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INFECTION AND IMMUNITY, May 2011, p. 2098–2111 Vol. 79, No. 50019-9567/11/$12.00 doi:10.1128/IAI.00983-10Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Nitric Oxide-Mediated Intracellular Growth Restriction of PathogenicRhodococcus equi Can Be Prevented by Iron�

Kristine von Bargen,1 Jens Wohlmann,1 Gregory Alan Taylor,2 Olaf Utermohlen,3 and Albert Haas1*Institute for Cell Biology, University of Bonn, Bonn, Germany1; Geriatric Research, Education, and Clinical Center,

VA Medical Center, Durham, North Carolina 277052; and Institute for Medical Microbiology, Immunology andHygiene, and Center of Molecular Medicine, Medical Center, University of Cologne, Cologne, Germany3

Received 9 September 2010/Returned for modification 2 October 2010/Accepted 16 February 2011

Rhodococcus equi is an intracellular pathogen which causes pneumonia in young horses and in immu-nocompromised humans. R. equi arrests phagosome maturation in macrophages at a prephagolysosomestage and grows inside a privileged compartment. Here, we show that, in murine macrophages activatedwith gamma interferon and lipopolysaccharide, R. equi does not multiply but stays viable for at least 24 h.Whereas infection control of other intracellular pathogens by activated macrophages is executed byenhanced phagosome acidification or phagolysosome formation, by autophagy or by the interferon-inducible GTPase Irgm1, none of these mechanisms seems to control R. equi infection. Growth control bymacrophage activation is fully mimicked by treatment of resting macrophages with nitric oxide donors,and inhibition of bacterial multiplication by either activation or nitric oxide donors is annihilated bycotreatment of infected macrophages with ferrous sulfate. Transcriptional analysis of the R. equi iron-regulated gene iupT demonstrates that intracellular R. equi encounters iron stress in activated, but not inresting, macrophages and that this stress is relieved by extracellular addition of ferrous sulfate. Ourresults suggest that nitric oxide is central to the restriction of bacterial access to iron in activatedmacrophages.

Rhodococcus equi is a soil organism which belongs to thegroup of mycolic acid-producing actinomycetes (42) and isclosely related to Mycobacterium tuberculosis. The facultativeintracellular pathogen R. equi can cause pneumonia in younghorses and immunocompromised humans when inhaled withcontaminated dust (42) and primarily infects phagocytic mono-cytes and macrophages (15). Newly phagocytosed bacteria re-side in a macrophage compartment which passes normallythrough the early phase of phagosome maturation and which isarrested in between an early and a late maturation stage. Theresulting compartment, the R. equi-containing vacuole (RCV),is characterized by some markers of late phagosomes, such asRas-like protein from rat brain (Rab) 7, bis(monoacylglycerol)phosphate (BMP), and lysosome-associated membrane pro-teins (LAMP) 1 and 2. However, the RCV acquires neitherhydrolytic enzymes nor proton-pumping vacuolar ATPase (v-ATPase), both of which are characteristic for late phagosomes,nor does it acidify or fuse with lysosomes. The bacteria multi-ply in this unusual vacuole, and the host cell is eventually lysedby necrosis (10, 22). R. equi virulence largely depends on vir-ulence-associated plasmids which may also determine hostspecificity (38). Virulence for foals requires a plasmid encodingthe central virulence factor, virulence-associated protein A(VapA) (12, 17).

Whereas a resting macrophage is susceptible to infectionwith different intracellular pathogens, its antimicrobial activity

is strongly enhanced by activation through proinflammatoryhost signals, e.g., interferons (IFN), or by various pathogen-associated molecular patterns, e.g., lipopolysaccharides (LPS)(35). Accordingly, R. equi cells multiply in resting macro-phages, yet their multiplication is completely inhibited whenmacrophages are activated before infection (5). Activation in-duces a dramatic change in gene expression and increasesmicrobicidal and antigen presentation capacities of the phago-cyte (48).

Mechanisms which execute the killing functions in activatedmacrophages include enhanced phagolysosome formation, in-duction of autophagy, or production of toxic radicals. Vacuolescontaining pathogenic mycobacteria (47, 50, 58), Legionellapneumophila (45), or Coxiella burnetii (11) are forced back intothe normal degradation pathway when macrophages are acti-vated before infection. Proteins that may mediate IFN-�–LPS-induced enhanced phagosome maturation include the IFN-�-induced immunity-related GTPases (IRG). One of them,Irgm1 (formerly known as LRG-47) (56), is a central player forcontrolling M. tuberculosis infections in mice. Irgm1�/� miceare unable to control M. tuberculosis replication. Bone marrow-derived macrophages (BMMs) from these animals are partlypermissive for M. tuberculosis multiplication even when acti-vated, and the bacteria are less frequently found in phago-lysosomes (26). Irgm1 is also involved in infection control viaautophagy (13, 51). Induction of autophagy in macrophagesleads to increased formation of M. tuberculosis-containingphagolysosomes and to killing of the bacteria (13).

Production of nitric oxide by activated macrophages is cru-cial for infection control of Leishmania and Burkholderia mallei(18, 29), whereas killing of Listeria monocytogenes largely de-pends on superoxide (39). Both compounds seem to be impor-

* Corresponding author. Mailing address: Cell Biology Institute,University of Bonn, Ulrich-Haberland-Str. 61 A, 53121 Bonn, Ger-many. Phone and fax: 0049 (0)228 73 6340. E-mail: [email protected].

� Published ahead of print on 7 March 2011.

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tant for elimination of Francisella tularensis (23). Likewise,control of R. equi infection by activated macrophages has beenascribed to killing of bacteria by peroxynitrite, a strongly bac-tericidal compound that results from reaction of superoxidewith nitric oxide radicals (5).

Here, we analyzed the compartmentation and survival of R.equi in activated macrophages and the mechanisms behindintracellular growth restriction of R. equi by macrophage acti-vation.

MATERIALS AND METHODS

Cultivation of macrophages and bacteria. J774E macrophage-like cells werecultivated in Dulbecco’s modified Eagle’s medium (DMEM) containing 10%fetal calf serum and 1% GlutaMax (all from Invitrogen, Karlsruhe, Germany),designated “DMEM complete” here. The knockout and transgenic mice used,including Irgm1�/�, GFP-LC3 (where GFP is green fluorescent protein), andgp91phox�/� mice, have been described (3, 34, 41). Bone marrow-derived mac-rophages (BMMs) were isolated and cultivated as described previously (10). Allexperiments involving mice were performed according to the German law foranimal protection. The procedures were additionally approved by the local gov-ernment according to the laws of the State of North Rhine-Westfalia, referencenumber 8.87-50.10.45.08.219.

If not stated otherwise, macrophages were activated by treatment with culturemedium containing 500 U/ml recombinant IFN-� (Tebu-bio, Offenbach, Ger-many) overnight and additionally with 250 ng/ml lipopolysaccharide (LPS; fromSalmonella enterica serotype Typhimurium; Sigma-Aldrich, Taufkirchen, Ger-many) 2 h before infection. IFN-� and LPS were left on activated macrophagesthe entire experiment. Rhodococcus equi 103� (where “�” denotes the presenceof a virulence-associated plasmid) has been isolated from a foal with R. equipneumonia (6). The isogenic but avirulent strain R. equi 103� has been curedfrom the virulence-associated plasmid (10). Strains will be denoted as “103�”and “103�” here. Bacteria were grown on brain heart infusion (BHI; BD Diag-nostic Systems, Sparks, MD) agar plates at 30°C for routine cultivation. Beforeinfection experiments, bacteria were grown in BHI broth at 37°C overnight at 200rpm on a rotary shaker to induce virulence gene expression.

