Iron Limitation Triggers Early Egress by the Intracellular Bacterial Pathogen Legionella pneumophila.

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Iron Limitation Triggers Early Egress by the Intracellular Bacterial Pathogen Legionella pneumophila Tamara J. O’Connor,a Huaixin Zheng,b Susan M. VanRheenen,c* Soma Ghosh,a Nicholas P. Cianciotto,b Ralph R. Isbergc,d Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USAa; Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USAb; Department of Molecular Biology and Microbiology, Tufts University Medical School, Boston, Massachusetts, USAc; Howard Hughes Medical Institute, Chevy Chase, Maryland, USAd

Legionella pneumophila is an intracellular bacterial pathogen that replicates in alveolar macrophages, causing a severe form of pneumonia. Intracellular growth of the bacterium depends on its ability to sequester iron from the host cell. In the L. pneumophila strain 130b, one mechanism used to acquire this essential nutrient is the siderophore legiobactin. Iron-bound legiobactin is imported by the transport protein LbtU. Here, we describe the role of LbtP, a paralog of LbtU, in iron acquisition in the L. pneumophila strain Philadelphia-1. Similar to LbtU, LbtP is a siderophore transport protein and is required for robust growth under iron-limiting conditions. Despite their similar functions, however, LbtU and LbtP do not contribute equally to iron acquisition. The Philadelphia-1 strain lacking LbtP is more sensitive to iron deprivation in vitro. Moreover, LbtP is important for L. pneumophila growth within macrophages while LbtU is dispensable. These results demonstrate that LbtP plays a dominant role over LbtU in iron acquisition. In contrast, loss of both LbtP and LbtU does not impair L. pneumophila growth in the amoebal host Acanthamoeba castellanii, demonstrating a host-specific requirement for the activities of these two transporters in iron acquisition. The growth defect of the ⌬lbtP mutant in macrophages is not due to alterations in growth kinetics. Instead, the absence of LbtP limits L. pneumophila replication and causes bacteria to prematurely exit the host cell. These results demonstrate the existence of a preprogrammed exit strategy in response to iron limitation that allows L. pneumophila to abandon the host cell when nutrients are exhausted.

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egionella pneumophila is an intracellular bacterial pathogen. In its natural environment of fresh water and soil (1, 2), L. pneumophila is a parasite of a broad assortment of free-living amoebae (3, 4). Human exposure to this pathogen occurs through the inhalation of contaminated water aerosols (5). Upon gaining access to the lung, L. pneumophila replicates within alveolar macrophages (6, 7), resulting in a severe and often fatal form of pneumonia (8, 9). The intracellular life cycle of L. pneumophila is highly conserved across multiple hosts. Upon engulfment into a membranebound compartment, L. pneumophila avoids digestion by preventing trafficking along the endocytic pathway to the lysosome (10, 11). In parallel, L. pneumophila recruits host endoplasmic reticulum-derived material to remodel its vacuole into a ribosome-studded compartment (12, 13). Roughly 4 to 6 h postinfection (hpi), when vacuole remodeling is complete, the bacteria begin to replicate. This process is dependent on a type IVb secretion system, termed Dot/Icm (14, 15), that translocates bacterial proteins across the vacuole membrane (16) to manipulate a variety of host cellular processes. L. pneumophila replication continues for 14 to 16 h, at which point the bacteria lyse out of the host cell and spread to neighboring cells (6). Egress from the host cell coincides with activation of several virulence traits, including motility, through induction of the flagellar regulon (17). While the timing of these events is highly conserved in multiple hosts, the signals that trigger egress and the mechanisms by which bacteria exit the vacuole and lyse the host cell remain unclear. A fundamental requirement for bacterial survival and replication is the acquisition of essential nutrients. Macrophages actively sequester essential nutrients such as iron from invading bacterial pathogens as a means to combat infection (18). The ability of L. pneumophila to acquire iron from the host cell is a critical deter-

August 2016 Volume 84 Number 8

minant in the outcome of infection (19). As a consequence, L. pneumophila employs several mechanisms for iron acquisition and assimilation during intracellular growth in cultured macrophages and a lung infection model (20–27). One mechanism employs legiobactin, an iron-chelating siderophore that has been extensively characterized in the L. pneumophila strain 130b (28). The machinery for the synthesis and transport of legiobactin consists of four proteins, LbtA, LbtB, LbtC, and LbtU, encoded by a single operon (29, 30). Legiobactin is synthesized in the bacterial cytoplasm by the siderophore synthetase LbtA and then exported by the membrane protein LbtB (23). Once bound to iron, legiobactin is recognized and imported by the outer membrane siderophore transport protein LbtU (29). Legiobactin is then transported across the inner membrane by LbtC to the bacterial cytoplasm (30), where it is relieved of iron and recycled. Mutants defective in siderophore synthesis and utilization exhibit reduced growth in a

Received 20 October 2015 Returned for modification 13 November 2015 Accepted 11 May 2016 Accepted manuscript posted online 16 May 2016 Citation O’Connor TJ, Zheng H, VanRheenen SM, Ghosh S, Cianciotto NP, Isberg RR. 2016. Iron limitation triggers early egress by the intracellular bacterial pathogen Legionella pneumophila. Infect Immun 84:2185–2197. doi:10.1128/IAI.01306-15. Editor: C. R. Roy, Yale University School of Medicine Address correspondence to Tamara J. O’Connor, [email protected]. * Present address: Susan M. VanRheenen, Merck Research Laboratories, Merck & Co. Inc., Upper Gwynedd, Pennsylvania, USA. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /IAI.01306-15. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Infection and Immunity

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murine model of pneumonia (28, 29), demonstrating an important role for this system in iron sequestration during infection. Here, we examine the role of the L. pneumophila Philadelphia-1 gene lpg2959 (called lbtP) in L. pneumophila pathogenesis. lbtP encodes a paralog of the siderophore transporter LbtU. We demonstrate the importance of lbtP, but not lbtU, for growth of L. pneumophila Philadelphia-1 in macrophages and a central role for its encoded protein in iron acquisition during infection. Moreover, we identify iron starvation at later stages of the infection cycle as a signal that triggers bacterial egress from the host cell. MATERIALS AND METHODS Bacterial strains, cultured cells, and growth media. All Legionella strains were generated in the Legionella pneumophila Philadelphia-1 strain Lp02 (31). L. pneumophila strains were cultured at 37°C in liquid N-(2-acetamido)-2-aminoethanesulfonic acid (ACES)-buffered yeast extract (AYE) medium or on solid charcoal ACES-buffered yeast extract (CYE) medium (32, 33) containing 0.4 mg/ml L-cysteine sulfate (Sigma) and 0.135 mg/ml ferric nitrate (Sigma) (unless indicated otherwise) and, when appropriate, 0.1 mg/ml thymidine (Sigma) (CYET), 40 ␮g/ml kanamycin, 50 ␮g/ml streptomycin, 5 ␮g/ml chloramphenicol, or 5% sucrose. Plasmids were introduced into L. pneumophila by electroporation (34) or by mating with the Escherichia coli Tra⫹ helper strain RK600 (35) or JB138 (a kind gift from J. Vogel, Washington University) as previously described (36). For growth within Acanthamoeba castellanii, the L. pneumophila chromosomal thy mutant allele in each bacterial strain examined was replaced with the thy⫹ allele by allelic exchange using pJB3395 (a kind gift from J. Vogel, Washington University) as previously described (35). The E. coli strain DH5␣ ␭pir was used for all plasmid cloning. E. coli strains were grown in liquid Luria broth (LB) or on solid LB plates supplemented with 50 ␮g/ml ampicillin, 25 ␮g/ml chloramphenicol, or 50 ␮g/ml kanamycin when appropriate. All bacterial strains are summarized in Table S1 in the supplemental material. Primary bone marrow-derived macrophages from A/J mice were isolated and cultured as previously described (31). A. castellanii (ATCC 30234) was cultured as previously described (37). Construction of L. pneumophila deletion mutants. Null mutations in individual genes were generated in L. pneumophila Philadelphia-1 strain Lp02 using a double recombination strategy employing the suicide vector pSR47s as previously described (38). Primer pairs for plasmid construction are listed in Table S1 in the supplemental material. All plasmids were sequenced prior to use. For each mutant, 12 to 16 individual isolates were screened by PCR, and intracellular growth phenotypes of three independent isolates were compared. Construction of lbtP and lbtU expression plasmids. L. pneumophila Philadelphia-1 strain Lp02 genomic DNA was isolated using a DNeasy blood and tissue kit (Qiagen). For in trans complementation experiments, lbtP (lpg2959) was amplified by PCR from genomic DNA using lbtPF and lbtPR primers (see Table S1 in the supplemental material) and then cloned as a BamHI-XbaI fragment into similarly digested pJB908 to generate pSV83. For subcellular localization experiments, lbtP and lbtU were amplified by PCR from genomic DNA using lbtPF2 and lbtPR2 and lbtUF and lbtUR primers, respectively (see Table S1), and then cloned as SacIXbaI fragments into similarly digested pTO10 to generate pTO11 and pTO12, respectively (see Table S1). pTO10 is a derivative of pJB908 in which a fragment encoding the FactorXa-V5-6xHIS protease cleavage site and epitope tags was generated by annealing primers V5-HIS-top and V5-HIS-bottom (see Table S1) and cloned into XbaI-SphI-digested pJB908. This vector enables isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible expression of C-terminally epitope-tagged fusion proteins in Legionella. All plasmids were sequenced prior to use. In vitro growth assays. L. pneumophila strains grown on solid media were resuspended in AYE media to an absorbance at 600 nm (A600) of 0.1 to 0.2, equivalent to 0.1 ⫻ 109 to 0.2 ⫻ 109 bacteria/ml. Bacterial growth at 37°C, with shaking, was monitored over 16 h by diluting culture aliquots

