that thisstrain harbors predicted genetic determinants capable of cod-ing for additional iron acquisition systems (2, 21). Neverthe-less, the relevance of iron acquisition functions expressed by A.baumannii in a biological system has not been explored.

This report shows that the ability of A. baumannii ATCC19606T to persist inside A549 human alveolar epithelial cells andcause their apoptosis depends on the expression of activeacinetobactin-mediated iron utilization functions and the pro-duction of the acinetobactin siderophore. Similarly, the infectionand killing of mice and G. mellonella larvae depend on the abilityof A. baumannii ATCC 19606T cells to express active acinetobac-tin biosynthesis and transport functions. These observations indi-cate that acinetobactin-mediated iron acquisition is an importantA. baumannii virulence factor, which could be used as a target fortherapeutic purposes.

MATERIALS AND METHODSBacterial strains, plasmids, cell lines, and culture conditions. All bacte-rial strains and plasmids used in this work are listed in Table 1. Bacterialstrains were routinely maintained in Luria-Bertani (LB) broth or agar (40)at 37°C with appropriate antibiotics. Iron-rich and iron-chelated condi-tions were attained by supplementing LB broth with FeCl3 dissolved in0.01 M HCl or with the synthetic iron chelator 2,2=-dipyridyl (DIP) to afinal concentration of 100 �M. A549 human alveolar epithelial cells weremaintained at 37°C in the presence of 5% CO2 using Dulbecco modifiedEagle medium (DMEM) supplemented with 10% heat-inactivated fetalbovine serum, 100 IU of penicillin, and 100 �g/ml streptomycin. Mono-layers were cultured to 70% confluence before use in bacterial attachmentand invasion assays.

Interaction of bacteria with A549 alveolar epithelial cells. To deter-mine short-term bacterial attachment and invasion, 1 � 105 A549 cellswere infected with 2 � 106 bacteria for 1 h at 37°C as described before (26).Bacteria attached to the surfaces of A549 cells were visualized using scan-

ning electron microscopy (SEM), and micrograph observations were con-firmed by counting the number of visible bacteria attached to A549 cellsusing five epithelial cells located in different fields from three differentbiological replicates. The number of intracellular bacteria was determinedby plating appropriate dilutions of lysates obtained from gentamicin(Gm)-treated A549 infected cells (26). To determine intracellular bacte-rial persistence, A549 monolayers were infected with 5 � 103 bacteriasuspended in 2 ml of phosphate-buffered saline (PBS) solution for 3 h at37°C. Coinfections using two bacterial strains were done using a 2-mlmixture containing 5 � 103 cells of each strain. The infected monolayerswere used to prepare lysates after Gm treatment as described before (26).To determine the total number of intracellular bacteria, appropriate lysatedilutions were plated on LB agar containing no antibiotics. To determinethe number of intracellular s1 or t6 mutant cells, appropriate dilutions ofA549 lysates were inoculated onto LB agar plates containing 40 �g/ml ofkanamycin (Km). In all cases, the numbers of CFU were recorded afterovernight (10 to 12 h) incubation at 37°C. Duplicate assays were done atleast three times using fresh samples each time, and the data were statis-tically analyzed using the Student t test; P values � 0.05 were consideredstatistically significant.

Microscopy analysis of infected A549 cells. The presence of intracel-lular bacteria was visualized with confocal laser scanning microscopy(CLSM) of A549 cells infected with the ATCC 19606T-GFP strain or thes1-GFP or t6-GFP derivatives harboring pMU125, which codes for theproduction of green fluorescent protein (GFP). Bacterial cells (5 � 103)suspended in PBS were added to 1 � 105 epithelial cells and incubated at37°C for 4 h. Afterward, the monolayers were washed three times withprewarmed PBS, trypsinized, suspended in 1 ml of PBS, and stained withpropidium iodide before being mounted onto a glass depression slide. Thesamples were visualized with a Zeiss 710 CLSM and analyzed using theZen 2009 software package. The production of GFP was detected usingthe green filter set between 508 nm and 520 nm. The number of intracel-lular bacteria was determined by counting the number of green spots in 5A549 cells in at least five different fields from each sample examined byCLSM. Counts were compared using the Student t test; P values � 0.05were considered significant. Experiments were done at least twice usingfresh biological samples each time.