Live-cell determination of intracellular R. equi. For determination of intracel-lular survival of R. equi, J774E cells were seeded into 24-well plates at 4 � 104

cells per well 2 days before infection with R. equi at a multiplicity of infection(MOI) of 0.25 for 30 min at 37°C. Samples were rinsed with phosphate-bufferedsaline (PBS) twice, and fresh DMEM complete containing 10 �g/ml gentamicinwas added. In samples for 48 h of incubation, cells were supplied with freshmedium at 24 h. At the times indicated, medium was removed and macrophageswere lysed in 1 ml PBS-0.1% (vol/vol) Triton X-100. Serial dilutions were platedonto 0.5� LB (Lennox) agar. Plates were incubated at 30°C for 30 h and CFUwere counted. Numbers of less than 10 were neglected for analysis as long as theywere not the only colonies grown in this dilution series. The mean number ofCFU for each sample and time was determined and expressed as a percentage ofthe 0 h values.

Quantification of fusion of phagosomes with lysosomal or endocytic compart-ments. J774E macrophages were seeded onto coverslips in a 24-well plate at 2 �105 cells per well.

(i) Phagosome-lysosome fusion. Macrophages were incubated with DMEMcomplete containing 30 �g/ml ovalbumin TexasRed (Invitrogen) overnight andrinsed with warm PBS, and the fluorescent conjugate was chased into lysosomesby incubation in fresh medium for 2 h. Cells were infected with ATTO-488(Atto-Tec, Siegen, Germany)-labeled live or heat-killed (15 min, 85°C) R. equi103� or 103� (aliquots of 2 � 108 bacteria, incubated in 50 �g/ml of the fluorin 0.1 M NaHCO3 for 45 min on ice, followed by one wash in 20 mM Tris/HCl[pH 8] and two washes in PBS) at an MOI of 40 for 20 min, rinsed with PBStwice, and incubated in fresh medium without bacteria. After 2 h and 5 h,macrophages were fixed with PBS-3% formaldehyde.

(ii) Phagosome-endosome fusion. After overnight incubation, macrophageswere infected with ATTO-488-labeled R. equi 103� or 103� or heat-killed (10min, 70°C) Escherichia coli DH5� labeled as described above at an MOI of 40 for15 min, rinsed with PBS twice, and incubated in fresh medium for 2 h. Mediumwas replaced by DMEM complete containing 150 �g/ml dextran Texas Redconjugate (Invitrogen). After 2 h or 4 h, cells were rinsed with PBS and fixed inPBS-3% formaldehyde.

All samples were mounted onto glass slides in Mowiol, and colocalization of

bacteria with lysosomal or endocytosed material was quantified using a confocallaser scanning microscope, model LSM510 (Zeiss, Oberkochen, Germany).

Microscopic and fluorimetric determination of phagosome pH. For determi-nation of phagosome colocalization with LysoTracker, J774E cells were seededat 1.5 � 105 cells/well onto glass coverslips and incubated overnight. They wereinfected with R. equi 103� at an MOI of 1 for 30 min, rinsed with PBS twice, andincubated in fresh DMEM complete containing 10 �g/ml gentamicin. After 1.5 hor 23.5 h, medium was replaced by DMEM complete containing 100 nM Lyso-Tracker (Invitrogen) and 3.3 �M of the DNA stain Syto13 (Invitrogen). Cellswere incubated for 30 min, rinsed with PBS, and mounted onto glass slides with5 mg/ml low-melting-point agarose (Sigma-Aldrich) in PBS. They were analyzedimmediately using a confocal laser scanning microscope. Quantitative ratio-metric determination of phagosome pH was as described in reference 54. Briefly,R. equi was labeled with a pH-insensitive fluor, allowing for the quantification ofingested bacteria, and with a pH-sensitive fluor, allowing for the quantification ofpH. J774E cells in 24-well plates were infected with labeled bacteria for 15 minand chased in fresh medium. For each time, fluorescence in two wells of infectedJ774E cells was determined in assay buffer in an FLX800 microplate reader(Bio-Tek Instruments, Neufahrn, Germany). After quantification of fluorescenceof the last sample, 10 �M of the K�/H� antiporter nigericin was added toequilibrate phagosome pH with that of the assay buffer as the internal control.The system was calibrated by incubating infected macrophages with K�-contain-ing buffers of defined pH containing 10 �M nigericin. For pH determinations ofphagosomes containing killed R. equi cells, bacteria were treated before infec-tions with heat (15 min, 85°C), UV irradiation (1 h on ice), formaldehyde (3% inPBS, 1 h at 4°C), or gramicidin (300 �g/ml in PBS, 2 h at 37°C). Bacteria werefluorescently labeled after killing, and bacteria with formaldehyde treatmentwere fluorescently labeled before killing. Efficient killing was tested by platingsamples on LB agar plates.

Transmission electron microscopy. To analyze the ultrastructural appearanceof the RCV in resting and activated macrophages, 5 � 106 J774E cells wereseeded into 53-mm cell culture dishes and incubated overnight. They wereinfected with R. equi 103� at an MOI of 20 for 30 min, rinsed with PBS twice,and incubated in fresh medium containing 150 �g/ml gentamicin. After 1 h,medium was replaced by DMEM complete containing 10 �g/ml gentamicin.

After different times of incubation, cells were fixed in PBS containing 2%formaldehyde and 0.5% glutaraldehyde for 1 h and prepared for electron mi-croscopy as described previously (10).

Microscopic analysis of intracellular multiplication. J774E (1.5 � 105 cellsper well) or bone marrow-derived (1 � 105 cells per well) macrophages wereseeded in a 24-well plate containing coverslips and incubated overnight. Theywere infected with R. equi 103� at an MOI of 1 for 30 min, rinsed with PBStwice, and incubated in fresh medium containing 10 �g/ml gentamicin. Macro-phages were treated with different buffers or chemicals before and/or afterinfection. Hanks’ balanced salt solution (HBSS; 0.137 M NaCl, 5.4 mM KCl, 0.25mM Na2HPO4, 0.44 mM KH2PO4, 1 mM MgSO4, 1.3 mM CaCl2, 4.2 mMNaHCO3, 5.6 mM glucose)-treated cells were rinsed with PBS twice and incu-bated with HBSS 2 h before and always after infection (then with 10 �g/mlgentamicin). 3-Methyladenine (3MA; Sigma-Aldrich; dissolved in medium withheating) was added at 10 mM for 2 h before and at 1 mM after infection; foranalysis of intracellular multiplication of 103�, 3MA was added only afterinfection at 10 mM. Earle’s balanced salt solution (EBSS; 117.2 mM NaCl, 5.3mM KCl, 1.0 mM NaH2PO4, 1.0 mM MgSO4, 1.8 mM CaCl2, 5.6 mM glucose,26.2 mM NaHCO3)-treated cells were rinsed with PBS twice and incubated withEBSS 1 h before and 7 h after infection (then with 10 �g/ml gentamicin), beforethe buffer was replaced by DMEM complete containing 10 �g/ml gentamicin.Rapamycin (200 nM; Merck, Darmstadt, Germany; dissolved in dimethyl sulfox-ide [DMSO]) was added 1 h before and always after infection, and DMSO wasadded as the corresponding control. Macrophages pretreated with 100 �M S-nitroso-N-acetyl-D,L-penicillamine (SNAP; Sigma-Aldrich; dissolved in PBS-30mM NaOH) were incubated with the compound in DMEM complete 24 h beforeinfection, rinsed with PBS twice, and incubated in medium containing 10 �g/mlgentamicin without SNAP after infection. Alternatively, SNAP was added onlyafter infection at 100 �M. The reactive oxygen species inhibitor N-acetyl-L-cysteine (Enzo Life Sciences, Lorrach, Germany; dissolved in sterile water) wasadded at 5 mM 2 h before and always after infection. Compounds that wereadded only after infection were 50 nM wortmannin (Merck; solved in DMSO),500 �M NG-methyl-L-arginine (NMLA; Sigma-Aldrich; order number M7033;dissolved in PBS), 50 �M diethylenetriamine-nitric oxide adduct (DETA-NO;Sigma-Aldrich; order number D185; dissolved in PBS-10 mM NaOH), and dif-ferent concentrations of FeSO4 (Roth, Karlsruhe, Germany; order numberP015-1; dissolved in medium). At 2 h and 24 h of infection, samples were fixedwith PBS-3% formaldehyde and stained with the DNA stain Syto13 in PBS at