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in 1⫻ phosphate-buffered saline and measuring the A600 in a Molecular Devices SpectraMax M5 spectrophotometer at regular intervals. For iron deprivation experiments, the iron chelator deferoxamine mesylate (DFX) (Sigma) was added to the culture medium lacking iron supplementation at the indicated concentrations just prior to the addition of bacteria. Bacterial growth at 37°C, with shaking, was monitored over 40 h by measuring the A600 every hour in a Synergy HT plate reader (BioTek) using KC4 data analysis software (BioTek). Iron sensitivity assays. L. pneumophila strains were grown for 3 days at 37°C on solid CYE medium supplemented with 250 ␮g/ml ferric pyrophosphate (Sigma) (23). Bacteria were suspended in resuspension buffer (50 mM morpholinepropanesulfonic acid [MOPS], 2 mM monobasic potassium phosphate, and 50 mM sodium chloride, pH 6.5) to an A600 of 0.3, which corresponds to approximately 0.3 ⫻ 109 bacteria/ml. Tenfold serial dilutions of the bacterial suspension were spotted onto solid CYE medium containing 250, 12.5, 5, and 1.25 ␮g/ml ferric pyrophosphate or medium lacking ferric pyrophosphate, and bacterial growth at 37°C was monitored over a 5-day period. Siderophore production and utilization assays. To assess siderophore production, L. pneumophila strains were cultured in deferrated, chemically defined medium (CDM) at 37°C for 24 h, and then cell-free supernatants were obtained by filtration as previously described (23). Iron-chelating activity within the supernatants was determined with the chrome azurol S (CAS) assay, using DFX as a standard control as previously described (23) or by assessing their ability to rescue the growth of an L. pneumophila 130b ⌬feoB mutant (22) on CYE medium lacking iron supplementation as previously described (29). Siderophore utilization was assessed as previously described (29), but with minor modifications. For each L. pneumophila strain tested, 1 ⫻ 104 bacteria were plated on solid non-iron-supplemented CYE medium. Seventy-five microliters of siderophore-containing supernatants obtained from the wild-type L. pneumophila Philadelphia-1 strain Lp02 (as described above) or deferrated CDM then were added to wells cut in the center of the agar plate. The plates were incubated at 37°C for 8 days, and growth of bacteria was compared. For rhizoferrin utilization experiments, L. pneumophila strains were plated on non-iron-supplemented CYE medium containing 8 ␮M DFX, and 50 ␮l of 40 ␮g/ml of purified rhizoferrin (EMC Microcollections, Tubingen, Germany) was added to wells cut in the center of the agar plate as previously described (27). Iron uptake assays. Iron uptake in the presence of legiobactin was measured as previously described (29). Briefly, bacterial strains were cultured in non-iron-supplemented AYE medium to mid-log phase, equivalent to an A660 of 1.0. Bacteria were harvested by centrifugation at 5,000 ⫻ g, rinsed, and resuspended in 30 ml of deferrated CDM at an A660 of 0.3. Bacterial cultures were incubated at 37°C with shaking for 18 h. Bacteria were harvested by centrifugation as described above and their supernatants collected. Bacterial pellets were resuspended in their respective legiobactin-containing supernatants at an A660 of 1.0. 55FeCl3 (PerkinElmer, Boston, MA) in 10 mM HCl was added to 9 ml of bacterial suspension to a final concentration of 1 ␮Ci/ml (37 kBq/ml) and then incubated at room temperature for 2 h. At 0-, 60-, and 120-min time points, 3 separate 1-ml aliquots of the suspension were filtered through 0.45-␮m-pore-size nitrocellulose membrane (Millipore, Billerica, MA) to collect the bacteria. Filters were washed with 5 ml of a 0.5% solution of thioglycolic acid (Sigma) to remove extracellular iron, and the mean radioactivity in counts per minute over a 5-min period was determined using a scintillation counter. Subcellular localization assays. Subcellular fractionation protocols were adapted from those previously described (39). L. pneumophila strains were resuspended in 20 ml of AYE medium to an A600 of 0.2, equivalent to approximately 0.2 ⫻ 109 bacteria/ml. Bacteria were cultured overnight at 37°C with shaking to postexponential phase, equivalent to an A600 of 3.5 to 4.0. All subsequent steps were performed at 4°C. Bacteria were harvested by centrifugation at 5,000 ⫻ g for 10 min and resuspended in 0.5 ml of ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA,