Protein analysis. To detect the production of the BauA acinetobactinreceptor protein during the infection of A549 cells, monolayers weregrown in 10 25-ml tissue culture flasks in 6 ml of supplemented DMEM.Epithelial cells were infected with 3 � 104 ATCC 19606T bacteria, whichwere grown overnight at 37°C in LB broth for 3 h at 37°C in the presence

TABLE 1 Bacterial strains and plasmids used in this study

Bacterial strain or plasmid Relevant characteristic(s) or usea

Source orreference

Bacterial strainsA. baumannii

ATCC 19606T Clinical isolate; type strain ATCC19606T-GFP ATCC 19606T derivative producing GFP encoded by a gene on pMU125; Ampr 19s1 basD::aph; ATCC 19606T acinetobactin production-deficient derivative; Kmr 20s1-GFP s1 derivative producing GFP encoded by a gene on pMU125; Ampr This workt6 bauA::EZ::TN�R6K�ori/KAN-2�; ATCC 19606T acinetobactin uptake-deficient derivative; Kmr 20t6-GFP t6 derivative producing GFP encoded by a gene on pMU125; Ampr This work

E. coliTop10 Used for DNA recombinant methods InvitrogenDH5� Used for DNA recombinant methods Gibco-BRL

PlasmidspWH1266 E. coli-A. baumannii shuttle vector; Ampr Tcr 27pMU125 pWH1266 harboring gfp; Ampr 19

a Ampr, ampicillin resistance; Kmr, kanamycin resistance; Tcr, tetracycline resistance.

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of 5% CO2 and then washed three times with prewarmed PBS,trypsinized, and collected by centrifugation. Cells were then lysed withsterile distilled water, and the internalized bacteria were collected by cen-trifugation, washed once with sterile PBS, and used to prepare bacterialtotal cell lysates as described before (43). Bacterial proteins were size frac-tionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) using 12.5% gels, transferred to nitrocellulose, and probedwith a polyclonal antibody against BauA as described before (20).

Evaluation of apoptosis using the TUNEL assay and epifluores-cence microscopy. A549 cell apoptosis was examined with the terminaldeoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling(TUNEL) assay (Promega, Madison, WI) as previously described (26).Briefly, infected epithelial cells were fixed with 4% paraformaldehyde for1 h and then washed three times with PBS. Cells were permeabilized with0.1% Triton X in PBS before the TUNEL reaction was performed. Thereaction was stopped with 2� SSC (1� SSC is 0.15 M NaCl plus 0.015 Msodium citrate) provided in the kit. Samples were viewed using an Olym-pus AX70 epifluorescence microscope and were analyzed using theMetaview software package. The apoptosis rate was determined by count-ing the number of apoptotic foci (green spots) in 50 A549 cells in fivedifferent fields from each sample examined by epifluorescence micros-copy. Counts were compared using the Student t test; P values � 0.05 wereconsidered significant. Experiments were done at least twice in duplicateusing fresh biological samples each time.

G. mellonella infection and killing assays. Final-instar larvae (Van-derhorst, Inc., St. Mary’s, OH) were stored in darkness at 25°C and usedwithin 7 days of receipt. Infection experiments were performed as previ-ously described (35). Briefly, caterpillars were evaluated for health andused in experiments based on three criteria: lack of melanization, move-ment in response to touch, and having a 250-mg- to 350-mg-mass range.Bacteria from overnight LB broth cultures were collected by centrifuga-tion, washed, and resuspended in PBS alone or in PBS containing 100 �MFeCl3 as an exogenous iron source. All bacterial samples were adjusted toan appropriate cell density as determined by optical density at 600 nm. Allbacterial inocula were confirmed by plating serial dilutions on LB agar anddetermining the number of CFU after overnight incubation at 37°C. Thecuticle of the larva was swabbed gently with ethanol, and the hemocoel atthe last left proleg was injected with 5-�l inocula containing from 100 to106 bacterial cells � 0.25 log using a syringe pump (New Era Pump Sys-tems, Inc., Wantagh, NY) with a 26-gauge needle.

For infection assays, larvae were injected with 1 � 105 cells of the A.baumannii ATCC 19606T type strain or the s1 or t6 isogenic derivative.Larvae injected with sterile PBS were used as negative controls. After in-cubation at 37°C in darkness for 18 h, 30 responsive larvae from eachexperimental group were placed on ice for 10 min and briefly washed with70% ethanol. Each larva was repeatedly washed with and homogenized in1 ml of sterile distilled water containing 50 �g/ml vancomycin (Van).Homogenates were serially diluted with sterile distilled water containing50 �g/ml Van, and 100-�l aliquots were plated on nutrient agar platescontaining 50 �g/ml Van. CFU counts were done with an AlphaImager2200 using the AlphaEase version 5.5 software package (Alpha InnotechCorporation-Cell Biosciences, Santa Clara, CA). Counts were comparedusing the Student t test; P values � 0.05 were considered significant.

For killing assays, each test series included control groups of nonin-jected larvae or larvae injected with 1 � 102 or 1 � 105 bacteria, sterilePBS, or PBS containing 100 �M FeCl3. The test groups included larvaeinfected with the parental strain ATCC 19606T or the isogenic derivatives1 or t6, all of which were injected in the presence or absence of 100 �MFeCl3. After injection, the larvae were incubated at 37°C in darkness, andthe larvae were assessed at 24-h intervals over 6 days. Caterpillars wereconsidered dead and removed from the study if they displayed no re-sponse to probing. The results of the trial were omitted if more than twodeaths occurred in the control groups. The experiments were repeated sixtimes using 10 larvae per experimental group, and the resulting survivalcurves were plotted using the Kaplan-Meier method (28). P values � 0.05

were considered statistically significant for the log rank test of survivalcurves (SAS Institute Inc., Cary, NC).