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1.67 �M for 30 min. Cells were rinsed with PBS four times and mounted ontoslides in Mowiol for microscopic analysis. The numbers of intracellular bacteriawere counted for at least 50 infected macrophages per sample. Since numbers ofbacteria in large clusters cannot be properly quantified, samples were analyzedfor robust bacterial multiplication by determining the percentage of macro-phages containing more than 10 bacteria (5).

Control for effects of treatments interfering with autophagic pathways. GFP-LC3 bone marrow-derived macrophages were seeded at 1 � 105 cells per welland activated with IFN-�–LPS where indicated. After overnight incubation, onesample of each resting or activated macrophage was left untreated. To investi-gate the effects of these treatments on LC3 localization, resting macrophageswere rinsed twice with PBS and were incubated in the starvation buffers HBSS orEBSS or with 200 nM rapamycin in DMEM complete. Activated macrophageswere incubated in 50 nM wortmannin or in 10 mM 3MA in DMEM complete.After 2 h of incubation, all samples were fixed with 3% formaldehyde in PBS,embedded in Mowiol, and investigated with a laser scanning microscope usingthe same microscope settings for all samples.

Quantification of nitric oxide. For analysis of nitric oxide production, J774Emacrophages were seeded into 24-well plates at 8 � 104 cells per well 2 daysbefore infection. Resting or activated macrophages were infected at an MOI of1 for 1 h and chased in fresh medium containing 10 �g/ml gentamicin. At thetimes indicated, medium was transferred into tubes and centrifuged at 140 � gfor 5 min. The supernatants were stored at �20°C until analysis for nitrite withthe Griess reagent system (Promega, Madison, WI) by following the manufac-turer’s instructions. For determination of nitric oxide release from nitric oxidedonors, these were dissolved as described above. SNAP was diluted in DMEMcomplete with or without 100 �M ferrous sulfate at 100 �M and incubated in15-ml polypropylene tubes at 37°C. At different times, samples were taken andstored at �20°C until analysis with Griess reagent. DETA-NO was diluted inDMEM complete at 50, 100, or 200 �M and incubated and analyzed as describedfor SNAP. A concentration of 50 �M DETA-NO was chosen for infectionexperiments to achieve a comparable final concentration of nitrite as an indica-tion for similar amounts of released nitric oxide.

Viability and VapA expression of SNAP-treated R. equi. R. equi was grown at30°C overnight, pelleted, and resuspended in DMEM complete with 100 �MSNAP or solvent alone at an optical density at 600 nm (OD600) of 0.1. Bacteriawere incubated in a rotary shaker at 200 rpm and 37°C, and OD600 was recordedover time. After 24 h of incubation, expression of VapA in samples was analyzedby immunoblotting (see Western blot analysis) after heating equal numbers ofbacteria to 95°C for 10 min in 2� Laemmli buffer (5� stock contains 60 mMTris-HCl [pH 6.8], 25% glycerol [vol/vol], 2% sodium dodecyl sulfate [wt/vol],5% �-mercaptoethanol [vol/vol], 0.1% bromphenol blue [wt/vol]), followed byremoval of debris by centrifugation. A 0.3 OD600 culture equivalent was appliedto a 15% SDS-polyacrylamide gel for each sample. To determine the effect ofnitric oxide on R. equi growth under conditions of limited iron concentrations, R.equi 103� was grown in minimal medium [30 mM K2HPO4, 16.5 mM KH2PO4,78 mM (NH4)2SO4, 0.85 mM sodium citrate, 1 mM MgSO4, 0.02% glycerol, 0.1mM thiamine, 20 mM acetate, supplemented with 1� trace metal solution (60)adjusted to different concentrations of ferrous sulfate] containing 100 �M fer-rous sulfate at 30°C overnight. Bacteria were pelleted, washed with PBS once,and used for inoculation of minimal media with different iron concentrations atan OD600 of 0.1. Suspensions were mixed with no or 100 �M SNAP. Opticaldensity was determined before and after 24 h of incubation at 37°C in a rotaryshaker.

Western blot analysis. Western blot analysis was as described in reference 10.Antibodies used were mouse anti-VapA (Mab10G5; 1:5.000; kindly supplied byShinji Takai, Kitasato University, Aomori, Japan [55]) and Odyssey goat anti-mouse IRDye 800CW (1:10.000; Li-Cor, Bad Homburg, Germany). Westernblots were analyzed with the Odyssey infrared imaging system (Li-Cor).

Determination of infection cytotoxicity. J774E macrophages were seeded into24-well plates at 8 � 104 cells per well 2 days before infection. They were infectedwith R. equi 103� at an MOI of 30 for 1 h. To remove extracellular bacteria,macrophages were rinsed with PBS twice and incubated in DMEM completecontaining 150 �g/ml gentamicin for 1 h before replacing the medium withDMEM complete containing 10 �g/ml gentamicin. Where indicated, cells wereadditionally treated with a mix of antibiotics (100 �M each hygromycin B,penicillin G, and apramycin, all from Sigma-Aldrich) at different times of chaseto kill intracellular bacteria. After 24 h of chase, an uninfected sample was lysedwith 1% Triton X-100 (vol/vol). Release of cytosolic lactate dehydrogenase(LDH) activity from this sample was set as 100%, and activities in all sampleswere expressed relative to this. Sample supernatants were collected, centrifugedat 6,150 � g for 5 min, and analyzed for LDH release with the cytotoxicity

FIG. 1. Virulent R. equi cells survive in activated J774E macro-phages for at least 24 h. (A) Resting and activated J774E macrophageswere infected with R. equi 103� at an MOI of 0.25 for 30 min andincubated in fresh medium containing gentamicin to kill extracellularbacteria. Infected macrophages were lysed at different times of infec-tion, and serial dilutions were plated. CFU were counted and calcu-lated as percentage of samples plated immediately after infection.Data represent means and standard deviations of results from fourindependent experiments. (B) Resting and activated J774E cells wereinfected with R. equi 103� at an MOI of 30 and incubated in freshmedium containing a low concentration (10 �g/ml) of gentamicin tokill extracellular bacteria. At different times of infection, a mix ofantibiotics (AB), including hygromycin B, penicillin G, and apramycin(each at 100 �M), was added to kill intracellular bacteria. After 24 h,the supernatants of all samples were collected and release of cytosoliclactate dehydrogenase was quantified. Cytotoxicity is expressed rela-tive to an uninfected sample lysed with Triton X-100. Data are meansand standard deviations of results from four independent experiments.(C) J774E macrophages were infected with R. equi 103� at an MOI of0.25 for 30 min and chased in fresh medium containing 10 �g/mlgentamicin to kill extracellular bacteria (Control) or gentamicin andthe mix of high-dose antibiotics (AB) described in the legend to panelB. Cells were lysed at different times of infection, and serial dilutionswere plated. CFU were counted and calculated as percentage of sam-ples plated at 0 h postinfection. Data represent means and standarddeviations of results from three or four independent experiments. *,P � 0.05.