Iron Starvation Induces Egress by L. pneumophila

and 1 mM dithiothreitol). To the cell suspension, 0.5 ml of ice-cold sucrose buffer (50 mM Tris-HCl, pH 7.5, 1 M sucrose, 1⫻ protease inhibitor cocktail [Roche]) was added. Spheroplasts were generated by adding 1 ml of 5 mM EDTA, pH 8.0, 0.1 mg/ml lysozyme and incubating on ice for 30 min. Magnesium sulfate (MgSO4) then was added to a final concentration of 20 mM, and the spheroplasts were harvested by centrifugation at 5,000 ⫻ g for 10 min. The supernatant or periplasmic fraction (P) was transferred to a sterile tube, and the cell pellet was resuspended in 5 ml of ice-cold lysis buffer containing 1⫻ protease inhibitor cocktail. Cells were lysed on ice by sonication using four 15-s sonic bursts of increasing intensity from 25 to 45%. Intact cells were removed by centrifugation at 5,000 ⫻ g for 10 min. The lysate, or total protein fraction (T), was centrifuged at 100,000 ⫻ g for 1 h at 4°C, and the supernatant or cytoplasmic protein fraction (C) was collected. The pellet or membrane fraction (M) was resuspended in 1 ml of ice-cold lysis buffer containing 1% Triton X-100 by gentle agitation overnight at 4°C. The membrane suspension was centrifuged at 100,000 ⫻ g for 1 h at 4°C as described above, and the Triton X-100 soluble proteincontaining supernatant, or inner membrane fraction (I), was collected. The pellet or outer membrane fraction (O) was resuspended in 0.5 ml of lysis buffer by gentle agitation for 2 h at 4°C. Protein fractions were quantified by Bradford assay (Bio-Rad). Twenty-microgram aliquots of fractions O, I, and C and 10 ␮g of fraction P were boiled for 10 min in 1⫻ Laemmli buffer and analyzed by Western analysis. Filters were probed with mouse anti-V5 antibody (1:500; Life Technologies) or rabbit antiisocitrate dehydrogenase (ICDH) (1:10,000; a kind gift from Linc Sonenshein, Tufts University School of Medicine) primary antibody and then probed with horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1:10,000; Life Technologies) secondary antibody. The Western blots were developed using ECL Plus Western blotting detection reagents (GE Healthcare Life Sciences). Intracellular growth assays. Growth of L. pneumophila in A/J bone marrow-derived macrophages and Acanthamoebae castellanii was performed as described previously (31, 40), with the following exception: A. castellanii was plated at 2 ⫻ 105 cells per well in a 96-well tissue culture plate and challenged with L. pneumophila strains at a multiplicity of infection (MOI) of 0.01 to 0.03. For iron-dependent growth assays in macrophages, macrophages were infected with L. pneumophila strains harboring pTO585 (see Table S1 in the supplemental material), and growth was monitored every hour for 36 h by measuring fluorescence in a Tecan M200Pro spectrophotometer. pTO585 is a derivative of pPpacS-EGFP (41) in which tdtomato was amplified using primers TO226 and TO227 (see Table S1) and cloned as an NdeI-PstI fragment into similarly digested pPpacS-EGFP to replace egfp. For iron depletion experiments, DFX was added to the culture media at a final concentration of 0, 50, 100, or 200 ␮M DFX at 1, 8, 10, or 12 h postinfection as indicated. For iron-dependent growth assays in A. castellanii, cells were rinsed 3 times with A. castellanii buffer (40) lacking iron supplement and equilibrated at 35°C for 1 h prior to infection. Bacterial strains constitutively expressing the enhanced green fluorescent protein (EGFP) from pPpacS-EGFP (41) (see Table S1) were rinsed with A. castellanii buffer lacking iron supplement and then used to infect A. castellanii at an MOI of 4 for 1 h. Cells were then rinsed with A. castellanii buffer lacking iron, and bacterial growth was monitored every hour for 36 h by measuring fluorescence in a Tecan M200Pro spectrophotometer. For gentamicin protection assays, 2 ⫻ 105 macrophages were infected with L. pneumophila strains at an MOI of 1 for 1 h and rinsed three times with macrophage culture media, and then gentamicin was added to a final concentration of 10 ␮g/ml. At the indicated time points, cell monolayers were rinsed three times with culture media lacking gentamicin to remove the antibiotic and then lysed with detergent to harvest bacteria as described previously (31). Cell lysates were then plated on bacteriological media and total bacteria were determined based on enumerating CFU. In parallel, replicate experiments lacking gentamicin in the culture media were performed to monitor total bacterial growth. Infectious center assays. For A/J macrophages, 4 ⫻ 105 cells were plated on coverslips in a 24-well tissue culture plate and incubated over-


night at 37°C. L. pneumophila strains were grown at 37°C in AYE to postexponential-phase A600 of 3.7 to 4.0, and motility was judged by visual inspection using an inverted microscope equipped with a 40⫻ lens. Bacteria then were used to challenge macrophages at an MOI of 1 for 1 h. Cells were rinsed 3 times with culture medium. At 1, 8, 10, 12, and 14 h postinfection, cells were fixed in 4% paraformaldehyde in 1⫻ PBS for 30 min at room temperature in the dark. Cells then were permeabilized with icecold methanol and stained with a rabbit ␣-Legionella antibody (1:5,000) (12) in 1⫻ PBS containing 4% goat serum (Gibco) followed by goat ␣-rabbit IgG Alexa Fluor 488 (1:1,000) (Molecular Probes). For iron supplementation experiments, culture medium was supplemented with 200 ␮M ferric ammonium citrate (Sigma) subsequent to 3 medium rinses 1 h postinfection. For iron depletion experiments, DFX was added to a final concentration of 200 ␮M at 8, 10, or 12 h postinfection as indicated. Extracellular bacterial accumulation and reinfection assays. A/J bone marrow-derived macrophages were plated at 1 ⫻ 105 cells per well in a 96-well tissue culture plate and challenged with L. pneumophila strains at an MOI of 1. After 1 h of challenge, cells were rinsed 3 times with fresh culture media and then incubated in culture medium containing 10 ␮g/ml gentamicin for 1 h to remove extracellular bacteria. Cells then were rinsed 3 times with fresh culture medium and incubated in medium lacking gentamicin. At 2, 21, 24, and 27 h postinfection, culture medium was collected and cells were very gently rinsed once with 100 ␮l of fresh medium to harvest extracellular bacteria. Cells were lysed with 0.02% digitonin at 37°C for 10 min to release intracellular bacteria as previously described (31). Culture media and cell lysates were plated on bacteriological medium, and extracellular and intracellular bacteria were enumerated based on CFU. For reinfection assays, extracellular bacteria harvested at 21 h postinfection were diluted 5- to 10-fold and used to challenge freshly plated A/J bone marrow-derived macrophages, and bacterial growth was monitored over 24 h using an intracellular growth assay plating for bacterial CFU as described above.

RESULTS

Loss of lpg2959 impairs intracellular growth of L. pneumophila in macrophages. Transposon insertions in lpg2959 (42) were previously identified in a screen for L. pneumophila Philadelphia-1 mutants that have altered timing, relative to the parental strain, of intracellular growth in bone marrow-derived macrophages from A/J mice (43). To determine if lpg2959 was required for growth in macrophages, a null deletion mutation of lpg2959 was generated in L. pneumophila Philadelphia-1, and the ability of the corresponding mutant to replicate in macrophages was examined. Growth of the ⌬lpg2959 mutant was impaired compared to that of the wild-type strain but was not as defective for growth as a translocation-deficient, dotA strain (34) (Fig. 1A). The defect was specific to intracellular growth, as the ⌬lpg2959 mutant strain grew as well as, if not slightly better than, the wild-type strain in bacteriological medium (Fig. 1B). The growth defect could be rescued by supplying lpg2959 in trans on a self-replicating plasmid, demonstrating that the impeded replication of the ⌬lpg2959 mutant was not due to polar effects on neighboring genes or additional mutations in the strain background. These results demonstrate the importance of lpg2959 for intracellular growth of L. pneumophila in a macrophage host. Lpg2959 is a paralog of the L. pneumophila strain 130b siderophore transporter LbtU. lpg2959 encodes a protein of unknown function. The N-terminal portion of the protein consists of a glutamate-rich region homologous to the bacterial protein FlxA (44) (Fig. 2A; see also Fig. S1A in the supplemental material). FlxA is a protein of unknown function but is conserved across many bacterial species. In E. coli, FlxA is a member of the flagellar regulon but is dispensable for motility (44). The C-terminal do-