Mouse sepsis model. Mice were housed under specific-pathogen-freeconditions with free access to food and water. For infection studies, apreviously described murine model of A. baumannii sepsis was employed(30). Briefly, inocula were prepared by inoculating 20 ml of Mueller-Hinton broth with a single colony of the indicated A. baumannii strainsand incubating for 24 h at 37°C. Cultures were adjusted to the appropriateconcentration with saline solution and combined 1:1 (vol/vol) with a 10%solution of porcine mucin (Sigma, St. Louis, MO; final mucin concentra-tion of 5%). Dilutions of the inocula were plated on blood agar to deter-mine final CFU values. For infection, female C57BL/6 mice between 14and 16 weeks of age were injected intraperitoneally with 0.5 ml of theinocula using a 26-gauge needle (n � 6 mice/group). Tissue bacterialloads were determined 16 h after infection with the indicated inocula ofthe 19606T, s1, and t6 strains (n � 8 mice/group). Mice were euthanizedwith an overdose of thiopental, and their spleens were aseptically removedand homogenized after the addition of 2 ml of saline solution. Serial di-lutions of tissue homogenates were plated on blood agar plates for quan-tification of viable bacteria after incubation at 37°C for 24 h. Colonycounts were compared using the Mann-Whitney U test with a P value �0.05 considered statistically significant. Animal survival was scored every12 h for 7 days following infection. Survival between groups was com-pared using the log rank test with a P value � 0.05 considered statisticallysignificant. All experiments involving the use of mice were approved bythe University Hospital Virgen del Rocío Committee on Ethics and Ex-perimentation.

RESULTSInitial interaction of A. baumannii with human epithelial cellsis independent of acinetobactin-mediated iron acquisitionfunctions. Based on our previous work showing the interactionof A. baumannii ATCC 19606T with A549 human respiratorycells (26), the role of the acinetobactin-mediated system inthese interactions was tested using this type strain and the iso-genic derivatives s1, affected in the expression of the BasDacinetobactin biosynthesis function, and t6, affected in theproduction of the BauA acinetobactin outer membrane recep-tor protein. SEM analysis showed that infection of A549 cellswith 2 � 106 bacteria of each strain for a short time, 1 h,resulted in comparable cytotoxic effects, such as cell roundingand absence of surface appendages compared with the mor-phology of monolayers incubated in sterile medium (Fig. 1A toD). Visual inspection of micrographs of infected A549 cells andcounting the number of visible bacteria attached to the A549cells (Fig. 1E) showed comparable numbers of bacteria of eachstrain attached to the surfaces of epithelial cells. Furthermore,there were no statistically significant differences in the countsof intracellular bacteria of each tested strain (Fig. 1F). Takentogether, these observations indicate that the acinetobactin-mediated iron acquisition system does not play a major role inthe initial interaction of the ATCC 19606T strain with humanrespiratory epithelial cells.

Bacterial intracellular persistence depends on the expressionof the acinetobactin-mediated system. To determine whetherbacterial persistence after the initial interaction with A549 cellsdepends on the expression of acinetobactin-mediated iron acqui-sition functions, we repeated the studies described above but us-ing a 3-h infection time. This approach failed to produce mean-ingful results perhaps because of the high infection dose used inthose experiments. However, clear differences were observedwhen the monolayers were infected for 3 h with a lower bacterial

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dose, 5 � 103 cells. Under these conditions, the ATCC 19606T

parental strain persisted significantly better than the s1 and t6mutants, which showed a 2.7- and a 12-fold reduction in the num-ber of intracellular bacteria, respectively, as determined after plat-ing cell lysates on LB agar (Fig. 2, samples P La, s1 La, and t6 La).Figure 2 also shows that coinfection of A549 monolayers with 5 �103 cells of the parental strain and the s1 mutant resulted in a2-fold increase in the number of intracellular bacteria comparedwith the number of intracellular bacteria recovered from mono-layers infected with only 5 � 103 cells of the parental strain (Fig. 2,compare P La and P�s1 La samples), with half of them being Kmresistant (Fig. 2, compare P�s1 La and P�s1 La-K samples).However, the number of Km-resistant colonies recovered frommonolayers coinfected with the parental strain and the s1 mutantwere 2.8-fold higher than the number of colonies recovered fromepithelial cells singly infected with s1 mutant bacteria (Fig. 2, com-pare P�s1 La-K and s1 La samples). This result indicates that the

parental strain was able to trans-complement the s1 mutant byproviding it with acinetobactin produced intracellularly. In con-trast, no biological complementation was observed with the t6mutant, which produces but does not transport ferric acinetobac-tin, when coinfected with parental cells. Plating lysates of A549monolayers coinfected with ATCC 19606T and t6 bacteria on LBagar containing Km resulted in a 13.8-fold reduction in CFUcounts compared with the counts obtained by plating these lysateson LB agar (Fig. 2, compare P�t6 La and P�t6 La-K samples).Furthermore, the t6 cell counts obtained on LB agar from singlyinfected monolayers were comparable with those obtained on LBagar containing Km from monolayers coinfected with ATCC19606T and t6 bacteria (Fig. 2, compare t6 La and P�t6 La-Ksamples). The expression of the acinetobactin-mediated iron ac-quisition system by intracellular bacteria was further tested byexamining the production of the acinetobactin outer membranereceptor protein BauA. Immunoblotting of bacterial total lysates

