energy-generating enzymes of burkholderia cepacia and ... · including protease peptone yeast...

JOURNAL OF BACTERIOLOGY, May 2003, p. 3167–3178 Vol. 185, No. 100021-9193/03/$08.00�0 DOI: 10.1128/JB.185.10.3167–3178.2003Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Energy-Generating Enzymes of Burkholderia cepacia and TheirInteractions with Macrophages

Vasu Punj, Rachna Sharma, Olga Zaborina, and A. M. Chakrabarty*Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, Illinois 60612

Received 10 January 2003/Accepted 25 February 2003

We previously demonstrated that several clinical and environmental isolates of Burkholderia cepacia secretedATP-utilizing enzymes to the medium; the secretion of these enzymes by cystic fibrosis lung isolate strain 38was shown to be greatly enhanced in the presence of �2-macroglobulin. Fractionation of the growth mediumof cystic fibrosis isolate strain 71 belonging to genomovar I demonstrated the presence of two additionalproteins, homologues of Pseudomonas aeruginosa azurin and cytochrome c551, which are normally involved inelectron transfer during denitrification. A Q-Sepharose column flowthrough fraction of the growth medium ofB. cepacia strain 71 enriched with the azurin and cytochrome c551 homologues triggered apoptosis in macro-phages and mast cells, leading to their death. Incubation of the Q-Sepharose column flowthrough fraction withantiazurin and anti-cytochrome c551 antibodies greatly reduced cell death. We cloned and hyperexpressed agene from B. cepacia strain 71 that encodes the homologue of P. aeruginosa azurin. Such azurin homologueswere detected in the growth medium of several strains belonging to genomovars I, III, and VI but not in thegrowth medium of strains belonging to other genomovars. The growth medium of the strains that elaboratedthe azurin homologue had high cytotoxicity towards macrophages. Purified azurin homologue was shown toinduce apoptosis in macrophages in a caspase-dependent manner and was localized in both the cytosol andnucleus when incubated with or microinjected into macrophages. This is an interesting example of theinteraction of a bacterial protein normally involved in cellular energetics with macrophages to effect their cell death.

In the last decade, Burkholderia cepacia has emerged as amajor pathogen in the lungs of patients suffering from cysticfibrosis and in patients with chronic granulomatous disease(11, 21, 26, 45). The increasing incidence of infection with B.cepacia in cystic fibrosis patients is reminiscent of similar in-fections caused by mucoid strains of Pseudomonas aeruginosa(12, 32, 37). However, unlike P. aeruginosa, the populationstructures of B. cepacia as a group are complex and weredivided initially into five genomovars (52), which were furtherdifferentiated into new species (27, 53). More recently,polyphasic studies and improved 16S ribosomal DNA-basedPCR assays have allowed sensitive and specific identification ofvarious bacterial species, such as Achromobacter xylosoxidansand Ralstonia pickettii in addition to B. cepacia and other pre-viously recognized members of the B. cepacia complex presentin the sputum and lungs of cystic fibrosis patients (3, 24). Thepresence of such hitherto unrecognized bacterial species aswell as other Burkholderia species such as B. anthina and B.pyrrocinia (54), has added a degree of complexity to an under-standing of the taxonomy, epidemiology, and pathogenesis ofthe multitude of strains isolated from the lungs of cystic fibrosispatients that are now collectively called B. cepacia complex(28).

Many environmental B. cepacia and Burkholderia sp. strainsare efficient biodegraders of toxic chemicals (5, 20, 25, 43, 47)as well as very effective in controlling plant pathogenic soilfungi and nematodes (11, 13). Thus, they have been proposedfor environmental release for purposes of toxic chemical biore-

mediation and enhanced agricultural productivity. Very little,however, is known about the potential pathogenicity of suchstrains recommended for application in an open environment,leading to the expression of profound concerns about the wis-dom of such releases (15, 22). Of particular concern are recentreports that the virulent genomovar III and other strains canbe isolated from soil and cannot be easily differentiated fromenvironmental isolates (2, 23).

The virulence factors elaborated by B. cepacia are largelyunknown (11), although hemolysins and exopolysaccharides(14, 19, 40) have been implicated in its pathogenicity. Werecently demonstrated that, similar to clinical isolates of P.aeruginosa, clinical isolates of B. cepacia secrete a number ofATP-utilizing enzymes that modulate the level of external ATPeffluxed from phagocytic cells, leading to their death. Severalenvironmental strains were found to be deficient in the releaseof these enzymes that acted as potent cytotoxic factors in thepresence of millimolar concentrations of ATP. We furtherdemonstrated that secretion of the ATP-utilizing enzymes by acystic fibrosis isolate, strain 38, of B. cepacia was greatly en-hanced in the presence of a mammalian protein such as �2-macroglobulin (33). Nothing is known about whether the �2-macroglobulin-mediated enhancement of secretion is uniqueto this strain or occurs in other clinical or environmentalstrains or whether secretion of other potential virulence factorsmay also be modulated by �2-macroglobulin. It is also notknown if B. cepacia secretes ATP-utilizing enzymes that act inan ATP-inducible manner or secretes other cytotoxic factorsthat may operate by an ATP-independent pathway, as reportedfor P. aeruginosa (58, 59).

In this paper, we report the secretion of two redox proteins,homologues of P. aeruginosa azurin and cytochrome c551, and

* Corresponding author. Mailing address: Dept. of Microbiologyand Immunology, University of Illinois College of Medicine, 835 SouthWolcott Avenue, Chicago, IL 60612. Phone: (312) 996-4586. Fax: (312)996-6415. E-mail: [email protected].

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demonstrate that secretion of the azurin homologue correlateswith the cytotoxicity demonstrated by the growth medium of B.cepacia genomovars I, III, and VI. Additionally, we demon-strate the induction of apoptosis in macrophages by purifiedpreparations of the azurin homologue, establishing it as a po-tential virulence factor.