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FIG 1 lpg2959 deletion mutant is defective for growth in macrophages. (A) Growth of the wild type (WT) and the dotA and lpg2959 deletion mutant strains harboring the empty control vector pJB908 or the lpg2959 deletion mutant harboring the lpg2959 complementation plasmid plpg2959 in A/J mouse bone marrow-derived macrophages. Bacterial growth, based on recovered CFU on solid media from lysed macrophages, was monitored over 72 h and encompassed 3 consecutive rounds of infection. Plotted is the total bacterial yield at the indicated time points normalized to the wild-type strain by percent uptake 2 h postinfection. (B) Growth of the L. pneumophila strains described for panel A in nutrient-rich bacteriological media. Bacterial growth based on absorbance at 600 nm was measured at regular intervals over a 16-h period. Data are representative of 2 independent experiments with 3 technical replicates each. Error bars indicate ⫾ standard deviations. An asterisk indicates a P value of ⬍0.05 by Student’s t test relative to the wild-type strain.

main of Lpg2959 is homologous to that of Lpg1326 (24% identity, 45% similarity) (Fig. 2A; see also Fig. S1B), an ortholog of the L. pneumophila 130b protein LbtU (lpw13361) (45) that imports the iron-bound siderophore legiobactin (27, 29). The genes responsible for siderophore production and transport in the L. pneumophila 130b strain are conserved in the L. pneumophila Philadelphia-1 strain (lpg1323-lpg1326) (23, 27, 29, 30) (Fig. 2B), indicating conserved utilization of a siderophore for iron acquisition in L. pneumophila Philadelphia-1. lpg2959 does not localize to this operon (Fig. 2B) but is highly conserved across multiple strains of L. pneumophila (10/10 completely sequenced genomes at ⬎92% identity [42, 46–52] and the L. pneumophila 130b strain [45]) (Fig. 2). Based on sequence similarity between LbtU and Lpg2959, we have designated lpg2959 legiobactin transporter paralog, or lbtP. An ⌬lbtP mutant is sensitive to iron deprivation in vitro. The sequence similarity of LbtP to LbtU suggested a role for LbtP in

iron acquisition, so the effects of loss of LbtP function on growth in iron-depleted bacteriological medium was determined. The L. pneumophila Philadelphia-1 wild-type strain Lp02 and isogenic ⌬lbtP and ⌬lbtU mutants were cultured in liquid AYE medium that lacked iron supplementation but contained increasing concentrations of the iron chelator deferoxamine mesylate (DFX). The growth of each strain was assessed by monitoring absorbance at 600 nm over a 36-h period (Fig. 3A). The wild-type strain grew robustly in moderate and low levels of iron (0 to 12.5 ␮M DFX) but failed to grow at the highest concentration of DFX (25 ␮M). In comparison, the ⌬lbtU mutant was indistinguishable from the wild-type strain, growing robustly in all but the highest concentration of DFX. In contrast, the ⌬lbtP mutant grew more slowly at lower concentrations of DFX (6.25 and 12.5 ␮M), demonstrating that the ⌬lbtP mutant is more sensitive to iron deprivation than both the wild-type and ⌬lbtU mutant strains. The behavior of the ⌬lbtP mutant is consistent with a role for LbtP in iron acquisition

FIG 2 Lpg2959 is a paralog of the L. pneumophila siderophore transporter LbtU. (A) Domain map of Lpg2959, which consists of an N-terminal FlxA domain and a C-terminal LbtU homology domain. (B) The lbtU and lpg2959 (lbtP) genetic loci are conserved between L. pneumophila strains Philadelphia-1 and 130b. lbtU is a member of the lbtABCU operon encoding proteins responsible for the synthesis and transport of the L. pneumophila siderophore legiobactin. lpg2959 (lbtP) is located distal from the lbtABCU operon and is among several transmembrane domain proteins of unknown function.

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FIG 3 LbtP is a siderophore transport protein important for iron acquisition. (A) The ⌬lbtP mutant strain shows enhanced sensitivity to iron limitation in

bacteriological media. Growth of the wild type (WT) and ⌬lbtU and ⌬lbtP mutant strains was monitored over time in the presence of increasing concentrations of the iron chelator DFX in media lacking iron supplementation. (B) LbtU and LbtP are differentially important for growth under iron-limited conditions. Tenfold serial dilutions of the wild type and the ⌬lbtU, ⌬lbtP, and ⌬lbtU ⌬lbtP mutant strains harboring the empty control vector pJB908, the lbtP complementation plasmid plbtP, or the lbtU complementation plasmid plbtU were spotted (from top to bottom) on solid CYE medium containing decreasing amounts of the iron supplement ferric pyrophosphate or medium lacking iron supplement. Bacterial growth after 5 days of incubation at 37°C is shown. (C) LbtP and LbtU are differentially important for iron uptake. L. pneumophila strains were grown in deferrated chemically defined medium (CDM) and then incubated with 55FeCl3 in the presence of their corresponding legiobactin-containing culture supernatants for 0, 60, and 120 min and assessed for the incorporation of radiolabeled iron. Data represent the means from 4 independent experiments with 3 technical replicates each normalized to the wild-type strain at 0 min. Error bars indicate ⫾ standard deviations. An asterisk indicates a P value of ⬍0.005 by Student’s t test relative to the wild-type strain. (D) LbtP and LbtU localize to the L. pneumophila outer membrane. LbtU and LbtP were expressed as C-terminal V5 epitope-tagged fusion proteins in the wild-type strain. The distribution of LbtU and LbtP in subcellular fractions of lysed bacteria was then analyzed by Western analysis. O, outer membrane; P, periplasm; I, inner membrane; C, cytoplasm. (E) Legiobactin utilization is dependent on lbtU and lbtP. The indicated L. pneumophila strains were plated on iron-supplemented CYE (CYE⫹Fe) or non-iron-supplemented CYE (CYE-Fe), and the center well was supplemented with either deferrated CDM lacking iron (CDM-Fe) or siderophore-containing culture supernatants of the L. pneumophila wild-type strain Lp02 (WT Sups). Bacterial growth after 4 or 8 days of incubation at 37°C is shown. Data shown in panels A, B, D, and E are representative of at least two independent experiments.



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and a more dominant contribution to iron assimilation than LbtU in L. pneumophila Philadelphia-1. To determine whether LbtU and LbtP functions overlap, the relative sensitivities of the ⌬lbtU and ⌬lbtP mutants and an ⌬lbtP ⌬lbtU double mutant were examined. Bacterial strains were cultured in AYE medium containing 100% of the standard iron supplement and then serially diluted and plated on solid CYE media containing decreasing amounts of iron supplement (100%, 5%, 2%, and 0.5%) or medium lacking iron supplement (0%) (Fig. 3B). At high levels of iron (CYE plus 100% Fe), the ⌬lbtU and ⌬lbtP mutants grew as well as the wild-type strain. At moderate levels of iron supplementation (CYE plus 2% Fe), the ⌬lbtU mutant grew as well as the wild-type strain, consistent with the phenotypes observed when grown in liquid media (Fig. 3A). In contrast, the ⌬lbtP mutant failed to grow at lower serial dilutions than the wild-type strain. Growth of the ⌬lbtP mutant could be restored by reintroducing lbtP on a self-replicating plasmid. These results suggest that LbtP, but not LbtU, is important for growth under conditions of moderate iron availability. Furthermore, the robust growth of the ⌬lbtU mutant demonstrates that LbtP is sufficient to support bacterial growth under conditions of moderate iron availability in the absence of LbtU. A mutant lacking both lbtU and lbtP was more defective for growth than the ⌬lbtP single-deletion mutant (Fig. 3B). Even under conditions of very moderate iron depletion (CYE plus 5% Fe) in which the ⌬lbtP and ⌬lbtU single-deletion mutants grew as well as the wild-type strain, the double mutant showed a stark reduction in growth. These results demonstrated that while LbtU can be dispensable in the presence LbtP, it makes a significant contribution to iron acquisition in the absence of LbtP. Growth of the ⌬lbtU ⌬lbtP double mutant could be completely restored to wildtype levels by reintroducing lbtU alone on a self-replicating plasmid (Fig. 3B). At low levels of iron (CYE plus 0.5% Fe), both the lbtU mutant and lbtP mutant displayed impaired growth relative to the parental strain, and the difference in the growth defects observed for the ⌬lbtP and ⌬lbtU single-deletion mutants was less robust, confirming a role for both LbtU and LbtP under iron starvation conditions. At very low levels of iron (CYE plus 0% Fe), the ⌬lbtP and ⌬lbtU mutants were indistinguishable from one another. At this level of iron depletion, the wild-type strain also showed a severe growth defect whereby the ⌬lbtP and ⌬lbtU mutants were only marginally more defective for growth. The behavior of the wild-type strain demonstrates that the presence of both LbtP and LbtU are insufficient to sequester enough iron to support bacterial growth at very low levels of iron. In comparison, the ⌬lbtP ⌬lbtU double mutant failed to grow completely. Collectively, these results demonstrate that in the Philadelphia-1 strain, LbtP plays a primary role in iron sequestration. In a strain lacking LbtP, the presence of LbtU can partially compensate for its absence. LbtP is important for iron uptake through siderophore transport. The sensitivity of bacteria lacking LbtP to iron deprivation was consistent with the protein being involved in iron acquisition, so the effect of loss of LbtP function on iron uptake was assessed. Each bacterial strain examined was resuspended in chemically defined medium (CDM) supplemented with radiolabeled FeCl3 as the sole iron source, and iron uptake was monitored over a 2-h period (Fig. 3C). Robust accumulation of radioactive iron was observed for the wild-type strain at 60 and 120 min. In contrast, the ⌬lbtU mutant showed reduced levels of iron uptake