FIG 1 Interaction of A. baumannii bacteria with A549 alveolar epithelial cells. (A) Sterile medium. (B to D) Adhesion of parental A. baumannii ATCC 19606T

cells (B) or cells of the s1 acinetobactin production (C) or t6 utilization (D) isogenic derivatives examined by SEM after A549 cell monolayers were infected with2 � 106 bacteria for 1 h. Bars, 1 �m. (E) Surface-attached bacterial counts collected from the SEM micrographs of infected monolayers, examples of which areshown in panels B to D. Data were collected using five A549 cells in different fields from three different biological replicates. Values are means � 1 standarddeviation (error bars). (F) Intracellular CFU counts obtained after lysates of Gm-treated A549 cells were plated on LB agar and incubated overnight at 37°C. Datarepresent three independent experiments done in duplicate each time. Values are means � 1 standard deviation (error bars).

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showed that ATCC 19606T cells produce BauA when they are in-side A549 cells but not when incubated in supplemented DMEMculture medium used to grow the monolayers (Fig. 2, inset). Thisresponse mimics that obtained when bacterial cells are cultured inLB broth under iron-chelated and iron-rich conditions, respec-tively. Considering these results and the fact that the infecting

bacteria were cultured in LB broth, medium that has enough ironto repress the production of BauA (33), the detection of BauA inbacteria isolated from infected epithelial cells indicate that thisprotein is produced in response to the iron-limiting conditionsimposed by the A549 intracellular environment.

Taken together, all the observations made using the A549epithelial cell infection assays indicate that the acinetobactin-mediated iron transport system is not only expressed by A. bau-mannii ATCC 19606T intracellular bacteria but also show that thisiron acquisition system is needed for bacterial intracellular persis-tence.

Microscopy analysis of A. baumannii-A549 cell interactions.The interaction of bacteria with A549 epithelial cells was also ex-amined by CLSM using isogenic strains producing GFP, encodedby a gene on the recombinant plasmid pMU125. This approachshowed that after infection, numerous 19606T-GFP green fluores-cent bacteria are located in the A549 cytoplasmic compartment(Fig. 3B). In contrast, very few and almost no green fluorescentcells of the s1-GFP and t6-GFP isogenic mutants could be detectedwithin the cytoplasm of A549 cells in single-strain infection exper-iments, respectively (Fig. 3C and D). The microscopy observa-tions were confirmed by visually counting the number of intra-cellular fluorescing bacteria. There was a 2.8- and a 8.5-foldreduction in the number of green foci representing s1-GFP andt6-GFP cells compared with the 19606T-GFP parental strain, re-spectively, in epithelial cells infected with a single strain (Fig. 3E).These observations confirm the ability of ATCC 19606T bacteriato localize and persist within the cytoplasmic space of the A549alveolar epithelial cells in a manner that depends on the expressionof an active acinetobactin-mediated iron uptake system.

Apoptosis of A549 alveolar epithelial cells in response to bac-terial infection. Our previous report showed that A. baumanniiATCC 19606T induces apoptosis in A549 epithelial cells (26). Totest whether the expression of iron acquisition functions might beinvolved in this process, the capacity of the s1 and t6 derivatives toinduce apoptosis was compared to that of the ATCC 19606T pa-rental strain using TUNEL assays. As expected from our previouswork (26), infection of A549 monolayers with A. baumannii

FIG 2 Role and expression of the acinetobactin-mediated iron acquisitionfunctions by intracellular bacteria. A549 monolayers were incubated with ster-ile medium (Sm) or infected for 3 h with 5 � 103 cells of the ATCC 19606T

parental strain (P) or cells of the s1 or t6 mutant. Monolayers were also coin-fected with 5 � 103 cells of the parental strain mixed with equal number of cellsof the s1 or t6 isogenic derivative. CFU counts were obtained after Gm-treatedA549 cell lysates were plated on LB agar (La) and LB agar containing 40 �g/mlKm (La-K) to determine total bacterial and mutant cell counts, respectively.(Inset) Detection of the acinetobactin outer membrane receptor protein BauA.A. baumannii ATCC 19606T whole-cell lysates were prepared from cells cul-tured in supplemented DMEM (lane 3) or from intracellular bacteria recov-ered after infection of epithelial cells (lane 4). Protein samples loaded in lane 1or 2, which were prepared from bacteria cultured in LB broth containing 100�M FeCl3 or 100 �M DIP, respectively, were used as a control for differentialproduction of BauA in response to free-iron availability. Horizontal bars withnumbers indicate the fold change before the slash and the P value for thecompared samples after the slash. Values are means � 1 standard deviation(error bars).