MATERIALS AND METHODS

Bacterial strains and media. B. cepacia strains were grown in several media,including protease peptone yeast extract (PPY; 10 g of peptone, 1.5 g of yeastextract, 10 g of sucrose, 5 g of sodium chloride in 1 liter of water), tryptone beefextract medium (TB; 10 g of Bacto tryptone, 3 g of Bacto beef extract in 1 literof water), and Luria broth (LB) medium. A list of the strains used in variousexperiments is shown in Table 1. All cultures were seeded directly from glycerolstocks to avoid variation in phenotypic characteristics as a result of genomicplasticity. All cultures were grown on rotary shakers at 32°C.

Column chromatographic fractionation of culture supernatant. B. cepaciastrain 71 was grown in TB medium to an A600 of 1.1 at 32°C. The cells werecentrifuged; the supernatant was filtered through 0.22-�m filters and concen-trated with an Amicon YM-10 membrane. This supernatant was fractionated onhydroxyapatite, ATP-agarose, and Q-Sepharose columns as described previously(59). The Q-Sepharose column flowthrough fraction and a portion of eachcolumn flowthrough fraction were concentrated, and 2 �g of protein from eachsample was used for the cytotoxicity assay. Part of the Q-Sepharose fraction wasrun on sodium dodecyl sulfate–4 to 20% polyacrylamide gel electrophoresis(SDS–4 to 20% PAGE) gel, followed by transfer to a polyvinylidene difluoridemembrane for N-terminal amino acid sequence analysis with an automatedEdman AB1477A protein sequencer.

Cloning of azurin homologue of B. cepacia. The azurin-encoding gene (azu)was amplified by PCR with B. cepacia genomic DNA as a template with theprimers 5�-GCCAAGCTTATGCTACGTAAACTC-3� (forward) and 5�-GCCCTGCAGCGCGCCCATGAAAAAGCC-3� (reverse). The HindIII and PstI re-striction enzyme sites were included within the primer to facilitate cloning. ThePCR-amplified product was cloned in vector pUC19. The recombinant plasmidwas transformed into Escherichia coli JM109 host cells, which were used forhyperexpression of the B. cepacia azu gene induced in the presence of 1 mMisopropyl-�-D-thiogalactopyranoside (IPTG). The hyperexpressed azurin was pu-rified from the periplasmic fraction of E. coli according to the method describedpreviously (18).

Macrophage cytotoxicity assay. Macrophages derived from J774 cell lines weregrown in RPMI 1640 medium containing L-glutamine, buffered with 10 mMHEPES, and supplemented with 10% fetal bovine serum. Cytotoxicity was de-termined by measuring the release of lactate dehydrogenase from macrophagesas described earlier (39). Cytotoxicity of the azurin homologue in J774 cells wasdetermined with the Cell Titer 96 Aqueous One solution cell proliferation assay(Promega Corporation, Madison, Wis.) as recommended by the manufacturer.Briefly, J774 macrophage cells were plated on 96-well culture plates (104 cells perwell) and incubated overnight at 37°C. Subsequently, the cells were treated withthe azurin homologue in various doses for various times. Untreated cells andcells treated with Triton X-100 were used as negative and positive controls,respectively. At the end of the incubation, 20 �l of the assay solution containinga tetrazolium compound (MTS; [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxyme-thoxyphenyl)-2-(4-sulfophenyl)]-2H-tetrazolium) and an electron-coupling re-agent (phenazine methosulfate) was added to each well, and the cells were

incubated for another 3 h at 37°C in a humidified 5% CO2 incubator. Theamount of soluble formazan produced by cellular reduction of MTS was mea-sured by reading absorbance at 490 nm, as described by Vairano et al. (51).

Antibody production and immunoblot analysis. The antigenic profile of theazurin sequence was analyzed with ABI antigen prediction software (56). Anti-bodies were raised corresponding to the predicted highly antigenic amino acidsequence CKQFTVNLSHPGNLPKN (amino acids 46 to 52). Immunization ofrabbits was carried out according to standard protocols, and the titer of anti-bodies in the serum was determined by indirect enzyme-linked immunosorbentassay.

Immunoblot analysis was performed by electrotransfer of proteins after SDS-PAGE to a Sequi Blot 0.2-�m polyvinylidene difluoride membrane (Bio-Rad)followed by incubation with primary antibodies. Western blotting was done withanti-rabbit immunoglobulin G labeled with horseradish peroxidase and detectedwith the ECL system (Amersham Biosciences).

Detection of DNA fragmentation by TUNEL assay. For the terminal de-oxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling (TUNEL)assay, the ApoAlert DNA fragmentation assay kit (Clontech, Palo Alto, Calif.)was used, and the assay was performed as recommended by the manufacturer.Briefly, macrophages were seeded at a density of 105 cells per ml on LabTekchamber slides for 2 h. The macrophages were then treated with 200 �g of azurinhomologue per ml for different time intervals. Simultaneously, cells were treatedwith phosphate-buffered saline (PBS) (untreated) or 50 �M benzyloxycarboxylVal-Ala-Asp fluoromethyl ketone (ZVAD-FMK; Clontech), a cell-permeatinggeneral caspase inhibitor. The cells were washed and fixed in 4% paraformalde-hyde–PBS and permeabilized with chilled 0.2% Triton X-100–PBS for 5 min onice. The slides were washed with PBS and equilibrated with equilibration buffer.

The tailing reaction was performed with the ApoAlert DNA fragmentationassay kit. A total of 50 �l of terminal deoxynucleotidyltransferase (TdT) mixture,consisting of 44.5 �l of equilibration buffer, 5 �l of nucleotide mixture, and 0.5�l of TdT enzyme, was evenly spread on the treated area and incubated in ahumid chamber at 37°C for 1 h. The reaction was terminated by incubating theslides with 2� SSC (1� SSC is 0.15 M NaCl plus 0.015 M sodium citrate) for 15min at room temperature. The cells were then stained with 1 �g of propidiumiodide per ml and washed. After the assay, a drop of antifade solution was added,and the treated portion of the slide was covered with a coverslip and the edgeswere sealed with clear nail polish. Slides were viewed within 2 h under an LSM510 confocal laser microscope equipped with a 40� objective and a dual filter setfor green fluorescence (488 nm) and red fluorescence (568 nm).