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relative to the wild-type strain at both time points (29). In comparison, the ⌬lbtP mutant showed a gross defect in iron uptake relative to both the wild-type strain and the ⌬lbtU mutant. The severe iron uptake defect of the ⌬lbtP mutant compared to that of the ⌬lbtU mutant was consistent with the increased sensitivity of the ⌬lbtP mutant to iron depletion in liquid and on solid medium (Fig. 3A and B). The iron uptake defect of the ⌬lbtP mutant could be restored to wild-type levels by supplying the ⌬lbtP mutant with a copy of the lbtP gene in trans (Fig. 3C). Surprisingly, the ⌬lbtP ⌬lbtU double mutant was no more defective for iron uptake than the ⌬lbtP single-deletion strain. This could be due to a mechanism of iron uptake that functions in the absence of LbtU and LbtP but does not allow L. pneumophila to effectively assimilate the iron and thus does not support L. pneumophila replication on bacteriological medium. These results demonstrate that LbtP and, to a lesser extent, LbtU are important for iron uptake in L. pneumophila Philadelphia-1 and that the severity of the growth defects of the ⌬lbtP and ⌬lbtU mutants in iron-replete medium was due to their differential abilities to efficiently sequester iron from the surrounding environment. In L. pneumophila strain 130b, LbtU localizes to the bacterial outer membrane, where it functions to transport the iron-loaded siderophore legiobactin into the cell (29). The homology of LbtP with LbtU and the role of LbtU in iron uptake suggested that LbtP also functions as a siderophore transporter. Consistent with this idea, LbtP localizes to the L. pneumophila outer membrane, similar to LbtU (Fig. 3D). In addition, both the ⌬lbtP and ⌬lbtU mutants are capable of synthesizing and secreting active siderophore (see Fig. S2 in the supplemental material). To examine the role of LbtP in siderophore uptake, its ability to utilize iron-loaded siderophore as a source of iron was examined. Culture supernatants of the wild-type strain grown on deferrated CDM, and thus containing siderophore, were assessed for their ability to rescue growth of the ⌬lbtU, ⌬lbtP, and ⌬lbtP ⌬lbtU mutant strains on solid bacteriological media containing basal levels of iron but lacking iron supplementation (Fig. 3E). While all strains exhibited robust growth on solid CYE supplemented with iron (Fig. 3E, top), none of the strains grew on iron-depleted media supplemented with non-iron-containing CDM (Fig. 3E, middle). Wild-type strain culture supernatants were able to rescue the growth of the wild-type strain (Fig. 3E, bottom), demonstrating its ability to utilize siderophore to sequester low levels of iron present in CYE medium. Consistent with the results observed for the L. pneumophila strain 130b ⌬lbtU deletion mutant (29), growth of the L. pneumophila Philadelphia-1 ⌬lbtU deletion mutant was not rescued by wild-type culture supernatants. The inability of exogenously added siderophore to rescue growth of the ⌬lbtU deletion strain despite an intact copy of lbtP indicates that the level of iron available under these conditions was insufficient to support bacterial growth, similar to the reduced growth of the ⌬lbtU mutant on solid media under conditions of very low levels of iron (Fig. 3B). Similarly, growth of the ⌬lbtP deletion mutant could not be rescued by wild-type strain culture supernatants. However, growth of the complemented strain, the ⌬lbtP mutant overexpressing lbtP, could be restored by wild-type strain culture supernatants, demonstrating a role of LbtP in siderophore utilization. Consistent with the behavior of the ⌬lbtU and ⌬lbtP single mutants, the ⌬lbtU ⌬lbtP double mutant also was unable to utilize iron-loaded siderophore to support growth on iron-depleted media. Recently, it was determined that legiobactin is equivalent in




structure to rhizoferrin, a siderophore first identified in fungi (27). Using purified rhizoferrin as the sole iron source, similar results were obtained (see Fig. S3 in the supplemental material). Collectively, these results demonstrate that LbtP is a siderophore transporter that functions in iron acquisition. LbtU is dispensable for growth in macrophages. The ⌬lbtP mutant shows a significant growth defect in macrophages (Fig. 1), consistent with the importance of iron acquisition during intracellular growth and a role for this protein in iron sequestration through siderophore transport (Fig. 3). Given the role of LbtU in siderophore transport and its importance in vitro for L. pneumophila growth under conditions of severe iron limitation or in the absence of LbtP (Fig. 3B), the importance of LbtU for growth in macrophages was examined. In contrast to lbtP, deletion of lbtU did not impair bacterial replication within macrophages (Fig. 4A). Moreover, deletion of lbtU in the ⌬lbtP mutant strain background did not further attenuate the growth defect of the ⌬lbtP singledeletion mutant (Fig. 4A) despite the enhanced sensitivity of the ⌬lbtU ⌬lbtP double mutant to iron limitation on bacteriological media (Fig. 3B). These results demonstrate that LbtP, but not LbtU, is required for robust intracellular growth of L. pneumophila Philadelphia-1 in macrophages and that LbtP is the dominant paralog required for iron acquisition for this strain in this model infection system. In the environment, L. pneumophila replicates within an assortment of free-living amoebae, natural hosts of this bacterium. While LbtP is the main contributor to iron acquisition in macrophages, LbtU may play a more central role in amoebae. To assess the importance of these proteins for replication in the amoebal host A. castellanii, growth of the ⌬lbtU, ⌬lbtP, and ⌬lbtU ⌬lbtP mutants was compared to that of the wild-type strain and an avirulent dotA mutant strain. In contrast to the results in macrophages, lbtU and lbtP both were dispensable for growth in A. castellanii (Fig. 4B). Moreover, the combined deletion of both transporters did not adversely affect L. pneumophila intracellular replication in this host. Under standard assay conditions, the A. castellanii culture medium is supplemented with iron (40), which could mask a phenotype of the ⌬lbtP or ⌬lbtU mutant. Moreover, subtle phenotypes at a single stage of the infection cycle may not be revealed using endpoint assays that measure cumulative growth. To test this, we compared the growth of the wild-type and mutant strains in the absence of iron supplement. To do this, strains constitutively expressing the enhanced green fluorescent protein were used to challenge A. castellanii and bacterial growth was measured by monitoring fluorescence in real time over a single round of infection. Consistent with previous observations (Fig. 4B), in either the presence or absence of iron supplement, both the rate of growth and cumulative number of bacteria at the end of the infection cycle for the ⌬lbtU, ⌬lbtP, and ⌬lbtU ⌬lbtP mutants were similar to that of the wild-type strain (Fig. 4C); these results further demonstrate the dispensability of lbtU and lbtP for growth of L. pneumophila Philadelphia-1 in A. castellanii. In contrast, the total number of bacteria that accumulated at the end of the growth cycle under iron starvation conditions was significantly reduced (P value of ⬍0.05 based on Student’s t test) compared to that under iron-supplemented conditions for all strains (Fig. 4C). These results demonstrate that under conditions of iron deprivation, L. pneumophila replication within A. castellanii is arrested early compared to conditions under which iron is more abundant.