FIG 3 CLSM analysis of A549 monolayers infected with A. baumannii ATCC 19606T isogenic strains. (A to D) Monolayers were infected with ATCC19606T-GFP (B), s1-GFP (C), or t6-GFP (D) cells, using uninfected monolayers (A) as a negative control. Production of GFP was detected using a green filter.All monolayers were stained with propidium iodide, which was detected using a red filter. All images were taken at a magnification of �630, and Z stack analysiswas performed at 0.63 �M increments. Bar, 20 �m. (E) Graphic representation of intracellular bacterial counts. Horizontal bars with numbers indicate the foldchange before the slash and the P value for the compared samples after the slash. Values are means � 1 standard deviation (error bars).

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ATCC 19606T cells resulted in detectable apoptosis, as it was ob-served in the presence of HCl (Fig. 4, micrographs and bar graph).An apoptotic response was also observed when the monolayerswere infected with the acinetobactin production mutant s1, al-though the number of apoptotic foci was reduced by 2-fold com-pared with the response obtained with the ATCC 19606T parentalstrain. A drastic apoptosis reduction (24-fold) was detected whenthe A549 epithelial cells were infected with acinetobactin receptormutant t6 bacterial cells. Taken together, these results indicatethat bacteria expressing fully active acinetobactin-mediated ironacquisition functions cause more cell damage than bacteria defi-

cient in either biosynthesis or transport of the acinetobactin sid-erophore.

Infection and killing of G. mellonella larvae. On the basis of areport describing the use of the greater wax moth G. mellonella toexamine antibiotic efficacy against A. baumannii (35), we testedthe role of acinetobactin-mediated iron acquisition functions inthis viable and convenient host model, which has been used tostudy other pathogens such as Pseudomonas aeruginosa (32),Burkholderia mallei (41), and Burkholderia cepacia (42). As shownin Fig. 5A, the ATCC 19606T strain not only survived but alsomultiplied 18 h after the larvae were infected. In contrast, the

FIG 4 A549 apoptotic response to bacterial infection determined by TUNEL assays and fluorescence microscopy. A549 monolayers were infected with cells ofthe ATCC 19606T parental strain (19606), the s1 acinetobactin synthesis mutant, or the t6 acinetobactin transport mutant. A549 cells incubated in the presenceof sterile medium (Sm) and sterile medium supplemented with 2 M HCl were used as negative and positive controls, respectively. Samples were examined byfluorescence microscopy at a magnification of �400 or �600. (Right) Bar graph of the number of apoptotic foci determined from the cognate micrographscaptured with fluorescence microscopy. Horizontal bars with numbers indicate the fold change before the slash and the P value for the compared samples afterthe slash. Values are means � 1 standard deviation (error bars).

FIG 5 G. mellonella infection and killing assays. (A) For infection assays, caterpillars were injected with 1 � 105 bacteria of the ATCC 19606T parental strain(19606) or the s1 or t6 iron-deficient isogenic derivative and incubated at 37°C in darkness for 18 h. Dilutions of whole-larva lysates were plated on nutrient agar,and colony counts were determined after overnight incubation at 37°C. Horizontal bars with numbers indicate the fold change before the slash and the P valuefor the compared samples after the slash. Values are means � 1 standard deviation (error bars). (B to E) For killing assays, caterpillars were infected with high (1 �105 bacteria [B and C]) or low (1 � 102 bacteria [D and E]) bacterial doses in the absence (B and D) or presence (C and E) of 100 �M Fe3Cl. For negative controls,caterpillars were injected with comparable volumes of PBS (B and D) or PBS plus 100 �M Fe3Cl (C and E). The inset in panel B shows the melanization of infectedcaterpillars (I) and no pigmentation of uninfected (U) caterpillars.

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number of bacteria recovered from infected larvae was reduced by2.2- and 5.0-fold when caterpillars were injected with the s1 and t6isogenic mutants, respectively. The inoculation of G. mellonellawith A. baumannii ATCC 19606T resulted in killing of caterpillarsin a time- and dose-dependent manner in a process that includedmelanization as described before using different A. baumanniiclinical strains (35). Infection of caterpillars with 1 � 105 ATCC19606T bacteria resulted in 40% death after 24 h inoculation, withmore than half of the larvae dying by the sixth day, a response thatis significantly different from that obtained with the PBS control(P � 0.0001) (Fig. 5B). The killing caused by the s1 and t6 deriv-atives was also significantly different (P � 0.0001) from this con-trol. However, both mutants were less virulent than the parentalstrain, with t6 killing significantly fewer worms (P � 0.002) thanATCC 19606T, but s1 killing being intermediate and almost sig-nificantly different (P � 0.06) from that of the parental strain (Fig.5B). The addition of 100 �M FeCl3 to the inocula made the killingrates of both mutants comparable to that of the ATCC 19606T