Preparation of cytosolic macrophage extracts for caspase assays. The cytoso-lic extracts were prepared, and caspase-3 and caspase-9 activities were deter-mined in these extracts as described by Zhou et al. (60). Briefly, cells werewashed with ice-cold phosphate-buffered saline and lysed in cell lysis buffer (50mM HEPES [pH 7.4], 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-pro-panesulfonate [CHAPS], 5 mM dithiothreitol, 0.1 mM EDTA). After centrifu-gation at 12,000 � g at 4°C for 10 min, the supernatant (cytosol) was used todetermine caspase activities. Caspase-3 activity was determined with the Apo-Alert caspase-3 assay kit (Clontech) in accordance with the manufacturer’srecommendations, based on spectrophotometric detection of the chromophorep-nitroaniline (pNA) released from the substrate Ac-DEVD-pNA (N-acetyl-Asp-Glu-Val-Asp-p-NO2-aniline) at 405 nm. For caspase-9, the caspase-9 color-imetric assay kit (Chemicon International, Temecula, Calif.) was used, whichinvolves release and quantitation of pNA from the substrate Ac-LEHD-pNA(N-acetyl-Leu-Glu-His-Asp-p-NO2-aniline). Specific inhibitors of caspase-3(DEVD-FMK) and caspase-9 (Ac-LEHD-CHO; Calbiochem-Novabiochem

TABLE 1. Bacterial strains used in this study

Strain Burkholderia species Genomovar Source or reference

PC783 B. cepacia complex I J. LiPuma, Univ. of MichiganHI2209 B. multivorans II J. LiPuma, Univ. of MichiganHI2147 B. cenocepacia III J. LiPuma, Univ. of MichiganHI2210 B. stabilis IV J. LiPuma, Univ. of MichiganHI2221 B. vietnamiensis V J. LiPuma, Univ. of MichiganAU0649 B. cepacia complex VI J. LiPuma, Univ. of MichiganHI2468 B. ambifaria VII J. LiPuma, Univ. of MichiganHI2725 B. anthina VIII J. LiPuma, Univ. of MichiganPC85 B. pyrrocinia IX J. LiPuma, Univ. of MichiganCEP 509 (referred to as strain 71 in the text) B. cepacia complex I 27

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Corp., La Jolla, Calif.) were used to determine the specific activation of the twocaspases in cells treated with the azurin homologue.

Subcellular fractionation of macrophages. Macrophages were treated with 200�g of the azurin homologue per ml for 0, 3, 6, and 12 h. Cytosolic extracts andnuclear extracts were prepared essentially by the procedure of Deveraux et al.(7). Briefly, cells were pelleted by centrifugation after washing with cold buffer A(20 mM HEPES [pH 7.5], 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mMdithiothreitol, and protease cocktail inhibitor). Subsequently, cells were resus-pended in the same buffer, incubated for 20 min on ice, and disrupted by 25passages through a 26-gauge needle. Cell extract was clarified first by low-speedcentrifugation and then by centrifugation at 15,000 � g at 4°C for 30 min.

Intracellular localization of azurin homologue. Macrophages (107 cells) wereused for subcellular fractionation after azurin treatment (200 �g/ml) for varioustime intervals. A total of 20 �g of protein from each time point was loaded onSDS-PAGE gels and blotted on a 0.2-�m polyvinylidene difluoride membrane(Bio-Rad, Hercules, Calif.). The azurin was detected with antiazurin antibody.

Microinjection of azurin homologue in J774 macrophages. Macrophages werecultured overnight on 22-mm glass coverslips coated with collagen adhered to adish. The azurin homologue labeled red with Alexa fluor 568 (Molecular Probes,Eugene, Oreg.) was microinjected into the cytoplasm of single cells with acomputer-controlled microinjector (AIS 2) system. All microinjection experi-ments were performed with a 0.5-s injection time and 100 hPa of pressure witha Zeiss 200 M microscope. Approximately 25 to 50 cells were injected in eachdish. After microinjection, cells were further incubated at 37°C for various times.Cells were then fixed, nuclear DNA was stained blue with 4�,6�-diamidino-2-phenylindole (DAPI), and fluorescent images were taken with a Zeiss LSM 510confocal laser microscope.

Detection of cytochrome c release. The cytosolic extracts were prepared frommacrophages either untreated or treated with the azurin homologue for varioustime periods. These extracts were subjected to SDS-PAGE electrophoresis andtransferred to polyvinylidene difluoride membranes, and the Western blot wasdeveloped with anti-cytochrome c monoclonal antibody. To determine the re-lease of mitochondrial cytochrome c by confocal microscopy, macrophages weregrown on coverslips overnight, treated with 200 �g of azurin homologue per ml,and incubated for different time intervals. The release of cytochrome c wasdetected by immunofluorescence following the procedure of Pervaiz et al. (36)with minor modifications. Briefly, cells were fixed with methanol-acetone (1:1,vol/vol) and incubated with blocking solution (3% bovine serum albumin) over-night at 4°C. The cells were then incubated for 2 h with mouse monoclonalanti-cytochrome c antibody (clone 6H2.B4; BD Biosciences, San Diego, Calif.).After three washes with PBS–1% fetal bovine serum, cells were exposed tofluorescein isothiocyanate-conjugated anti-mouse immunoglobulin G (SigmaChemical Co., St. Louis, Mo.). Cells were washed extensively, mounted withantifade Vectashield solution with DAPI, and analyzed by confocal microscopy.Cytosolic cytochrome c (fluorescing green) showed a diffuse staining patterncompared to punctate mitochondrial cytochrome c staining in untreated cells.