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detection kit (Roche Diagnostics, Mannheim, Germany) by following the instruc-tions of the manufacturer.

Immunofluorescence microscopy. Fixed and quenched (PBS-50 mM NH4Clfor 30 min) macrophages on coverslips were incubated in blocking buffer (PBScontaining 5% bovine serum albumin and 0.1% saponin, both from Sigma-Aldrich). Samples were sequentially stained with first and secondary antibodiesin blocking buffer, embedded in Mowiol, and analyzed using the confocal laserscanning microscope LSM510 (Zeiss) and the same microscope settings for allsamples in any one experiment. Antibodies used were goat anti-Irgm1 (1:50;Santa Cruz Biotechnology), mouse anti-R. equi 103� serum (1:60; producedagainst complete fixed R. equi 103�), donkey anti-mouse-Cy3 (1:50; Dianova,Hamburg, Germany), and donkey anti-goat-Cy3 (1:50; Dianova).

RNA isolation from intramacrophage bacteria. R. equi was grown in 10-mlBHI broth portions at 37°C for 16 h at 200 rpm. Cultivation at iron-limitedconditions was in minimal medium with acetate containing 2,2-dipyridyl (Sigma-Aldrich, Munich, Germany). Concentrations for activation with IFN-� and LPSovernight were as above, and IFN-� and LPS were left on J774E macrophagesduring the entire experiment. Infection was performed in 6-cm dishes at aconcentration of 2 � 106 macrophages per dish at an MOI of 10 for 30 min at37°C, followed by two rinses in PBS and the addition of fresh DMEM containing10 �g/ml gentamicin. For iron-rich conditions, medium was additionally supple-mented with 50 �M FeSO4. Cells were kept at 37°C and 10% CO2. After theindicated time periods of infection, macrophages were rinsed with warm PBS andwere lysed in 1 ml 0.5% Triton X-100 in water. The obtained solution was shakenon ice for 10 min and spun at 4,000 rpm for 5 min to harvest intracellularbacteria. RNA from these bacteria or bacteria grown in minimal medium wasisolated with the ZR fungal/bacterial RNA miniprep kit (Hiss DiagnosticsGmbH, Freiburg, Germany) according to the manufacturer’s instructions.

RT-PCR and real-time PCR. Concentrations of extracted RNAs were normal-ized, and RNA was used for reverse transcription (RT) with the SuperScriptVILO cDNA synthesis kit (Invitrogen, Darmstadt, Germany) by following themanufacturer’s recommendations. Resulting cDNA was directly used as thetemplate for amplification using the Maxima SYBR green/ROX quantitativePCR (qPCR) Master Mix (Fermentas, St. Leon-Rot, Germany) with the follow-ing primers: 16srRNA200F and 16srRNA200R (for 16SrRNA) (31) and SID4-193F and SID4-193R (for iupT) (32). Amplification and analysis were performedin the LightCycler 480 SW 1.5 (Roche Applied Science, Mannheim, Germany)instrument.

Statistics. Data are expressed as means � standard deviations. Significance ofdifferences was analyzed by a two-tailed unpaired Student’s t test. Differenceswere termed as significant when P values were �0.05.

RESULTS

R. equi survives for at least 24 h in activated macrophages.Activated macrophages have an increased microbicidal capac-ity (35). We investigated the viability of virulent R. equi 103�in resting and IFN-�–LPS-activated J774E macrophages by

CFU quantification. Bacteria multiplied in resting macro-phages over the course of 48 h (Fig. 1A). In contrast, in acti-vated J774E macrophages, R. equi did not multiply but sur-vived for at least 24 h (recovery of 106% of bacterial load at0 h) and was reduced to less than 20% of the initial load by 48 h(Fig. 1A).

Infection with viable but not with dead R. equi at high MOIis cytotoxic for macrophages (22). Therefore, levels of R. equicytotoxic effects on macrophages can be used as a measure ofbacterial intracellular viability. Cytotoxicity of infection with103� was little but significantly decreased in IFN-�–LPS-acti-vated J774E cells compared with that of resting cells at 24 hafter infection (39% versus 47%; P 0.02) (Fig. 1B). Toanalyze whether necrosis of macrophages was due to the in-tracellular survival of 103� or due to activation-mediated cy-totoxic host cell responses to infection, bacteria were killedintracellularly with a mix of antibiotics at different times afterinfection. The combination and concentrations of antibioticsused here killed 94% of all intracellular R. equi within 8 h, asdetermined by CFU counting (Fig. 1C). At 24 h postinfection,necrotic death of infected resting and activated J774E cellstreated with antibiotics was reduced compared to that of un-treated cells depending on treatment time (Fig. 1B), strength-ening the interpretation that R. equi retains metabolic activityin activated macrophages for at least several hours.

R. equi compartmentation does not change upon activation.To test whether control of R. equi infection by activated J774Ecells was correlated with altered intracellular vesicle traffick-ing, we compared key features of maturation of R. equi-con-taining phagosomes in resting and activated macrophages. Fre-quencies of fusion of RCVs with lysosomes was unaltered aftermacrophage activation, regardless of whether virulent, aviru-lent, or heat-killed R. equi were contained (Fig. 2A). More-over, RCVs in activated macrophages were still as accessible tonewly endocytosed molecules as in resting cells (Fig. 2B).

Phagosome acidification by v-ATPase precedes phagosome-lysosome fusion and is part of the macrophage killing arsenal.To determine the effect of macrophage activation on pH of theRCV, we used a calibrated microplate-based assay early afterinfection and a microscopic assay based on accumulation of

FIG. 2. Communication of the R. equi-containing vacuole with endosomes or lysosomes in resting or activated macrophages. (A) Lysosomesof resting or IFN-�–LPS-activated J774E macrophages were preloaded with the fluid phase marker ovalbumin-TexasRed (OvTR) and infectedwith ATTO-488-labeled R. equi 103� or 103� or heat-killed 103� (103�T). Samples were fixed at 2 h or 5 h postinfection. (B) Resting orIFN-�–LPS-activated J774E cells were infected with ATTO-488-labeled R. equi 103� or 103� or heat-killed E. coli (E.c.T) and incubated for2 h before medium was exchanged for medium containing dextran TexasRed (DexTR). Samples were incubated at 37°C for another 2 h or 4 hbefore fixation. Samples were microscopically analyzed for colocalization of phagosomes with the fluorescent tracers. Data represent means andstandard deviations of results from three or four experiments.



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LysoTracker fluor in acidic compartments for early and latetimes. The pH of phagosomes containing virulent R. equidropped initially to pH 6.1 before it slowly rose to neutral pH.At 3 h postinfection, RCV pH had stabilized at 7.4 (Fig. 3A).Phagosome pH neutralization strictly depended on the viru-lence plasmid and on bacterial viability, as phagosomes con-taining the plasmid-cured strain or heat-killed R. equi acidifiedto pH 5.0 and pH 5.2 at 3 h postinfection, respectively (Fig.3A). Regardless of whether R. equi had been killed by UVirradiation or formaldehyde or gramicidin addition, theirphagosomes acidified (Fig. 3B). Macrophage activation with

IFN-�–LPS before and during infection with viable, virulent R.equi had no significant effect on RCV pH compared to that inresting J774E cells (Fig. 3C). Even at 24 h after infection, onlyvery few RCVs were positive for LysoTracker in resting andactivated macrophages (Fig. 3D and E).