parental strain, particularly during the last days of the experimen-tal infection, with all of them being significantly different from thePBS control (P � 0.0001) (Fig. 5C). Interestingly, although theaddition of inorganic iron to the inocula increased the killing ratesduring the early days of the infection, the numbers of caterpillarskilled by all strains at the end of the experiment were statisticallyindistinguishable (P � 0.18) from that obtained without the sup-plementation of the inocula with FeCl3 (compare Fig. 5B and C).The lower infection dose of 1 � 102 ATCC 19606T bacterial cellsresulted in a modest 20% total population death by the sixth day,a value that is statistically different (P � 0.008) from that recordedwith the s1 and t6 mutants, which displayed a response indistin-guishable from that of the controls injected with sterile PBS (Fig.5D). However, the addition of an exogenous iron source resultedin comparable death rates by all three strains by the 48-h timepoint that remained close to 20%, a value that is significantly dif-ferent (P � 0.0007) from the PBS control, until the end of theexperimental infection (Fig. 5E). Taken together, these results in-dicate that the production of a fully active acinetobactin-mediated

iron acquisition system is required for full virulence of A. bauman-nii in the G. mellonella host.

Infection and killing of mice. In order to evaluate the role of theacinetobactin-mediated iron acquisition system in a mammalianhost, we employed a previously characterized sepsis mouse model(30). This model produces an acute sepsis in which A. baumanniirapidly disseminates throughout the body to different organ systems.Importantly, mortality in this model has been shown to be dependentupon both the infecting A. baumannii strain and the number of bac-teria in the inoculum, indicating that this model can be used to char-acterize differences in virulence between strains. Using this model,the ability of the ATCC 19606T, s1, and t6 strains to persist in infectedanimals was determined by measuring spleen bacterial loads 16 hafter infection. As shown in Fig. 6, infection with the parental ATCC19606T strain resulted in significantly higher spleen bacterial loadscompared to both the s1 and t6 strains (medians of 8.6 versus 7.3 and5.7, respectively; P � 0.001). Spleen bacterial loads in mice infectedwith the s1 strain showed a tendency toward being higher than inmice infected with the t6 strain; however, the difference did not reachstatistical significance (P � 0.093). These results indicate that theacinetobactin-mediated iron acquisition system plays a role in bacte-rial survival when tested in an in vivo experimental model.

In order to determine whether deficiencies in the acineto-bactin-mediated iron acquisition system also affect postinfectionmortality, mice were infected with differing inocula (1.7 � 104 to3.1 � 106 CFU) of the ATCC 19606T, s1, and t6 strains, and sur-vival was measured. As shown in Table 2, all mice infected with thehighest inoculum of the ATCC 19606T strain (1.9 � 106 CFU)succumbed to infection by 24 h. In contrast, infection with a sim-ilar inoculum of the s1 mutant (3.0 � 106 CFU) resulted in amortality of 66.7%, with a mean time to death of 36.0 � 13.9 h(P � 0.019). The t6 isogenic mutant also produced significantlyless mortality compared to the ATCC 19606T parental strain witha similar inoculum (3.1 � 106 CFU), with no mice succumbing toinfection within 7 days postinfection (P � 0.0009). Interestingly,comparison of mortality between the s1 and t6 strains also re-vealed that the s1 strain produced significantly higher mortalitywith a similar inoculum (approximately 3 � 106 CFU) thanthe t6 strain (P � 0.019). These results indicate that that theacinetobactin-mediated iron acquisition system is required forfull virulence in a mouse infection model and that lack of produc-

FIG 6 Postinfection tissue bacterial loads in mice infected with the ATCC19606T, s1, and t6 strains. C57BL/6 mice (n � 8 mice/group) were infectedwith �1 � 106 CFU of the indicated strain, and bacterial loads were measuredin spleen homogenates 16 h postinfection. Each symbol represents the bacte-rial load from an individual mice, with the horizontal line indicating the me-dian of the group. The asterisk indicates that the values for the ATCC 19606T

parental strain (19606) and the s1 and t6 iron-deficient isogenic derivativeswere statistically significantly different (P � 0.001).

TABLE 2 Virulence of A. baumannii strains in a mouse sepsis model

Strain Inoculum (CFU) Mortality (%)a MTTDb (h)

ATCC 19606T 1.9 � 106 100 24.0 � 0.01.0 � 105 33.3 42.0c

1.7 � 104 0 NA

s1 3.0 � 106 66.7 36.0 � 13.91.0 � 105 0 NA2.0 � 104 0 NA

t6 3.1 � 106 0 NA3.5 � 105 0 NA2.9 � 104 0 NA

a There were six mice in each group.b MTTD, mean time to death (hours). The mean � standard deviation are shown. NA,not applicable due to no deaths in the group.c Calculation of the standard deviation was not possible because there were only twomice.