Nucleotide sequence accession number. The DNA sequence encoding theazurin homologue has been given GenBank accession number AY238602.

RESULTS

Fractionation of growth medium of cystic fibrosis isolate B.cepacia strain 71 and assay for cytotoxicity of different frac-tions. We previously reported that several clinical isolates of B.cepacia secreted ATP-utilizing enzymes to the medium thatcaused macrophage and mast cell death through activation ofpurinergic receptors (33). To examine if B. cepacia producescytotoxic agents that operate through an ATP-independent(purinergic receptor-independent) pathway, we fractionatedthe growth medium (supernatant) of the cystic fibrosis isolateB. cepacia strain 71 (Table 1) by column chromatography. Thesupernatant growth medium remaining after centrifugation ofcells was passed through hydroxyapatite, ATP-agarose, andQ-Sepharose columns, and the flowthrough fractions fromeach column were assayed for ATP-utilizing enzyme activityand cytotoxicity, as measured by release of lactate dehydroge-nase from macrophages and mast cells.

Most of the ATP-utilizing enzymes were removed during

column chromatography, leaving the Q-Sepharose flowthrough(QSFT) fraction virtually free of ATP-utilizing enzymes. Thecytotoxicity of the supernatant, hydroxyapatite columnflowthrough, ATP-agarose column flowthrough, and QSFTfractions to J774 cell line-derived macrophages in the absenceand in the presence of 1.0 mM ATP is shown in Table 2. At 1.0mM, ATP itself had substantial cytotoxicity (22%), as reportedpreviously (39). The supernatant, hydroxyapatite flowthrough,and ATP-agarose column flowthrough fractions had low cyto-toxicity by themselves (in the absence of ATP), but the cyto-toxicities were significantly higher in the presence of ATP. TheQSFT fraction, on the other hand, had high cytotoxicity, whichcould not be further enhanced in the presence of ATP (Table2). Thus, the growth medium of B. cepacia strain 71 had ATP-independent cytotoxic activity as well.

To see if the ATP-independent cytotoxicity was limited tomacrophages or might also affect other phagocytic cells, we ex-amined the cytotoxicity of the strain 71 QSFT fraction towardsmast cells. The supernatant, hydroxyapatite flowthrough, andATP-agarose flowthrough fractions demonstrated low cytotox-icity, while the QSFT fraction showed high cytotoxicity in theabsence of ATP (data not shown), confirming that the cyto-toxic agent(s) present in the QSFT fraction is active againstmast cells as well.

Presence of homologues of azurin and cytochrome c551 inQSFT fraction. In order to determine the nature of the pro-teins present in the QSFT fraction that might be responsiblefor the death of macrophages and mast cells, we ran the super-natant, ATP-agarose flowthrough, hydroxyapatite flowthrough,and QSFT fractions on SDS-PAGE. While the supernatant,ATP-agarose flowthrough, and hydroxyapatite flowthroughfractions had multiple protein bands, the QSFT fraction hadthree major protein bands of 8 kDa, 21 kDa, and 75 kDa (datanot shown). N-terminal amino acid sequencing of the 8-kDa(EDPEVLFKNK) and the 21-kDa (AXXSVDIQGN, where Xis a residue of uncertain identity) bands showed 100% and 80%sequence identity with that of P. aeruginosa cytochrome c551

and the copper-containing redox protein azurin, respectively(55). We have yet to characterize the 75-kDa protein.

To analyze whether the azurin and cytochrome c551 homo-logues of B. cepacia were indeed responsible for the deathcaused by the QSFT fraction, we determined if a mixture ofantiazurin and anti-cytochrome c551 (2 �g of each) antibodies

TABLE 2. Cytotoxicity of supernatant and various flowthroughchromatographic fractions from hydroxyapatite, ATP-agarose,

and Q-Sepharose columnsa

FractionCytotoxicity (% cell death)

With ATP Without ATP

None (buffer) 22.14 � 4.2 NDb

Supernatant 54.07 � 4.4 13.62 � 4.5Hydroxyapatite FT 45.17 � 3.3 16.97 � 4.1ATP-agarose FT 52.69 � 2.5 19.98 � 3.2QSFT 43.03 � 5.1 45.82 � 5.2

a Cytotoxicity inducible with 1.0 mM ATP and ATP-independent cytotoxicitywere determined in J774 cell line-derived macrophages by release of the cyto-plasmic enzyme lactate dehydrogenase as described earlier (39). We used 2 �g ofprotein from each fraction. Numbers represent percent cell death � standarddeviation when triplicate determinants were available. FT, flowthrough.

b ND, not detectable.

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would have any effect on the cytotoxicity exhibited by the B.cepacia QSFT fraction. Polyclonal antibodies against purifiedazurin and cytochrome c551 were prepared for this purpose.When a mixture of azurin and cytochrome c551 was tested forcytotoxicity against J774 cell line-derived macrophages, highcytotoxicity was observed (Fig. 1, column A�C). When thismixture was incubated with a mixture of antiazurin and anti-cytochrome c551 antibodies (4 �g of protein) and then testedfor macrophage cytotoxicity, the cytotoxicity was greatly dimin-ished (Fig. 1, column A�C�Ab4), suggesting that antibodytreatment neutralizes the cytotoxicity mediated by azurin pluscytochrome c551.