To identify possible differences between intracellular life ofR. equi in resting and activated macrophages, the ultrastructureof infected cells was analyzed. Bacteria were internalized byresting and activated macrophages via distinct and slenderpseudopods (Fig. 4A and B). At 2 h postinfection, phagosomemembranes were tightly attached to R. equi (Fig. 4C and D).

FIG. 3. Diversion of phagosome pH by R. equi depends on possession of a virulence-associated plasmid and bacterial viability but not on hostactivation status. (A) J774E cells were infected with fluorescently labeled virulent (103�), avirulent (103�), or heat-killed R. equi (103�T) for15 min, and fresh medium was added. At different times after infection, fluorescence was quantified and used to calculate phagosome pH (pH at0 min corresponds to that of the external buffer). At 180 min (A and B) or 300 min (C), nigericin was added (black arrows) to equilibratephagosome pH with that of the surrounding buffer to control for correct calibration. (B) As described for panel A, but infection was with 103�that had been killed by gramicidin, formaldehyde (FA), or UV irradiation before infection. (C) As described for panel A, but infection was with103� only of resting versus IFN-�–LPS-activated macrophages. (D and E) Resting or activated J774E cells were infected with 103� at an MOIof 1 for 30 min, and fresh medium was added. At 1.5 h and 23.5 h, medium was exchanged for DMEM complete containing LysoTracker (red)and Syto13 (green), which was left on the cells for 30 min to visualize acidic compartments and bacteria. Samples were mounted onto slides, andcolocalization of bacteria with LysoTracker was quantified by laser scanning microscopy. Panel E shows representative micrographs of resultsshown in panel D (black bars, 10 �m). The filled arrowhead points to bacteria colocalizing with LysoTracker, and open arrowheads point tobacteria that do not. Data in panels A to D represent means and standard deviations of results from three independent experiments.



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As infection progressed, phagosomes became more spaciousand began to fill with small vesicles and membranes in bothresting and activated macrophages (Fig. 4E and F). At 24 hpostinfection, R. equi in resting and activated macrophages

resided in large vacuoles that were filled with vesicular elec-tron-dense and electron-lucent structures (Fig. 4G and H)(10). The high numbers of microorganisms observed in ac-tivated macrophages were not the result of bacterial multi-plication but were due to the high MOI of 20, which wasnecessary to visualize phagosomes by transmission electronmicroscopy. The cytoplasm of many macrophages in acti-vated samples was electron dense, yet this was independentof infection (Fig. 4H).

In all samples investigated, R. equi cells were enclosed byvacuoles with apparently intact single membranes. Even at 24 hpostinfection, when a considerable number of resting and ac-tivated macrophages were evidently necrotic and had lost mostof their cytosolic contents, many phagosomes still had an intactmembrane (Fig. 4I and J). In summary, no obvious differences

FIG. 5. Irgm1 is not involved in control of R. equi infection inactivated macrophages. (A) Resting or IFN-�- or IFN-�–LPS-acti-vated BMMs derived of B6 wild-type (B6-wt) or Irgm1�/� mice wereinfected with 103� at an MOI of 1 for 30 min and incubated for 2 h or24 h before fixation. DNA was stained with Syto13, and intracellularbacteria were counted to determine the percentage of macrophagescontaining more than 10 bacteria. For each sample and experiment,bacteria in at least 50 infected macrophages were analyzed. Datarepresent means and standard deviations of results from three or fourexperiments. *, P � 0.05. (B) Immunofluorescence of uninfected rest-ing or IFN-�–LPS-activated B6-wt and Irgm1�/� BMMs treated withanti-Irgm1 and using the same microscope settings for all samples(bars, 20 �m).

FIG. 4. Ultrastructural morphology of R. equi-containing phago-somes in resting and activated J774E macrophages. Resting or IFN-�–LPS-activated macrophages were infected with 103� for 1 h, andmedium was replaced by fresh medium without bacteria. Samples werefixed at 0 h (A, B), 2 h (C, D), 8 h (E, F), or 24 h (G, H) postinfectionand prepared for transmission electron microscopy. (I, J) At 24 h afterinfection, many macrophages were necrotic yet contained R. equiwithin phagosomes with intact membranes. Phagosome membranesare indicated by arrowheads. Bars, 1 �m.



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were observed between the ultrastructures of RCVs in acti-vated versus resting macrophages.

Growth restriction by activated macrophages is not due toimmunity-related GTPase Irgm1. The interferon-inducedGTPase Irgm1 is involved in protection against infection withmultiple intracellular bacteria, including M. tuberculosis (25).However, activated primary bone marrow-derived macrophages(BMMs) of Irgm1�/� mice were as effective in controlling infec-tion with R. equi 103� as were activated wild-type BMMs (Fig.5A). The knockout phenotype of Irgm1�/� macrophages andincreased Irgm1 expression in wild-type BMMs upon activationwere confirmed by immunofluorescence microscopy (Fig. 5B).

Growth inhibition by activated macrophages is not due toautophagy. Autophagy is a cellular mechanism which is in-duced upon macrophage activation and helps to control macro-phage infection with mycobacteria (7, 13). Autophagy can beinhibited by phosphoinositol 3-kinase inhibitors such as3-methyladenine (3MA) or wortmannin (WM). Indeed, 3MAlargely restored multiplication of R. equi in activated J774Ecells (Fig. 6A), but WM did not (Fig. 6A). If inhibition ofautophagy promoted infection, activation of autophagy could

possibly suppress it. However, multiplication of R. equi in rest-ing J774E cells was normal after induction of autophagy withrapamycin or by starvation in HBSS or EBSS (Fig. 6B). Con-versely, treatment of resting macrophages with 3MA did notpromote multiplication of avirulent R. equi 103� (Fig. 6C).The effects of the different treatments on macrophage au-tophagy were confirmed by analysis of the distribution of amarker for autophagic vacuoles, microtubule-associated pro-tein light chain 3 (LC3) in resting and activated BMMs ofGFP-LC3 transgenic mice (not shown).

To directly investigate RCV intersection with autophagic com-partments, RCV colocalization with GFP-LC3 was determined ininfected resting and activated GFP-LC3 BMMs. There was infre-quent but significant association of the RCV with LC3 early afterinfection, which was more pronounced in activated than in restingmacrophages (19.2 versus 4.9% colocalization at 10 min; P 0.049) (Fig. 6D and E). GFP-LC3 rarely colocalized with RCVs atlater times after infection (Fig. 6D).

Nitric oxide restricts R. equi multiplication in activatedmacrophages. Inducible nitric oxide synthase (NOS2; formerlyi-NOS) is central for control of R. equi infection by activated

FIG. 6. Autophagic pathways and R. equi infection control. (A and B) IFN-�–LPS-activated or resting J774E cells were infected with 103� atan MOI of 1 for 30 min and incubated in fresh medium for 2 h or 24 h. Cells were left untreated (control) or incubated with different compoundswhich manipulate autophagic pathways: the autophagy inhibitors 3-methyl adenine (3MA; 10 mM for 2 h before/1 mM after infection) orwortmannin (WM; 50 nM, with DMSO control) or with the autophagy inducers EBSS, HBSS, or rapamycin (RM; 200 nM, with DMSO control).Samples were fixed at 2 h and 24 h of infection and stained with Syto13, and the percentages of macrophages containing more than 10 bacteriawere determined. (C) Experimental setup as described for panel A, but resting J774E cells were infected with 103� or 103� and either not treatedor treated with 10 mM 3MA. (D) Resting or IFN-�–LPS-activated bone marrow-derived macrophages of GFP-LC3 mice were infected with R. equi103� as described for panel A and fixed at different times after infection. Samples were stained with antibodies against R. equi to visualize bacteria,and colocalization of R. equi with LC3 was determined in at least 50 infected macrophages per sample. (E) Representative fluorescence micrographof experiments shown in panel D from a 10-min, activated sample (black bar, 10 �M). Red, R. equi 103�; green, GFP-LC3. The inset shows acloseup of the LC3-positive phagosome. All data in panels A to D represent means and standard deviations of results from three to nineindependent experiments. *, P � 0.05.