FIG 4 lbtP is differentially important for growth in macrophages and amoebae, whereas lbtU is dispensable in both hosts. (A) The ⌬lbtP mutant shows a growth defect in bone marrow-derived A/J mouse macrophages relative to the wild type and ⌬lbtU deletion mutant. (B) The absence of lbtP and lbtU, individually or in combination, does not impair L. pneumophila growth in A. castellanii compared to that of the wild-type strain. (A and B) Bacterial growth based on recovered CFU on solid media from lysed host cells was monitored over 48 to 72 h, encompassing 2 to 3 consecutive rounds of infection. Plotted is the total bacterial yield at the indicated time points normalized to the wildtype strain by percent uptake 2 h postinfection. (C) Lack of iron supplement in A. castellanii culture medium does not impair growth of the ⌬lbtP, ⌬lbtU, or ⌬lbtP ⌬lbtU mutant relative to the wild-type strain but induces early growth arrest for all strains compared to growth in iron-supplemented medium. Plotted is the relative fluorescence units (RFU) measured at the indicated time points normalized to the wild-type strain at 2 h postinfection. Data are representative of 2 to 5 independent experiments with 3 technical replicates each. Error bars indicate ⫾ standard deviations.

The intracellular growth defect of the ⌬lbtP mutant in macrophages is due to decreased cumulative growth in response to iron deprivation. To determine the basis of the growth defect of the ⌬lbtP mutant in macrophages, the kinetics of intracellular replication of this mutant was examined in detail. Macrophages were challenged with either the wild-type strain or the ⌬lbtP mutant, and the number of bacteria per phagosome was enumerated at various time points over the course of a single round of infection (Fig. 5A). By 14 h postinfection, the wild-type strain showed robust growth and an accumulation of large vacuoles containing ⬎20 bacteria. In contrast, the number of large vacuoles observed for the ⌬lbtP mutant was significantly reduced. The lack of large vacuoles did not coincide with an overabundance of vacuoles containing one or a few bacteria, suggesting that the lack of large


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FIG 5 ⌬lbtP mutant undergoes premature growth arrest in macrophages in an iron-dependent manner. (A) Macrophages challenged with the ⌬lbtP mutant

exhibit fewer vacuoles containing large numbers of bacteria than the wild-type strain. Macrophages were infected with the wild type or the ⌬lbtP mutant strain harboring the empty vector pJB908 or the ⌬lbtP mutant harboring the lbtP complementation plasmid plbtP. At 8, 10, 12, and 14 hpi, cells were fixed and then stained with antibodies against L. pneumophila and visualized by fluorescence microscopy. (B) Iron supplementation of the macrophage culture medium rescues the growth defect of the ⌬lbtP mutant. Macrophages were challenged with the wild type or ⌬lbtP mutant strain constitutively expressing GFP in the presence or absence of iron supplement. At 14 hpi, cells were fixed and visualized by fluorescence microscopy. (C) Iron depletion induces early growth arrest of wild-type bacteria in macrophages in a temporal and dose-dependent manner. Macrophages were challenged with wild-type bacteria expressing GFP, and then 200 ␮M DFX was added at various time points (top) or various concentrations of DFX were added 10 hpi (bottom), and bacterial growth was measured by monitoring fluorescence. RFU, relative fluorescence units. (D) Iron depletion at intermediate stages of the infection cycle results in fewer vacuoles containing large numbers of bacteria. Macrophages were challenged with wild-type bacteria. At the indicated time points DFX was added, and at 14 hpi cells were processed as described for panel A. (A to D) Data are representative of 2 independent experiments with 3 technical replicates each. (A, B, and D) The number of bacteria per vacuole for 100 vacuoles per technical replicate was scored. Error bars indicate ⫾ standard deviations. An asterisk indicates a P value of ⬍0.05 by Student’s t test relative to the wild-type strain. **, P ⬍ 0.01; ***, P ⬍ 0.0001.

vacuoles was not due to an inability to generate a replication vacuole. The large vacuole formation defect could be rescued by reintroducing lbtP into the ⌬lbtP mutant on a self-replicating plasmid. These results indicate that LbtP is required to support L.

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pneumophila growth at later stages of the infection cycle, when the number of bacteria within the replication vacuole begins to accumulate at high levels. The role of LbtP in iron acquisition suggests that the early




growth arrest observed for the ⌬lbtP mutant is due to an inability to sequester sufficient amounts of iron to support bacterial replication. To determine if the early growth arrest of the ⌬lbtP mutant was due to iron limitation, large vacuole formation by the ⌬lbtP mutant was examined in macrophages cultured in iron-supplemented media. The presence of elevated iron in the macrophage culture media restored the formation of large vacuoles by the ⌬lbtP mutant (Fig. 5B). These data demonstrate that the early growth arrest phenotype of the ⌬lbtP mutant is dependent upon the availability of iron during intracellular replication. The dependence of ⌬lbtP mutant cumulative growth on iron supplementation suggested that iron availability is a determining factor in the extent of bacterial replication within the host cell. To test this, growth of the wild-type strain within macrophages under iron-limited conditions was examined. Macrophages were challenged with the wild-type strain constitutively expressing the fluorescent protein tdtomato. At 1 hpi, cells were rinsed, medium containing 200 ␮M DFX was added, and growth was monitored by measuring fluorescence over the course of a single infection cycle (Fig. 5C, top). The addition of iron chelator 1 hpi completely abolished L. pneumophila intracellular growth, consistent with the severe growth defect of the mavN iroT mutant, which is impaired in iron acquisition (24, 25). To circumvent the essentiality of iron at early time points for intracellular growth, macrophages were challenged with wild-type bacteria, and then, at 8, 10, or 12 hpi, DFX was added to the culture medium, allowing bacteria to establish a replication vacuole before iron deprivation was induced. At each time point, addition of the iron chelator limited bacterial growth, indicated by a decrease in the total number of bacteria that accumulated at later time points compared to the amount of bacteria in the absence of iron chelator (Fig. 5C, top). Moreover, the total number of bacteria that accumulated directly correlated with the timing of DFX addition, whereby the earlier DFX was added, the lower the total number of bacteria. While the rate of bacterial growth was similar under each condition, growth plateaued at different time points and different levels depending on the timing of adding iron chelator. These results were consistent with results in A. castellanii (Fig. 4C). Similarly, when various concentrations of DFX were added at a single time point, a dosedependent response in the total number of bacteria that accumulated was observed (Fig. 5C, bottom). Consistent with this observation, the number of large vacuoles containing ⬎20 bacteria was significantly reduced in the presence of DFX (Fig. 5D). Collectively, these results demonstrate that iron deprivation subsequent to establishing a replication vacuole induces the growth arrest of L. pneumophila. Premature growth arrest of the ⌬lbtP mutant coincides with early exit from the host. The dependence of the ⌬lbtP mutant phenotype on the abundance of iron indicated that bacteria within the replication vacuole are starved for this essential nutrient. Because the ⌬lbtP mutant survives the initial round of infection and exhibits an increase in bacterial numbers over consecutive rounds of infection (Fig. 1 and 4), we examined whether the ⌬lbtP mutant, upon arresting growth, remains within the host cell for extended periods of time. Macrophages were challenged with wildtype bacteria, a dotA translocation-deficient strain, and the ⌬lbtP mutant. After an initial infection period of 1 h, the macrophage culture medium was replaced with medium containing gentamicin. L. pneumophila is sensitive to gentamicin, so bacteria within the host cell are protected because gentamicin is not membrane