When the B. cepacia QSFT fraction was tested for cytotox-icity, high cytotoxicity was also observed (Fig. 1, columnQSFT). Pretreatment of the QSFT fraction with increasingamounts (0.5 to 4 �g of protein) of antiazurin and anti-cyto-chrome c551 antibodies greatly reduced this cytotoxicity (Fig. 1,columns QSFT�Ab 0.5 to Ab 4), demonstrating a major roleof azurin and cytochrome c551 in the QSFT fraction for im-parting cytotoxicity. Treatment with increasing amounts (0.5 to4 �g of protein) of preimmune serum, however, did not lead toappreciable loss of cytotoxicity (data not shown), confirmingthat the antiazurin and anti-cytochrome c551 antibodies led toneutralization of homologues of such proteins in the QSFT

fraction, thereby contributing to the loss of cytotoxicity. Wehave not yet determined the role played by cytochrome c in B.cepacia virulence. The cloning of its gene and its hyperexpres-sion are currently under investigation.

Cloning and hyperexpression of gene encoding B. cepaciaazurin homologue. While the loss of cytotoxicity of the QSFTfraction on treatment with antiazurin plus anti-cytochrome c551

antibodies provided insights into the potential role of theseredox proteins in cytotoxicity, it was necessary to test the in-volvement of the azurin and cytochrome c551 homologues in B.cepacia virulence directly. As a first step towards this goal, wecloned the azu gene encoding the azurin homologue from B.cepacia as described under Materials and Methods. The aminoacid sequence of the B. cepacia strain 71 (genomovar I) azugene product showed significant sequence identity with theazurin sequences of a number of other bacteria (Fig. 2), in-cluding some, such as Achromobacter xylosoxidans, which canalso be recovered from the sputum of cystic fibrosis patients(24).

We explored if the azurin homologue could be detected inthe growth medium of B. cepacia strain 71 during growth in Lbroth. Western blotting data demonstrated the presence of theazurin homologue in the growth medium filtered through a0.22-�m filter during early to mid-log phase (data not shown).

FIG. 1. Neutralization of macrophage cytotoxicity by a mixture of antiazurin and anti-cytochrome c551 antibodies. Macrophage cytotoxicity wasdetermined by the lactate dehydrogenase (LDH) release assay. A mixture of azurin and cytochrome c551 (A�C) was used as a positive control.The cytotoxicity associated with this combination was neutralized by treatment with antibodies against azurin and cytochrome c551 (A�C�Ab 4).When macrophages treated with various dilutions of antibody mixtures (Ab 0.5 to Ab 4) were treated with 2 �g of the QSFT fraction, a gradualdecrease in cytotoxicity was observed with increasing concentrations of antibody mixture. The numbers after Ab represent micrograms of protein.Incubation with preimmune serum, even at high concentrations, had no effect on cytotoxicity (data not shown).



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Then we explored if the azurin homologue could be detected inthe filtered growth medium of various B. cepacia genomovars,as depicted in Table 1. Of the nine genomovars tested, onlystrain 71 (genomovar I), strain PC783 (genomovar I), strainHI2147 (genomovar III), and strain AU0649 (genomovar VI)demonstrated the presence of an azurin homologue in thegrowth medium (Fig. 3A). All the other strains were negativein this respect.

Similar to the ATP-utilizing enzymes whose secretion wasstimulated in the presence of 1 mg of �2-macroglobulin per mlin the growth medium (33), the secretion of the azurin homo-logue was stimulated to various degrees during growth of thefour strains in the presence of 1 mg of �2-macroglobulin per ml(Fig. 3B). When the growth medium (5 �g of protein each)from the above strains was examined for cytotoxicity towardsmacrophages, significant cytotoxicity was observed only withthe growth medium of the strains that secreted azurin homo-logues in significant amounts (Table 3). The high cytotoxicity

thus reflects a high level of secretion of azurin when the cellswere grown in the presence of �2-macroglobulin.

B. cepacia azurin homologue induces apoptosis in macro-phages. Since our primary goal was to study the interaction ofredox proteins such as azurin with macrophages, it was impor-tant for us to evaluate the role of the B. cepacia azurin homo-logue in macrophage cytotoxicity. The cytotoxicity was deter-mined by the MTS assay as described under Materials andMethods. Recombinant B. cepacia azurin homologue demon-strated high cytotoxicity towards J774 cell line-derived macro-phages, and this cytotoxicity was concentration dependent(Fig. 4A). When the azurin homologue was used at 200 �g/mlfor various time periods, the maximal cytotoxicity was reachedbetween 24 and 32 h (Fig. 4B).

To define the nature of the macrophage cell cytotoxicity, wetested to see if the purified azurin homologue could cause celldeath through the induction of apoptosis. One of the hallmarksof apoptosis is DNA fragmentation; we used the TUNEL assay

FIG. 2. Multiple alignment of B. cepacia azurin protein homologue sequence with sequences of azurin from various organisms. Clustal Wsoftware was used to generate the alignment. �, amino acids conserved among all the proteins; ∧, amino acids conserved in at least three of sixorganisms; #, nonconserved amino acids (50). The signal sequence (amino acids 1 to 18) is highly variable among all the proteins.



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(8) to determine the extent of incorporation of fluorescentlytagged fluorescein-dUTP (Clontech ApoAlert DNA fragmen-tation assay kit) into the nuclear DNA of untreated and azurin-treated macrophages. Extensive dUTP incorporation occursonly in apoptotic cells with considerable nuclear DNA frag-mentation, giving rise to cells in which the nuclear DNA flu-oresces green. Untreated macrophages did not show any sig-nificant nuclear green fluorescence when incubated withfluorescein-dUTP in the TUNEL assay (Fig. 5A, untreated),showing little dUTP incorporation in nuclear DNA. Whenmacrophages were treated with the B. cepacia azurin homo-logue for 6 h, 12 h, and 16 h, increasing green fluorescence-tagged macrophages were seen (Fig. 5A, as indicated) underthe green channel (bottom, left); the red channel (top, left)

showed all macrophages, both healthy and apoptotic. The su-perimpositions of the red and green channels showed macro-phages with those incorporating dUTP in their DNA as yellow(lower right-hand channels in Fig. 5A). When macrophageswere treated with the azurin homologue in the presence of thegeneral caspase inhibitor ZVAD-FMK, very few cells wereseen to undergo apoptotic changes (Fig. 5B).