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tion of the BauA acinetobactin outer membrane receptor resultsin a more attenuated phenotype than a BasD acinetobactin bio-synthesis deficiency.

DISCUSSION

Although previous work showed that the ability of A. baumanniiATCC 19606T to grow under iron-chelated laboratory conditionsdepends on the expression of a fully active acinetobactin-mediated iron acquisition system (20, 31), which is able to scav-enge iron from transferrin and lactoferrin (48), this system’s rolein biological models that mimic the conditions this pathogen en-counters in the human host has never been tested experimentally.Our data show that in contrast to bacterial adhesion and internal-ization roles played by some siderophore receptors, such as theIroN and IreA receptors produced by extraintestinal pathogenicEscherichia coli (24, 39), the absence of the BauA acinetobactinouter membrane receptor in the t6 derivative does not affect bac-terial attachment and internalization in human alveolar epithelialcells after 1 h of infection. Similarly, the lack of acinetobactinproduction by the s1 mutant does not impair bacterium-A549 cellmonolayer interactions, with both mutants producing a cyto-pathic response similar to that detected with the ATCC 19606T

parental strain. This response could be due to the expression ofbacterial functions other than those related to siderophore-mediated iron acquisition, such as the production of the outermembrane protein OmpA or unidentified OmpA-independentfactors that cause alterations in cellular morphology and apop-totic death (26). In contrast to initial bacterium-A549 cell inter-actions, the ability of A. baumannii ATCC 19606T to persist withinthis epithelial cell type depends on the active production of acin-etobactin and transport of ferric acinetobactin complexes as dem-onstrated by the intracellular cross-feeding of the s1 acinetobactinproduction-deficient derivative, but not the t6 acinetobactintransport mutant, by parental cells in coinfection assays. The de-tection of BauA in intracellular bacteria further supports this con-clusion and confirms the role of the acinetobactin system as animportant factor needed for persistence in human cells. All theseobservations are congruent with published reports indicating thatthe intracellular persistence and growth of some bacterial patho-gens, such as Bacillus anthracis (8) and Mycobacterium tuberculosis(17), depend on the expression of active siderophore-mediatedsystems that provide bacteria with essential iron while residingwithin human macrophages.

Considering the role the acinetobactin-mediated iron uptakesystem plays in the ability of A. baumannii ATCC 19606T to persistintracellularly, the differences in the A549 cell apoptotic responsedetected with the parental and isogenic iron uptake mutants couldreflect the amount of intracellular bacteria actively producingapoptotic factors, such as OmpA (10, 11, 26), a condition thatcould determine the extent of cellular damage. Alternatively, thesedifferences may reflect the amount of acinetobactin being pro-duced and secreted by intracellular bacteria. The presence of fer-riacinetobactin could contribute to cell injury by catalyzing theformation of damaging hydroxyl radicals in a manner similar tothat described with P. aeruginosa ferripyochelin and A549 alveolarepithelial cells (5, 13). It is also possible that the capacity of intra-cellular bacteria to produce acinetobactin determines the extent ofdepletion of reduced glutathione and increase in iron limitationbecause of the presence of this siderophore. These types of re-sponses were observed when neuroblastoma cells were treated

with catechol (29), a chemical moiety present in the acinetobactinmolecule. Certainly, these are interesting avenues that we plan toexplore to gain a better understanding of the mechanisms bywhich acinetobactin could negatively affect the viability of hostcells.

The critical role of the acinetobactin system is also evidentwhen tested using more-complex experimental models that in-clude host responses that pathogens must evade to persist andprosper after infection. Our data show that, unless free inorganiciron is in excess, ATCC 19606T acinetobactin production and uti-lization mutants are impaired in their ability to persist in and killG. mellonella caterpillars, a response similar to that of Photorhab-dus temperata. The capacity of this bacterium to grow under ironlimitation and interact with insect hosts depends on the produc-tion and utilization of the catechol-based siderophore photobac-tin, conditions that are circumvented by adding inorganic iron tothe growth media or the inocula (12, 46). It is interesting to notethat the killing of caterpillars by the ATCC 19606T strain wheninjected with supplemental iron is faster than the response ob-tained with infections in which the inocula were not supple-mented with inorganic iron, although the final numbers of deadworms are comparable over a 6-day time period under both con-ditions (compare Fig. 5B and C). These results can be attributed tothe elimination of the initial growth constraints imposed by the G.mellonella iron sequestration functions. Similarly, the data ob-tained with the mouse sepsis model show that inactivation of acin-etobactin biosynthetic and uptake functions drastically affect thecapacity of A. baumannii ATCC 19606T to infect and kill this ver-tebrate host as shown by the data presented in Table 2 and Fig. 6.Taken together, all these observations lend support to our hypoth-esis that acinetobactin-mediated iron acquisition functions arenecessary for A. baumannii to facilitate pathogenesis in eukaryoticcells and organisms expressing nonspecific defense functions.Furthermore, our results show that the A549 tissue culture, G.mellonella, and mouse sepsis experimental infection systems arevalid and convenient models to study the pathobiology of A. bau-mannii.