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macrophages (5). However, macrophage activation boosts notonly production of nitric oxide after phagocytosis but also thatof superoxide (35). To distinguish effects that might be causedby nitric oxide or superoxide alone, we treated infected mac-rophages with different compounds that affect production ofeither radical in vitro. The addition of the nitric oxide donorS-nitroso-N-acetyl-D,L-penicillamine (SNAP) to the medium ofinfected resting J774E cells strongly inhibited multiplication ofR. equi (Fig. 7A). However, when J774E cells were treated withSNAP for 24 h before but not during and after infection, R.

equi multiplied approximately as often as in untreated restingcells (Fig. 7B).

Inhibition of nitric oxide synthesis in activated macrophageswith NG-methyl-L-arginine (NMLA) allowed R. equi multipli-cation, as had been described previously (5) (Fig. 7C). Incontrast, treatment with N-acetyl-L-cysteine, a membrane-per-meable compound which quenches reactive oxygen speciesstoichiometrically (59), did not restore R. equi intracellularmultiplication in activated macrophages (Fig. 7C), making akilling role of these compounds unlikely. This is in line with theresults from bone marrow-derived macrophages of wild-typeand transgenic mice (gp91phox�/� mice) which are deficient inthe major superoxide generator in immune cells, the NADPHoxidase complex. Activation with IFN-� and LPS reduced mul-tiplication of R. equi in resting macrophages of both genotypesas effectively as treatment with the nitric oxide donor SNAP(Fig. 7D). Thus, nitric oxide is essential for control of R. equiinfection, while NADPH oxidase activity is dispensable.

It has been reported that R. equi is resistant to high concen-trations of nitric oxide (5). However, since the kinetics of nitricoxide release, the redox state of nitric oxide, and the produc-tion of specific by-products can vary considerably between dif-ferent nitric oxide donors (9, 21), we examined whether therewas a direct bactericidal effect of SNAP or its products on R.equi. Bacteria were cultivated in DMEM complete in the pres-ence of SNAP for 24 h without any loss in viability (Fig. 8A).Expression of the essential virulence factor VapA by SNAP-treated R. equi was the same as that by untreated bacteria (Fig.8B), underscoring bacterial fitness.

SNAP and IFN-�–LPS-activated macrophages have similarquantities and kinetics of nitric oxide release. To compare theamounts of nitric oxide to which R. equi was exposed under thedifferent experimental conditions, we quantified nitric oxiderelease from (i) SNAP, (ii) resting J774E macrophages, (iii)J774E cells activated with IFN-� alone, or (iv) J774E cellsactivated with IFN-�–LPS. All conditions were tested withuninfected and infected macrophages. No nitric oxide produc-tion was detected in resting macrophages, regardless ofwhether these were infected or not (Fig. 9A). Activation withIFN-� alone led to production of small amounts of nitric oxide,which were massively increased in response to infection witheither virulent or avirulent R. equi (Fig. 9B). J774E cells acti-vated with IFN-�–LPS produced nitric oxide faster than cellsactivated with IFN-� alone, yet final nitrite concentrationswere lower than with IFN-�-activated cells (Fig. 9C). Evenuninfected macrophages produced large amounts of nitric ox-ide when they had been activated with IFN-� and LPS (Fig.9C). Infection with plasmid-cured R. equi reproducibly re-sulted in a slightly higher release of nitric oxide by macro-phages activated with IFN-�–LPS than infection with virulentbacteria (Fig. 9C; P 0.038 and 0.008 at 24 h and 48 h afterinfection, respectively).

In contrast to nitric oxide production by macrophages, nitricoxide release from SNAP was detectable already at 4 h (Fig.9D). However, from 8 h onward, both the kinetics and theconcentrations of nitric oxide were very similar to those pro-duced by IFN-�–LPS-treated macrophages (Fig. 9C and D).

Different nitric oxide donors do not only differ in kinetics ofrelease but also in the redox state of nitric oxide produced (9).We therefore investigated the effect of a nitric oxide donor

FIG. 7. Multiplication of R. equi in resting or activated macro-phages treated with compounds acting on release of nitric oxide orsuperoxide. (A) Resting J774E cells were infected with 103� at anMOI of 1 for 30 min in the absence of relevant reagents and incubatedin fresh medium containing the nitric oxide donor SNAP or solventalone (Control) for 2 h or 24 h. Samples were fixed and stained withSyto13 to quantify the percentage of macrophages with more than 10bacteria. (B) Infection and analysis of resting J774E cells was the sameas described for panel A, but samples were treated with 100 �M SNAPor solvent alone (Control) for 24 h before infection and in mediumwithout SNAP or solvent after infection (SNAP b.i., 24 h). (C) Infec-tion and analysis of IFN-�–LPS-activated J774E were the same asdescribed for panel A, but samples were incubated in the absence orpresence of 500 �M the NOS2 inhibitor NG-methyl-L-arginine(NMLA) after infection or treated with 5 mM the superoxide scaven-ger N-acetyl-L-cysteine (NAC) 2 h before and always after infection.(D) Experimental setup was as described for panel A but using restingor IFN-�–LPS-activated BMMs from B6 wild-type (B6-wt) or B6-derived NADPH oxidase knockout (gp91phox�/�) mice. Data shownin panels A to D represent means and standard deviations of resultsfrom three independent experiments. *, P � 0.05.



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from a compound class different from that of SNAP, diethyl-enetriamine-nitric oxide adduct (DETA-NO), on R. equi intra-cellular multiplication. Within 1 h, a concentration of 50 �MDETA-NO in DMEM complete already yielded nitric oxideamounts similar to those after 24 h of incubation of 100 �MSNAP (Fig. 9E). However, the restriction of R. equi growth inresting macrophages by DETA-NO was as pronounced as thatby treatment with SNAP (Fig. 9F).

Iron supplementation abrogates activation- or nitric oxide-mediated growth restriction. Iron availability is a limiting fac-tor during infection of activated macrophages (4). To test thepossible role(s) of iron limitation during the infection with R.equi, we took advantage of the R. equi iupT gene which isinvolved in catecholate siderophore synthesis and which ishighly upregulated in iron-depleted medium (32). Its transcrip-tion was clearly induced in R. equi inside activated macro-phages at 2 h of infection, whereas by 8 h of infection, theupregulation had ceased (Fig. 10). Remarkably, the strongupregulation at 2 h could be completely annihilated by theaddition of ferrous sulfate to the macrophage medium. Inresident macrophages, R. equi iupT expression was not upregu-lated at either time of infection. 2,2-Dipyridyl (2,2-bipyridin) isa membrane-permeable iron chelator which inhibits R. equigrowth in liquid medium at high concentrations (200 �M) butnot at lower concentrations (80 ��) (31). We observed that R.equi strain 103 did not multiply in minimal medium at 150 �M2,2-dipyridyl and, hence, used supplementation with 0, 50, or80 �M this compound as a control for iupT induction. Whereas50 �M 2,2-dipyridyl did not very much increase iupT transcrip-tion, a 14-fold relative induction was observed using 80 �M(Fig. 10). In summary, these data suggested a strong ironremoval response of R. equi early in infection of activated butnot resident macrophages.