One of the mitochondrial events associated with apoptosis isan alteration in the mitochondrial membrane potential, lead-ing to cytosolic translocation of cytochrome c from the inter-membrane space of mitochondria (1). We therefore measuredthe release of macrophage mitochondrial cytochrome c to thecytosol by confocal microscopy. In macrophages treated withbuffer (untreated) or after 6 h of treatment with the azurinhomologue (200 �g/ml), most of the cytochrome c was foundas granules in the mitochondria showing a punctate stainingpattern (Fig. 6A). After 12 h of treatment, however, the cyto-chrome c was found throughout the cytosol, showing diffusestaining in a few macrophages undergoing apoptosis (Fig. 6A).When the cytosolic fractions (30 �g of protein each) free ofmitochondria were examined with anti-cytochrome c antibod-ies by Western blotting, very little cytochrome c was detected

FIG. 3. Release of azurin homologue during growth of variousstrains of B. cepacia complex in L broth as detected by Westernblotting. Even though all the strains (Table 1) were tested, only thosethat released the azurin homologue are shown (A). These strains alsoshowed an enhanced level of azurin in the growth medium when theywere grown in the presence of �2-macroglobulin (1 mg/ml) (B). All thestrains were grown in the presence and absence of �2-macroglobulin toan optical density at 600 nm of �1.1. The cells were then centrifuged,supernatants were filtered through a 0.22-�m filter, and high-molecu-lar-weight proteins were separated by passage through CentriconYM100 (Amicon) and precipitated with ammonium sulfate. Excess saltwas removed by extensive washing in Centricon YM10, and then 30 �gof proteins was separated on SDS-PAGE and blotted with antiazurinantibodies to detect the presence of the azurin homologue as describedin Materials and Methods. The amount of azurin was quantified withNucleoTech Gel Expert 97 software.

FIG. 4. Cytotoxicity of the azurin homologue of the B. cepacia towards J774 cells as determined by MTS assay as described in Materials andMethods. (A) Cytotoxicity of macrophages treated for 24 h with various concentrations of the azurin homologue. (B) Cytotoxicity of macrophagestreated with 200 �g of the azurin homologue per ml for various times.

TABLE 3. Cytotoxicity of supernatants of various strains grown inthe presence of �2-macroglobulina

Strain Cytotoxicity(% cell death)

71 100.0PC783 61.7HI2147 65.9AU0649 38.2HI2209 6.4HI2210 10.6HI2221 8.7HI2468 13.2HI2725 6.3DC85 11.4

a The cytotoxicity of supernatant fractions (5 �g of protein) of each strain wasdetermined as described in Materials and Methods. The cytotoxicity of B. cepaciastrain 71 was set at 100%.



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in the cytosol after 3 or 6 h of treatment with the azurinhomologue, but some accumulation was observed after 12 h oftreatment (Fig. 6B). When the level of �-actin, a cytosolicprotein, was examined as an internal control by Western blot-ting, its level remained fairly constant throughout the treat-ment process, suggesting that 12 h of treatment of macro-phages with the azurin homologue elicited the release ofmitochondrial cytochrome c to the cytosol before the onset ofapoptosis mediated by the azurin homologue.

Since release of mitochondrial cytochrome c to the cytosoltriggers caspase cascade activation (1), we assayed the level oftwo members of the caspase cascade, caspase-3 and caspase-9,that are known to be activated during apoptosis due to releaseof mitochondrial cytochrome c (42, 46). Macrophages wereincubated for various time periods in the absence or in thepresence of the azurin homologue (200 �g/ml) and washedthoroughly, and cytosolic extracts were prepared as describedin Materials and Methods. Caspase-3 activity was assayed with60 �g of cytosolic protein extract with the colorimetric sub-strate Ac-DEVD-pNA at 37°C for 1 h. Caspase-9 was assayedsimilarly with Ac-LEHD-pNA as a substrate. Specific inhibi-

tors of both caspase-3 (Ac-DEVD-FMK) and caspase-9 (Ac-LEHD-CHO) were also included to determine the specificityof the caspase reactions. The results clearly demonstrated thatboth caspase-3 and caspase-9 (Table 4) activities increasedwith time in macrophage cytosolic extract when the macro-phages were treated with the azurin homologue. The caspaseactivities remained low when the macrophages were treatedwith buffer (untreated) and when the extracts from treatedmacrophages were assayed in the presence of caspase inhibi-tors.

Trafficking of azurin homologue to macrophage nucleus.Since incubation of macrophages with the purified azurin ho-mologue protein triggered apoptosis, it is clear that the azurinhomologue either exerts its effect as a surface-bound protein orenters the cell cytosol. The role of any macrophage surfacereceptor(s) in the internalization of the azurin homologue isunknown at present. To evaluate the localization of the azurinhomologue in J774 cell line-derived macrophages, we incu-bated the macrophages for 0, 3, 6, and 12 h, washed them,made extracts, and isolated various subcellular fractions. West-ern blotting was then performed with antiazurin antibody with

FIG. 5. TUNEL assay for detection of apoptosis-induced nuclear DNA fragmentation in azurin homologue-treated macrophages. The assay isbased on terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling, where Tdt catalyzes the incorporation of fluorescein-dUTP atthe free 3�-hydroxyl ends of fragmented DNA in cells undergoing apoptosis. The incorporation of fluorescein-dUTP into the fragmented nuclearDNA generates the green fluorescence detected by confocal microscopy. (A) J774 cell-line derived macrophages were grown on LabTek chamberslides and incubated for 6, 12, and 16 h. A negative control (untreated) without azurin treatment (treated with TM buffer for 16 h) was alsomaintained. Macrophages viewed under both red and green channels are shown. (B) Macrophages treated with azurin homologue (16 h) and 50�M general caspase inhibitor ZVAD-FMK, showing inhibition of DNA fragmentation induced by the azurin homologue.