The results obtained with the three infection models used inthis work indicate that the ability of A. baumannii ATCC 19606T

to persist during infection of eukaryotic cells and invertebrate andvertebrate hosts that impose iron-limiting conditions depends onthe production of a fully active acinetobactin-mediated iron ac-quisition system. This observation supports our previous findingshowing that the inactivation of acinetobactin biosynthetic ortransport functions was enough to drastically reduce bacterialgrowth in laboratory media supplemented with the synthetic ironchelator DIP (20). Our results are also in agreement with the ob-servation that DIP-mediated iron starvation induces the tran-scriptional expression of acinetobactin synthesis and transportgenes as well as genes located in siderophore clusters 1 and 5 (21).However, the drastic growth attenuation of the s1 and t6 mutantsin all three experimental models indicates that these gene clusters maynot code for fully active iron uptake systems. Accordingly, cluster 5contains only two predicted genes coding for potential enzymaticactivities needed for 2,3-dihydroxybenzoic acid (DHBA) biosynthe-sis, which could complement or overlap functions coded for geneslocated in the acinetobactin cluster or cluster 1. Although the lattercluster contains 11 predicted genes coding for putative siderophorebiosynthesis and transport functions, our data are compatible withthe hypothesis that this cluster may represent an incomplete system

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acquired from a source different from that related to the acinetobac-tin cluster and whose function in iron uptake remains to be tested andvalidated experimentally.

It is interesting to note that, with the exception of the datadisplayed in Fig. 1, all the results obtained with the three experi-mental models used in this work showed that, although signifi-cantly attenuated compared to the parental ATCC 19606T strain,the s1 acinetobactin synthesis mutant has an intermediate pheno-type with respect to the t6 acinetobactin receptor mutant. Thisoutcome could be due to an increased iron-chelated environmentcreated by t6 cells producing but not using acinetobactin andtherefore enhancing iron restriction and making residual intracel-lular iron unavailable for bacterial growth. This possibility is sup-ported by our previous observation that the growth of the s1 mu-tant is less affected than the growth of the t6 derivative whencultured in chelated laboratory media (49). A similar outcome wasobtained when Yersinia pestis derivatives defective in yersiniabac-tin biosynthesis and transport functions were compared usingiron-deficient media, with the latter derivative being more ironsensitive than a yersiniabactin biosynthesis mutant (25). It is alsopossible that the enhanced phenotype of the s1 derivative com-pared to that of the t6 mutant could be due to the fact that theformer mutant still produces acinetobactin intermediates andprecursors such as DHBA, which showed intrinsic iron acquisi-tion functions involved in virulence as in the case of Brucella abor-tus (3, 34). These precursors and intermediates could bind iron,although with less affinity than acinetobactin, and potentiallyserve as a source used by the s1 derivative to better persist andconsequently cause more host injury than the t6 mutant. How-ever, such a hypothesis implies that BauA could play a role in therecognition and transport of some of these intermediates and pre-cursors if one considers the fact that the t6 mutant grows lessefficiently than the s1 mutant when both derivatives are culturedunder iron-chelated conditions. These possibilities will be testedby examining the iron uptake behavior and virulence response ofan ATCC 19606T derivative in which inactivation of the entA genewould abolish the production of the acinetobactin precursorDHBA.

In summary, our results indicate that the success of A. bau-mannii ATCC 19606T as a pathogen capable of affecting inverte-brate and vertebrate hosts and human epithelia depends on theproduction of an active acinetobactin-mediated iron acquisitionsystem, which allows bacteria to persist as well as to cause cellapoptosis and host killing. This property is most likely not re-stricted to the ATCC 19606T strain, since several A. baumanniisequenced and annotated genomes show the presence of the acin-etobactin gene cluster. However, the presence of gene clusterscoding for additional siderophore-mediated iron acquisitionfunctions may indicate that the virulence of these isolates is inde-pendent of the expression of the acinetobactin-mediated system, apossibility that can be tested using the experimental infectionmodels described in this report in combination with appropriateisogenic derivatives affected in iron acquisition functions.

ACKNOWLEDGMENTS

This work was supported by funds from Public Health AI070174 and NSF0420479 grants and Miami University research funds.

We are grateful to E. Lafontaine (College of Veterinary Medicine, Uni-versity of Georgia) for providing the A549 cell line. We thank RichardEdelmann, Matt Duley, and the staff at the Miami University Center for

Advanced Microscopy and Imaging for their help with electron and light/fluorescence microscopy. We also thank Xiao-Wen Cheng (Departmentof Microbiology, Miami University) for assistance with the technical de-sign of G. mellonella experiments and Michael Hughes and the staff at theMiami University Statistical Consulting Center for the guidance providedduring development and data analysis of the G. mellonella killing assays,respectively.

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