To further analyze whether this iron removal stress couldcontribute to killing of R. equi by activated macrophages, weadded 100 �M ferrous sulfate extracellularly to infected IFN-�–LPS-treated J774E cells, which completely restored multi-plication of R. equi (Fig. 11A and B). The effect was concen-tration dependent and was not observed after the addition ofequimolar amounts of magnesium sulfate (Fig. 11A and B).

Importantly, the same phenomena were observed with SNAP-treated macrophages (Fig. 11A).

Restoration of R. equi intracellular multiplication by ferroussulfate could have been caused indirectly, e.g., by a decrease inmacrophage viability by ferrous sulfate or by a decreased nitricoxide production in the presence of iron ions, but neither wasthe case (Fig. 11C and D). Furthermore, ferrous ions canenhance the decomposition of SNAP (9), but here release ofnitric oxide from SNAP did not change in the presence of 100�M ferrous sulfate (not shown), excluding this as a cause of theobserved effects.

Another possible explanation for the observed effects was adirect action of nitric oxide on bacterial iron. To test thesensitivity of R. equi to nitric oxide under iron-limiting condi-tions, we grew the bacteria in minimal (Fig. 11E) rather thanrich (Fig. 8A) medium to be able to control for iron availabil-ity. Bacteria were grown in minimal medium containing 100�M ferrous sulfate overnight, pelleted, and cultivated in min-imal media with different iron concentrations in the presenceor absence of 100 �M SNAP for 24 h (Fig. 11E). At a concen-tration of 1 �M added ferrous sulfate, bacterial growth wassignificantly decreased by approximately 20% compared togrowth in medium with 250 �M iron (Fig. 11E), indicating thatiron was at a growth-limiting level. However, even at this lowlevel of free iron, growth of SNAP-treated R. equi was the sameas that in the untreated control samples (Fig. 11E).

DISCUSSION

This study analyzed the mechanisms by which activated mac-rophages inhibit growth of the intracellular pathogen R. equi. Ithad been proposed that IFN-�–LPS activation of macrophagesbefore infection leads to a fast killing of R. equi by peroxyni-trite (5). However, in our experimental system, R. equi survivedin activated macrophages for at least 24 h, followed by elimi-nation within the following 24 h. At this time, IFN-�–LPSactivation had already resulted in significant macrophagecytotoxicity. Therefore, the decrease in numbers of recoveredbacteria may either reflect their clearance or macrophage

FIG. 8. Effects of nitric oxide treatment on R. equi viability and VapA expression. (A) Sensitivity of R. equi 103� to treatment with nitric oxidewas assessed by cultivation in DMEM complete containing 100 �M SNAP or solvent only (Control) at 37°C. Culture optical density at 600 nm wasdetermined at various times. Data represent means and standard deviations of results from three independent experiments. (B) Samples fromcontrol and SNAP-treated 103� grown for 24 h and taken from the three experiments (1, 2, and 3) shown in panel A were applied to SDS-PAGE,Western blotted, and decorated with anti-VapA. Numbers indicate molecular weight in kDa, and “VapA” indicates the migration position ofVapA.



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death, leading to exposure of formerly intracellular R. equi togentamicin in the medium.

There are precedents for pathogens which do not multiply inactivated macrophages yet survive for considerable periods oftime, such as M. avium or L. pneumophila (45, 47). It has beensuggested that growth restriction of these pathogens is medi-ated by enhanced formation of phagolysosomes. Increasedphagosome-lysosome fusion and/or phagosome acidificationupon macrophage activation has been reported for severalother intracellular pathogens that do not grow in or are killedby activated host cells (19, 46, 50, 58), underscoring a closeconnection between the loss of their privileged intracellularcompartment and infection control.

The effects of macrophage activation on RCV properties

were investigated by comparison of hallmark features ofphagosome maturation in resting and activated J774E cells.The kinetics of pH in vacuoles containing virulent R. equimatched the RCV maturation profile (10): the initial acidifi-cation to the pH of early endosomes observed in this study isparalleled by the published normal acquisition and loss of earlymaturation stage markers (10). The subsequent failure of theRCV to acquire v-ATPase and to fuse with lysosomes (10)coincided with a rise in phagosome pH. Similar interferencewith phagosome acidification has been observed with othermembers of the mycolata, suggesting related mechanisms, i.e.,virulent but not avirulent strains of Nocardia asteroides residein neutral-pH vacuoles (1), and phagosomes containing patho-genic mycobacteria have a pH of 6.2 to 6.5 (44, 52, 63).

FIG. 9. Quantification of nitric oxide release by resting or IFN-�- or IFN-�–LPS-activated J774E macrophages or by SNAP. Resting (A) orIFN-� (B)- or IFN-�–LPS (C)-activated J774E cells were left uninfected or infected with 103� or 103� at an MOI of 1 for 30 min and incubatedin fresh medium. At the times indicated, supernatants were collected, and nitrite as a stable end product of nitric oxide was quantified using Griessreagent. (D and E) SNAP or DETA-NO were dissolved in DMEM at 100 �M (SNAP; Control; solvent only) or at 50, 100, or 200 �M (DETA-NO)and incubated at 37°C. Samples were taken at different times and analyzed for nitric oxide as described for panels A to C. All data are means andstandard deviations of results from three to five experiments. (F) Resting J774E cells were infected with 103� at an MOI of 1 for 30 min in theabsence of relevant reagents and then cultivated in fresh medium without (Control) or with the nitric oxide donors SNAP (100 �M) or DETA-NO(50 �M) for 2 h or 24 h. Samples were fixed and stained with Syto13 to quantify the percentage of macrophages with more than 10 bacteria. Datashown represent means and standard deviations of results from four independent experiments. A P value of �0.05 is indicated by the followingsymbols: #, uninfected versus 103�; °, uninfected versus 103�; *, 103� versus 103� in panels A to C or nitric oxide donor(s) versus the controlin panels D and F.



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Surprisingly, the unusual progression of R. equi phagosomepH was also observed in activated macrophages, and there wasno enhanced RCV-lysosome fusion. Unlike M. avium-contain-ing phagosomes (40), RCVs in activated macrophages re-mained accessible to endocytosed material. Hence, growth re-striction in activated macrophages was likely not caused byalterations in RCV compartmentation characteristics.

Activation of macrophages can induce a change in not onlyintracellular trafficking but also autophagy, a cellular starvationand stress response which restricts multiplication of intracel-lular mycobacteria (13). The autophagy inhibitor 3MA signif-icantly increased R. equi multiplication in activated macro-phages, yet no other established inhibitor (wortmannin) orinducer (rapamycin or starvation) of autophagy altered bacte-rial growth. Furthermore, no bacteria were observed in au-tophagic structures by transmission electron microscopy. Co-localization of the RCV with the autophagy marker LC3 wasinfrequent and occurred only early in infection, as has alsobeen observed with latex bead phagosomes in resting, non-starved cells (49), essentially excluding a specific activation-related role of LC3 in R. equi control. The results obtainedwith 3MA treatment therefore likely reflect an autophagy-independent activity of this compound similar to those ob-served by others (33).