FIG 6 Growth arrest of the ⌬lbtP mutant during intracellular growth coincides with early exit of bacteria from the host cell. (A) The ⌬lbtP mutant exhibits early egress from macrophages relative to the wild-type strain. A/J macrophages were challenged with the wild-type strain, an avirulent dotA mutant, or the ⌬lbtP mutant for 1 h. Macrophage culture medium was then replaced with medium containing gentamicin. Bacterial numbers, based on recovered CFU on solid media from lysed host cells after removal of the gentamicin by repeated washing of the cell layer, was monitored over 27 h, encompassing a single round of infection. (B) The reduction in ⌬lbtP mutant bacteria at later time points is not due to lack of bacterial growth. A/J macrophages were challenged as described for panel A but in the absence of gentamicin. (A and B) Plotted is the total bacterial yield at the indicated time points normalized to the wild-type strain by percent uptake 1 h postinfection. (C) Extracellular ⌬lbtP mutant bacteria and wild-type bacteria under iron depletion conditions accumulate in the culture media prior to wild-type bacteria in the presence of iron. Macrophages were challenged with wild-type or ⌬lbtP mutant bacteria. For iron depletion conditions, DFX was added to a final concentration of 200 ␮M at 10 hpi. At 21 or 24 hpi, bacteria in the culture media were harvested (extracellular bacteria), and bacteria within host cells were released by detergent lysis (intracellular bacteria) and enumerated as described for panel A. (D) ⌬lbtP mutant bacteria that exit host cells are virulent. Extracellular wild-type and ⌬lbtP mutant bacteria harvested at 21 hpi, as described for panel C, were used to challenge fresh macrophages, and growth was monitored based on recovered CFU as described for panel A. (A to D) Data are representative of 2 to 3 independent experiments with 3 technical replicates each. Error bars indicate ⫾ standard deviations. An asterisk indicates a P value of ⬍0.01 by Student’s t test relative to the wild-type strain.

permeable, but bacteria that exit the host cell into the surrounding culture media are killed. At regular intervals over a single round of infection, cell monolayers were thoroughly rinsed to remove the gentamicin, host cells were lysed, and the number of intracellular bacteria was determined by plating the corresponding lysates on bacteriological media (Fig. 6A). The wild-type strain exhibited a 10-fold increase in the number of bacteria in the first 21 h of infection. By 25 hpi, the number of wild-type bacteria recovered


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was dramatically reduced, indicative of bacterial egress from the host cell. A dotA avirulent strain was unable to form a replication vacuole and therefore did not exhibit cumulative replication over the course of the infection (34). Consistent with these observations, no growth was observed for the dotA mutant at any time point examined. Moreover, at 27 hpi, when the number of wildtype bacteria fell below that present at the start of the infection, the total number of dotA mutant bacteria was maintained, consistent with this mutant continuing to occupy an intracellular niche. Similar to the wild-type strain, the ⌬lbtP mutant exhibits robust growth through the first 15 h of infection. In contrast, the number of bacteria recovered 18 hpi was reduced compared to that for the wild-type strain, and by 21 hpi, when the majority of the wild-type bacteria still occupy an intracellular niche, the number of ⌬lbtP mutant bacteria recovered is dramatically reduced, indicative of exposure to gentamicin in the extracellular media. The reduction in bacteria at these later time points is specific to the gentamicin treatment, as a parallel set of infections demonstrated that in the absence of gentamicin in the macrophage culture media, the total number of ⌬lbtP mutant bacteria continues to rise between 18 and 24 hpi (Fig. 6B). These results suggest that the ⌬lbtP mutant exits the host cell prior to wild-type bacteria. To verify the early egress phenotype of the ⌬lbtP mutant, macrophages were challenged with wild-type bacteria or the ⌬lbtP mutant and the percentage of extracellular bacteria was determined at 21 and 24 hpi, corresponding to the timing of egress of the ⌬lbtP mutant and wild-type bacteria, respectively (Fig. 6A). The culture media of infected macrophages was harvested and plated on bacteriological media to monitor extracellular bacteria, and then host cells were lysed with detergent and cell lysates were plated on bacteriological media to enumerate intracellular bacteria. Consistent with the timing of egress of wild-type bacteria in the gentamicin protection assay (Fig. 6A), at 21 h, few wild-type bacteria had accumulated in the culture medium, whereas by 24 h, the percentage of extracellular wild-type bacteria had increased. In contrast, the number of ⌬lbtP mutant bacteria in the culture medium at 21 h was greater than that observed for the wild-type strain (Fig. 6C), consistent with the early egress phenotype of the ⌬lbtP mutant (Fig. 6A). Thus, the ⌬lbtP mutant prematurely exits the host cell relative to the wild-type strain, and this early egress temporally correlates with the early growth arrest phenotype of bacteria lacking LbtP (Fig. 5). The early egress phenotype of the ⌬lbtP mutant could be induced in wild-type bacteria through iron starvation, as the addition of DFX resulted in a greater number of extracellular bacteria at 21 h postinfection (Fig. 6C). Collectively, these results demonstrate that in response to iron deprivation, L. pneumophila arrests growth and exits the host cell. L. pneumophila virulence correlates with the transition from exponential phase to stationary phase and the activation of several virulence traits (53). While arrested growth and premature egress may allow L. pneumophila to avoid imminent death within the host cell resulting from starvation of an essential nutrient, exiting the host cell prior to activating virulence traits that ensure successful infection of the next host cell encountered could be detrimental to the bacterium. To assess whether ⌬lbtP mutant bacteria that prematurely exit macrophages are virulent, extracellular bacteria harvested 21 hpi (Fig. 6C) were used to challenge fresh macrophages, and growth of the ⌬lbtP mutant was compared to that of wild-type bacteria (Fig. 6D). While the ⌬lbtP mutant exhibited a growth defect relative to the wild-type strain, the phenotype was

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FIG 7 In response to iron limitation, L. pneumophila prematurely arrests growth and exits the host cell. Iron deprivation caused by the absence of the siderophore transport protein LbtP leads to the arrest of bacterial replication and triggers a preprogrammed egress strategy, allowing L. pneumophila to exit the host cell. Lp, Legionella pneumophila; LCV, Legionella-containing vacuole.

no more severe than that observed for ⌬lbtP mutant bacteria grown in bacteriological medium (Fig. 1A, 4A, and 6A). The intracellular replication of ⌬lbtP mutant bacteria upon reinfection demonstrated that the bacteria that exit the host cell early are virulent. DISCUSSION