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20 �g of protein from each fraction. We also microinjected theazurin homologue into the macrophage cells and used confocalmicroscopy to detect the presence of azurin in the cytosolic andnuclear fractions.

When the purified azurin homologue was incubated with themacrophages and subcellular fractions (cytosol and nuclear)were examined, azurin was found (Fig. 7A) in the cytosolicfraction both during early (3 h) and later (6 h and 12 h) periodsof incubation. In contrast, very little azurin homologue was

found in the nuclear fraction at 3 h but was detected in largeamounts at 6 h and 12 h of incubation (Fig. 7A). We alsomicroinjected the azurin homologue directly into the macro-phage cytosol to examine its nuclear trafficking. Confocal mi-croscopy of the microinjected macrophages confirmed thepresence of the azurin homologue in the cytosol at 15 min; by3 h, azurin was also found in the nucleus (Fig. 7B), suggestingthat the cytosolic azurin homologue can traffic to the nucleusduring incubation of the macrophages with purified protein or

FIG. 6. Time course of mitochondrial cytochrome c release into the cytosol of macrophages during treatment with the azurin homologue.(A) Macrophages were grown on coverslips and treated with the azurin homologue (200 �g/ml) for the indicated times; mitochondrial cytochromec localization was determined by confocal microscopy with anti-cytochrome c antibody as described in Materials and Methods. The nucleus wasstained blue with DAPI. (B) Cytosolic extracts of azurin homologue-treated and untreated (0 h) macrophages taken at the times indicated wereseparated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and subjected to Western blot analysis with mitochondrialanti-cytochrome c (Cyt C) antibody.

TABLE 4. Measurement of caspase activities in cytosolic extracts of macrophages treated or not with the azurin homologuea

Extract

Mean activity (nmol of pNA/h) � SD

Caspase-3 Caspase-9

6 h 12 h 16 h 6 h 12 h 16 h

Untreated control 2.6 � 1.2 2.8 � 1.6 3.1 � 0.6 7.8 � 1.2 6.5 � 0.7 5.9 � 0.3Treated 9.2 � 0.5 34.2 � 1.1 47.3 � 0.2 9.6 � 1.1 19.2 � 2.2 26.5 � 0.2Treated � inhibitor 3.3 � 0.2 2.6 � 0.9 8.3 � 1.1 8.2 � 1.0 9.2 � 1.3 7.3 � 1.3

a Caspase-3 activity was determined with the ApoAlert caspase-3 assay kit (Clontech). The release of p-nitroaniline (pNA) was measured spectrophotometrically at405 nm from the caspase-3 substrate Ac-DEVD-pNA with or without the caspase-3-specific inhibitor Ac-DEVD-FMK. Caspase-9 activity determination involved therelease of pNA from 200 �M Ac-LEHD-pNA as a substrate after various periods of treatment with the azurin homologue, with or without the caspase-9-specificinhibitor Ac-LEHD-CHO. In each case, cytoslic extracts of buffer-incubated (untreated) macrophages were used as a negative control. Results shown are means �standard deviation of triplicate experiments.



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after microinjection of the azurin homologue into the macro-phage cytosol.

DISCUSSION

One of the goals of this study was to understand how bac-terial enzymes that are normally involved in cellular energeticsinteract with mammalian cells such as macrophages, ultimatelytriggering their death. The role of ATP-utilizing enzymes inthe induction of macrophage cell death has been reportedpreviously for both P. aeruginosa (58) and B. cepacia (33). Boththe Pseudomonas and Burkholderia genera are well known fortheir nutritional versatility as well as for their ability to causefatal infections in the lungs of cystic fibrosis patients, yet thenature and outcome of such infections are often different. P.aeruginosa is an extracellular pathogen that causes chronicinfections in cystic fibrosis patients; in contrast, B. cepacia isknown to be capable of some intracellular replication (30, 31,41) and causes rapid morbidity and mortality in such patients.This suggests that the nature of the virulence factors or the wayin which they act must be different.

Both P. aeruginosa and B. cepacia elaborate ATP-utilizingenzymes that have been reported to cause macrophage celldeath; however, it is not known if they act similarly. For ex-

ample, ADP-ribosylating enzymes and toxins of P. aeruginosasuch as exotoxin A and exoenzyme S, as well as cholera toxinproduced by Vibrio cholerae, even though they have the com-mon ADP-ribosylating activity, all have different targets anddifferent modes of action.

We have reported that P. aeruginosa elaborates azurin andcytochrome c into the growth medium that induce cell deaththrough complex formation with and stabilization of tumorsuppressor protein p53 (57). The azurin homologue of B. ce-pacia has now been shown to induce apoptosis in macrophages.Given the differences in the mode of action of these two patho-gens, it would be interesting to know if both of them have thesame mode of action. An interesting example in this regard isthe elaboration of a pore-forming protein, listeriolysin O, se-creted by the intracellular pathogen Listeria monocytogenesand a related pore-forming protein, perfringolysin O, secretedby the extracellular pathogen Clostridium perfringens. The pres-ence of listeriolysin O in L. monocytogenes allows the bacte-rium to escape from the macrophage vacuole to reach themacrophage cytosol and live there but does not allow lysis ofthe plasma membrane of the macrophage and its killing. Lys-teriolysin O and perfringolysin O have 43% sequence identityand 70% sequence similarity. While highly homologous, theyhave very different modes of action, since extracellular C. per-

FIG. 7. Subcellular localization and trafficking of the azurin homologue. (A) Localization of the azurin homologue in the cytosol and nuclearfractions of macrophages treated with 200 �g of the protein per ml for the indicated times was determined by Western blotting with antiazurinantibodies. (B) Representative confocal microscopy images for trafficking of the azurin homologue from the cytosol to the nucleus of macrophages.Microinjection of Alexa Fluor 568-labeled azurin homologue is described in Materials and Methods. After microinjection, the macrophage cellswere incubated for the indicated time periods. The insets show magnified images of single injected cells after 15 min and 3 h. Azurin is labeledred (Alexa Fluor 568), while the nucleus is stained blue with DAPI. The arrows indicate the entry of the azurin homologue in the nucleus.



