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

Abstract

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 ΔlbtP 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.

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.

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.

FIG 3

LbtP is a siderophore transport protein important for iron acquisition. (A) The ΔlbtP mutant strain shows enhanced sensitivity to iron limitation in bacteriological media. Growth of the wild type (WT) and ΔlbtU and ΔlbtP 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 ΔlbtU, ΔlbtP, and ΔlbtU ΔlbtP 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.

FIG 4

lbtP is differentially important for growth in macrophages and amoebae, whereas lbtU is dispensable in both hosts. (A) The ΔlbtP mutant shows a growth defect in bone marrow-derived A/J mouse macrophages relative to the wild type and ΔlbtU 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 wild-type strain by percent uptake 2 h postinfection. (C) Lack of iron supplement in A. castellanii culture medium does not impair growth of the ΔlbtP, ΔlbtU, or ΔlbtP ΔlbtU 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.

FIG 5

ΔlbtP mutant undergoes premature growth arrest in macrophages in an iron-dependent manner. (A) Macrophages challenged with the ΔlbtP mutant exhibit fewer vacuoles containing large numbers of bacteria than the wild-type strain. Macrophages were infected with the wild type or the ΔlbtP mutant strain harboring the empty vector pJB908 or the ΔlbtP 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 ΔlbtP mutant. Macrophages were challenged with the wild type or ΔlbtP 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 μM 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.

FIG 6

Growth arrest of the ΔlbtP mutant during intracellular growth coincides with early exit of bacteria from the host cell. (A) The ΔlbtP 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 ΔlbtP 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 ΔlbtP 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 ΔlbtP 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 ΔlbtP mutant bacteria. For iron depletion conditions, DFX was added to a final concentration of 200 μM 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) ΔlbtP mutant bacteria that exit host cells are virulent. Extracellular wild-type and ΔlbtP 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.

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.