Egress plays a critical role in pathogen dissemination and the spread of infection. Despite this, our knowledge of the mechanisms governing pathogen escape from host cells and the signals that trigger egress is limited. Here, we describe LbtP, a novel siderophore import protein of L. pneumophila that is important for intracellular growth in macrophages, and its absence causes L. pneumophila to prematurely arrest replication and abandon the host cell. These results indicate that L. pneumophila activates an exit strategy in response to iron limitation (Fig. 7). Egress has long been attributed to the effects of high bacterial loads within the host cell. One mechanism proposed to govern L. pneumophila egress is quorum sensing (54); however, iron deprivation resulting from the loss of LbtP causes L. pneumophila to exit the host cell prior to the accumulation of large numbers of bacteria. Abandoning the host cell before reaching a maximum bacterial density suggests that L. pneumophila is able to activate this pathway irrespective of a threshold bacterial quorum. These results demonstrate that egress occurs in response to the exhaustion of iron resources. The signals that trigger egress may not be limited to iron deprivation. Consistent with this idea, amino acid starvation in macrophages induces a stringent response that activates several virulence traits associated with L. pneumophila transmission (53). Similarly, during growth in A. castellanii, arginine in the L. pneumophila vacuole becomes limited, leading to the induction of several virulence genes (55). The induction of virulence traits by both iron and amino acid limitation indicates that L. pneumophila employs an integrated regulatory network that allows it to respond to multiple signals. A similar strategy has been observed for the intracellular parasite Toxoplasma gondii, for which egress occurs in response to several environmental cues, including vacuole acidification (56), potassium efflux from the host cell (57), and




increased cellular calcium (58). A preprogrammed exit strategy that functions independently of bacterial load would allow L. pneumophila to activate egress in response to the exhaustion of host nutrients as a result of overburdening the host cell, nutritional immunity imposed by host defenses, variations in host metabolism that alter nutrient availability, or any host condition unable to support bacterial replication. The ability to abandon the host cell in response to a series of signals that alert the bacteria to the impending inhabitability of its intracellular environment would ensure the survival and propagation of L. pneumophila. The early egress of the ⌬lbtP mutant demonstrates a link between iron starvation and the mechanism by which L. pneumophila escapes the host cell. The transition from a replicative phase to a transmissive phase prior to egress coincides with the activation of several virulence traits, including motility and the production of flagellin (17). Cytosolic flagellin induces a proinflammatory response that leads to host cell death through the activation of Naip/Nlrc1 and caspase-1-mediated pyroptosis (59–62). L. pneumophila may exploit this pathway for egress, whereby activation of the flagellar regulon at late stages of infection triggers host cell lysis and the release of bacteria. Iron starvation of L. pneumophila grown in vitro induces transmission gene expression, including the flagellar regulon (24), providing a possible link between iron limitation and egress. In parallel, the FlxA domain of LbtP may function posttranscriptionally to link LbtP and flagellar activity. Flagellin-mediated cytotoxicity may not be the sole mechanism by which L. pneumophila escapes the host cell. While L. pneumophila is cytotoxic to A. castellanii, lysis of amoebae requires far greater numbers of bacteria than macrophages (54). Moreover, many of the cytosolic surveillance pathways responsible for pyroptosis in macrophages do not appear to be conserved in A. castellanii, suggesting an alternative pathway for cell lysis in this host. In macrophages, pyroptosis also can be induced by caspase-11 through the activity of NRLP3 and ASC (63). This pathway occurs independently of flagellin and caspase-1 (64) but requires a functional Icm/Dot secretion system (63). This may serve as an alternative pathway by which L. pneumophila induces egress and may depend on the activity of specific Icm/Dot translocated substrates (65). In support of this idea, loss of Icm/Dot translocated substrates has been shown to delay the release of L. pneumophila from A. castellanii (66). Thus, L. pneumophila may employ a complex regulatory network that integrates multiple environmental cues and exit strategies. The effects of the loss of LbtP only manifest at later stages of the infection cycle. The ability of the ⌬lbtP mutant to generate a replication vacuole and complete 3 to 4 rounds of replication (Fig. 5) suggests that iron acquisition through LbtP-independent mechanisms is sufficient to support bacterial growth in the early stages of the infection cycle. The importance of LbtP at later stages of the infection cycle may occur because host iron resources change over the course of an infection, perhaps coincident with the depletion of residual bacterial iron stores acquired during growth on ironsupplemented medium prior to infection. Alternatively, the effect of loss of LbtP function may accumulate over the course of the infection such that as the number of bacteria increases, iron resources are more rapidly depleted, causing iron levels to fall below that required for continued bacterial replication. Redundancy is a central theme in L. pneumophila pathogenesis (16, 67). While L. pneumophila encodes two paralogs of the legio-


bactin siderophore importer, there are clear distinctions between them. First, LbtP plays a dominant role over LbtU in iron acquisition in the Philadelphia-1 strain. Bacteria lacking LbtP are more defective for iron uptake, and growth of the ⌬lbtP mutant is more sensitive to iron depletion in vitro than a strain lacking LbtU (Fig. 3). The difference in iron sensitivity of the ⌬lbtP and ⌬lbtU mutants may be due to differential expression levels of lbtU and lbtP, as overexpression of lbtU is able to completely restore growth of the ⌬lbtU ⌬lbtP double mutant to wild-type levels (Fig. 3B). Second, the domain architecture of the two proteins is dissimilar. LbtP contains an N-terminal extension homologous to FlxA that is absent from LbtU. This extension may function as a regulatory plug that is characteristic of this family of transporters (68). Third, expression of lbtP and lbtU are not subject to the same regulatory circuits. lbtU is part of an operon that is regulated by the ironresponsive transcription factor Fur (24, 29, 30), whereas the lbtP promoter lacks a consensus fur box (24) and its regulation is not responsive to changes in iron levels, either in L. pneumophila 130b (24) or L. pneumophila Philadelphia-1 (T. O’Connor and R. R. Isberg, unpublished data). Finally, while LbtP is important for growth in macrophages, LbtU is dispensable under these conditions (Fig. 4), demonstrating differential requirements for each protein for intracellular growth of the Philadelphia-1 strain. This is not due to differential regulation of the two genes, as lbtU is upregulated over the course of the infection while lbtP transcript levels remain constant (69). Thus, while LbtU and LbtP share similar functions, their contributions to iron assimilation and L. pneumophila growth are distinct and dependent on the environment. Despite the distinct contributions of LbtU and LbtP, their activities appear to be interconnected. While LbtU is dispensable for growth on solid bacteriological media at moderate levels of iron depletion (Fig. 3B), its absence severely exacerbates the growth defect of the ⌬lbtP mutant. Thus, LbtU may partially compensate for loss of LbtP under conditions of moderate iron limitation. One mechanism by which this may be achieved is the upregulation of lbtU. Since expression of lbtU, but not lbtP, is iron dependent, reduced iron in the absence of LbtP would trigger production of LbtU. Increased levels of LbtU may allow L. pneumophila to continue to sequester iron in the absence of LbtP. Alternatively, in addition to legiobactin, LbtP may also import an as-yet unidentified siderophore. Consistent with this idea, the L. pneumophila gene frgA encodes a siderophore synthetase, functionally similar to LbtA but dispensable for legiobactin production (23, 70). Activation of the lbtABCU regulon in response to iron limitation and subsequent overproduction of legiobactin may outcompete a second siderophore for iron binding, rendering LbtU more important for iron acquisition under conditions where iron is severely limited. Thus, while the contribution of LbtU and LbtP to L. pneumophila growth varies depending on the environment, overlap between their individual functions may ensure adequate iron assimilation under a range of iron concentrations. ACKNOWLEDGMENTS This work was supported by the Howard Hughes Medical Institute and a Natalie V. Zucker Fellowship to T.J.O. and NIH grant AI034937, awarded to N.P.C. R.R.I. is an investigator of the Howard Hughes Medical Institute.


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FUNDING INFORMATION This work, including the efforts of Tamara O’Connor, was funded by Natalie V. Zucker Fellowship. This work, including the efforts of Nicholas P. Cianciotto, was funded by National Institutes of Health (AI034937). This work, including the efforts of Tamara O’Connor and Ralph R. Isberg, was funded by Howard Hughes Medical Institute (HHMI).

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Sulfonamide inhibition studies of two β-carbonic anhydrases from the bacterial pathogen Legionella pneumophila.

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