An important defense mechanism of activated macrophagesdepends on immunity-related GTPases (IRG), includingIrgm1 (LRG-47) (57). In contrast to the situation with Salmo-nella (14) or Mycobacterium (26), activated Irgm1�/� primarymacrophages inhibited R. equi multiplication as much as wild-type phagocytes. As Irgm1 has been correlated to autophagicmechanisms (13) and acts independently of nitric oxide pro-duction (26), this result supports the concept of an autophagy-independent, nitric oxide-dependent (as discussed below)mode of R. equi growth restriction. It also suggests that IRGproteins other than Irgm1 are not involved either, since Irgm1

is crucial for correct localization of at least two other immu-nity-related GTPase family members (16).

Nitric oxide production by NOS2 is critically required forcontrol of R. equi infection by activated macrophages (5).SNAP (100 �M) produced nitric oxide in similar quantities andwith similar kinetics as IFN-�–LPS-activated macrophages in-fected with 103�, suggesting that R. equi was exposed to sim-ilar concentrations of nitric oxide in SNAP-treated and inactivated macrophages. SNAP addition fully mimicked thegrowth-inhibiting effect of J774E activation, and so did addi-tion of the nitric oxide donor DETA-NO. DETA-NO differsfrom SNAP in that nitric oxide is released very fast and indifferent redox form(s) (9), suggesting that exact kinetics ofnitric oxide release and/or the amounts of particular nitricoxide redox variants are not relevant for inhibition of R. equiintracellular multiplication.

Growth restriction of R. equi by SNAP or by macrophageactivation was independent of NADPH oxidase activity, inagreement with earlier studies (5). However, NADPH oxidaseknockout mice succumb to R. equi infection (5). This mayresult from NADPH oxidase knockout effects on other cells,e.g., neutrophils which respond to infection with a strong,gp91phox-containing NADPH oxidase (20)-depending oxida-tive burst and which are indispensable for clearance of R. equiinfection (27). Also, treatment of activated macrophages withthe reactive oxygen species quencher N-acetyl-L-cysteine(NAC) did not restore R. equi intracellular growth. We con-clude that growth inhibition by SNAP was not mediated byperoxynitrite, whose production requires both nitric oxide andsuperoxide, but was rather caused by nitric oxide alone or itssuperoxide-independent reaction products.

The precise identity of the nitric oxide target remains elu-sive. Our and others’ (5) data show that, unlike M. tuberculosis(24) or L. pneumophila (53), R. equi is not sensitive to directtreatment with nitric oxide even at low iron supply. Further,treatment of macrophages with SNAP before infection did notrestrict R. equi intracellular growth, indicating that nitric oxideaction is required only after infection. R. equi iupT is a genewhose expression is strongly upregulated in iron-limiting con-ditions, and it is also upregulated in activated but not in resi-dent macrophages. Hence, nitric oxide production is likely partof an activation-mediated host iron restriction system. Supple-mentation with abundant iron from the extracellular spaceannihilates the macrophages’ effort to restrict iron, and henceintracellular R. equi cells do not upregulate the iron acquisitionmachinery. A multitude of data from other groups suggest ageneral regulatory effect of macrophage activation and partic-ularly nitric oxide on intracellular iron levels. IFN-� activationreduces free iron in macrophages and their phagosomes (36,53, 61), and nitric oxide affects cellular iron uptake and avail-ability (30, 43, 62). Growth inhibition by macrophage activa-tion of several intracellular pathogens, such as L. pneumophilaor S. Typhimurium, has been correlated with their limitedaccess to iron (2, 37).

A particularly surprising finding of this study was that R. equiapparently did not suffer from iron removal stress in residentmacrophages, as reflected in low iupT expression. It seems as ifintracellular R. equi cells manage to readily acquire iron insufficient quantities, possibly through a constitutively expressed

FIG. 10. Real-time PRC analysis of iupT expression inside residentor activated macrophages. R. equi was grown for 16 h in minimalmedium (Min. medium) with acetate and 0 �M (DIP 0), 50 �M (DIP50), or 80 �M (DIP 80) 2,2-dipyridyl or isolated from resting oractivated macrophages that had been infected for 2 h or 8 h in theabsence or presence (�FeSO4) of 50 �M ferrous sulfate. Expression ofiupT is shown relative to expression of 16S-rRNA (fold change expres-sion). The mean values from two independent determinations areshown.



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iron acquisition system (28) or through the baseline expressionof the iup genes or another inducible iron acquisition system.

In summary, this study shows that R. equi cells survive for atleast 24 h in activated macrophages, where they are able toestablish their unusual compartment. Unlike in the case withseveral other intracellular pathogens, such as M. tuberculosis,growth restriction by immune activation is not dependent onautophagy, Irgm1, or phagolysosome formation. Inhibition ofgrowth was completely mimicked by treatment of resting mac-rophages with nitric oxide donors, demonstrating the centralrole of nitric oxide and the dispensability of induction of hun-dreds of genes by IFN-�–LPS (8) in macrophage defenseagainst R. equi infection. Mechanisms restricting multiplicationof R. equi could be annihilated by the addition of ferroussulfate. Macrophage activation and nitric oxide synthesis likelyact in a regulatory way on the producing J774E cell itself by (i)making the macrophages more capable of storing iron in a waythat it cannot readily be accessed by R. equi, (ii) making the

macrophage able to prevent iron from being scavenged bybacterial siderophores, or (iii) inactivating a host-driven path-way to directly supply the intraphagosome space with iron.

ACKNOWLEDGMENTS

We thank Sabine Spurck and Ulrike Karow for expert technicalassistance, Shinji Takai for the anti-VapA antibody, Noburo Miz-ushima (Tokyo Metropolitan Institute of Medical Sciences) for theGFP-LC3 mice, Ralf Brandes (University of Frankfurt Faculty of Med-icine) for the gp91phox�/� mice, and Stefanie Riesenberg and JoachimSchultze (LIMES, Bonn) for access to and help with the Roche Light-Cycler. We acknowledge valuable discussions with Jonathan Howardand Simon Newman.

This study was supported by a fellowship of the German NationalAcademic Foundation to K.V.B., by an NIH grant (AI57831) and a VAMerit Review grant to G.T., and by a collaborative research center(SFB 670) grant from the Deutsche Forschungsgemeinschaft (GermanResearch Foundation) to O.U. and A.H.

FIG. 11. Inhibition of multiplication of R. equi in activated or in SNAP-treated resting J774E cells can be abrogated by the addition of ironsulfate. (A) Resting or IFN-�–LPS-activated J774E cells were infected with 103� at an MOI of 1 for 30 min and incubated in fresh mediumcontaining no (control) or different additives for 2 h or 24 h. These were 100 �M the nitric oxide donor SNAP, 100 �M ferrous sulfate (FeSO4),or 100 �M magnesium sulfate (MgSO4). Samples were fixed and stained with Syto13 to quantify the percentage of macrophages with more than10 bacteria. *, P � 0.05. (B) Experimental setup as described for panel A, but activated macrophages were treated with no (Control) or differentconcentrations of ferrous sulfate after infection. *, P � 0.05. (C and D) Effects of ferrous sulfate treatment on viability (C) and nitric oxideproduction (D) of J774E cells: IFN-�–LPS-activated macrophages were treated with different concentrations of iron sulfate. At 24 h of incubation,sample supernatants were collected and the release of lactate dehydrogenase (C) and nitric oxide (D) was quantified. (E) 103� was grown inminimal medium with 100 �M ferrous sulfate overnight and used for inoculation of minimal medium with different iron concentrations at an OD600of 0.1. Suspensions were mixed with no (Control; solvent only) or 100 �M SNAP. Optical density was determined before and after 24 h ofincubation at 37°C. A P value of �0.05 is indicated by the following symbols: *, versus control-250 �M ferrous sulfate; #, versus SNAP-250 �Mferrous sulfate. Data of all graphs represent means and standard deviations of results from three or four experiments.



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