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fringens does not need to live in the macrophage and thereforeperfringolysin O allows lysis of the macrophage plasma mem-brane, leading to the death of the cell. A small addition of 27amino acids with a PEST-like sequence in the L. monocyto-genes protein accounts for this difference, since transfer of thissequence to perfringolysin O transformed that toxic cytolysininto a nontoxic derivative that facilitated intracellular growth(6). It would be of great interest to know if the P. aeruginosaazurin and B. cepacia azurin homologues exhibit differences intheir mechanism of cytotoxicity towards macrophages.

We have reported that the secretion of ATP-utilizing en-zymes by B. cepacia strain 38 and others is greatly enhanced inthe presence of �2-macroglobulin (33). It is interesting that theamount of the azurin homologue present in the growth me-dium of B. cepacia strains (Fig. 3A) belonging to genomovarsI, III, and VI is also greatly increased in the presence of�2-macroglobulin (Fig. 3B), and there is a correlation betweenthe elaboration of the azurin homologue in the growth mediumand the amount of cytotoxicity exhibited by that growth me-dium (Table 3).

Even though both the ATP-utilizing enzymes and the azurinhomologue respond to a common mammalian protein such as�2-macroglobulin, which may enhance the secretory mecha-nism of both of these virulence factors, the mechanism of cellkilling by these factors is very different. The ATP-utilizingenzymes are active mostly in the presence of ATP, operatingthrough activation of the P2Z purinergic receptors (58). In-deed, similar modulation of purinergic receptor activation bysecreted ATP-utilizing enzymes of the parasitic nematodeTrichinella spiralis has recently been reported (9, 10). In con-trast, the azurin homologue triggers the release of mitochon-drial cytochrome c to the cytosol (Fig. 6), resulting in elevatedcaspase-3 and caspase-9 levels (Table 4) and nuclear DNAfragmentation (Fig. 5). Further details of the mode of action ofthe B. cepacia homologue are currently under investigation.

One of the most intriguing and difficult aspects of B. cepaciainfection in the lungs of cystic fibrosis patients is the diversityof various B. cepacia-like organisms, as described in Table 1. Itis by no means clear which microorganisms are truly virulentinfective agents and which are simply commensal carryovers oreven hardy contaminants growing in a somewhat weakenedimmune system. We have examined only a single strain fromeach genomovar (two strains from genomovar I), which istotally inadequate to allow us to draw any general conclusions.Nevertheless, our data imply some degree of correlation be-tween the azurin-mediated cytotoxicity exhibited by membersof genomovars I, III, and VI and the frequency of isolation ofsuch strains from the lungs of cystic fibrosis patients, particu-larly members of genomovar III. Many more members of theB. cepacia complex assigned to different genomovars need tobe examined for the ability to secrete the azurin homologueand for severity of infection before any meaningful conclusionscan be drawn about azurin’s potential as a virulence factor ofthe B. cepacia complex.

Finally, one may wonder why ATP-utilizing enzymes andredox proteins elaborated by the pathogens found in the cysticfibrosis lung, B. cepacia and P. aeruginosa, that are normallyinvolved in the energetics of the cell are also involved in mam-malian cell death. It is interesting that these pathogens pref-erably release these enzymes in response to a mammalian host

protein such as �2-macroglobulin or -casein (33, 58). Ananalogy to this is the release from mitochondria of similarenzymes such as cytochrome c and the apoptosis-inducing fac-tor AIF, an oxidoreductase flavoprotein, in the presence ofdeath signals such as withdrawal of growth factors, presence ofDNA-damaging agents, chemotherapeutics, etc., leading tocell death (1, 4, 35).

It is also interesting that adenylate kinase is secreted by P.aeruginosa as a virulence factor (29) and is surface exposed inStreptococcus agalactiae, the causative agent of sepsis, pneu-monia, and meningitis in neonates (49), but is also releasedsimultaneously with cytochrome c from the intermembranespace of mitochondria during apoptosis (16, 44). Mitochondriaare the storehouse of the energetics of eukaryotes, harboringboth the electron transport chain and the machinery for ATPsynthesis. Mitochondria, of course, are prokaryote-like struc-tures which are believed to have evolved hundreds of millionsof years ago when the ancestral eukaryotic cells entered into amutually beneficial partnership with the ancestors of thepresent-day bacteria that allowed the eukaryotic cells to utilizethe energy-generating machinery of the prokaryotes to takeadvantage of moving from an anaerobic to an increasinglyoxygen-rich environment (17, 34). Unlike some obligate endo-symbiotic bacteria of aphids, in which the genome has re-mained fairly stable for the past 50 to 70 million years (48), theprokaryotic ancestors of the protomitochondria eventually lostmany of their essential genes, transferring some to the eukary-otic nucleus and thereby becoming an obligate endosymbioticorganelle.

Since mitochondria are central to mammalian cell apoptosis,in which release of AIF or cytochrome c plays an importantrole, it appears that present-day prokaryotes such as B. cepacia,P. aeruginosa, and presumably others retain the ability to usetheir energy-generating machinery in the form of ATP-utilizingenzymes or redox proteins to effect mammalian cell death,much to their advantage in coping with a nonsymbiotic hostileenvironment (38). The present study thus provides additionalevidence of the interesting role of bacterial proteins that arenormally involved in cellular energetics in mammalian celldeath.

ACKNOWLEDGMENTS

This work was supported by PHS grant ES04050-17 from the Na-tional Institute of Environmental Health Sciences.

We thank J. LiPuma and E. Mahenthiralingam for providing thestrains mentioned in Table 1.

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