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Nat Commun
Nat Commun
Nature Communications
2041-1723
Nature Publishing Group UK London

39245746
52184
10.1038/s41467-024-52184-x
Article
Iron-depleting nutritional immunity controls extracellular bacterial replication in Legionella pneumophila infections
Torres-Escobar Ascención 1
Wilkins Ashley 14
Juárez-Rodríguez María D. 2
Circu Magdalena 1
Latimer Brian 3
http://orcid.org/0000-0001-9841-6802
Dragoi Ana-Maria 23
http://orcid.org/0000-0002-3294-7595
Ivanov Stanimir S. stanimir.ivanov@lsuhs.edu

1
1 https://ror.org/03151rh82 grid.411417.6 0000 0004 0443 6864 Department of Microbiology and Immunology, Louisiana State University Health Sciences Center—Shreveport, Shreveport, LA 71130 USA
2 https://ror.org/03151rh82 grid.411417.6 0000 0004 0443 6864 Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center—Shreveport, Shreveport, LA 71130 USA
3 grid.411417.6 0000 0004 0443 6864 Innovative North Louisiana Experimental Therapeutics program (INLET), Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center—Shreveport, Shreveport, LA 71130 USA
4 grid.419681.3 0000 0001 2164 9667 Present Address: Bacterial Physiology and Metabolism Unit, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840 USA
8 9 2024
8 9 2024
2024
15 78482 2 2024
29 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
The accidental human pathogen Legionella pneumophila (Lp) is the etiological agent for a severe atypical pneumonia known as Legionnaires’ disease. In human infections and animal models of disease alveolar macrophages are the primary cellular niche that supports bacterial replication within a unique intracellular membrane-bound organelle. The Dot/Icm apparatus—a type IV secretion system that translocates ~300 bacterial proteins within the cytosol of the infected cell—is a central virulence factor required for intracellular growth. Mutant strains lacking functional Dot/Icm apparatus are transported to and degraded within the lysosomes of infected macrophages. The early foundational work from Dr. Horwitz’s group unequivocally established that Legionella does not replicate extracellularly during infection—a phenomenon well supported by experimental evidence for four decades. Our data challenges this paradigm by demonstrating that macrophages and monocytes provide the necessary nutrients and support robust Legionella extracellular replication. We show that the previously reported lack of Lp extracellular replication is not a bacteria intrinsic feature but rather a result of robust restriction by serum-derived nutritional immunity factors. Specifically, the host iron-sequestering protein Transferrin is identified here as a critical suppressor of Lp extracellular replication in an iron-dependent manner. In iron-overload conditions or in the absence of Transferrin, Lp bypasses growth restriction by IFNγ-primed macrophages though extracellular replication. It is well established that certain risk factors associated with development of Legionnaires’ disease, such as smoking, produce a chronic pulmonary environment of iron-overload. Our work indicates that iron-overload could be an important determinant of severe infection by allowing Lp to overcome nutritional immunity and replicate extracellularly, which in turn would circumvent intracellular cell intrinsic host defenses. Thus, we provide evidence for nutritional immunity as a key underappreciated host defense mechanism in Legionella pathogenesis.

In this work, authors indicate a cooperation between nutritional immunity and interferon limits Legionella replication during infection and compromised nutritional immunity may increase severity by allowing the bacteria to replicate outside of host cells.

Subject terms

Bacterial pathogenesis
Bacterial immune evasion
Monocytes and macrophages
Antimicrobial responses
https://doi.org/10.13039/100000060 U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID) AI143839 Ivanov Stanimir S. https://doi.org/10.13039/100000057 U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS) P20GM134974-5749 Ivanov Stanimir S. Ike Muslow Predoctoral Fellowship from LSU Health Shreveport to AAWissue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Legionella species are environmental Gram-negative γ-proteobacteria that parasitize and replicate within free-living unicellular eukaryotes1,2. Legionella pneumophila (Lp) is the leading cause of a lower respiratory infection known as legionellosis that produces an atypical pneumonia with a significant disease burden3. Inhalation and deposition of aerosolized bacteria in the alveoli triggers the early flu-like symptoms of legionellosis—including dry cough, chills and fever—but disease progression varies from a mild self-limiting syndrome (aka Pontiac fever) to a debilitating severe pneumonia (aka Legionnaires’ disease), which can be lethal without appropriate antibiotics treatment4,5. Outbreaks from a single clinical isolate frequently manifest in both the mild and the severe disease forms6–13, implicating host-determinants as important drivers of disease progression. History of smoking, old age and ongoing immunosuppression therapy represent key risk factors for progression of legionellosis towards Legionnaires’ disease4,5, however the mechanistic links remain unresolved.

In human infections and animal models of disease, alveolar macrophages are the primary cellular niche for bacterial replication14–18. When internalized by phagocytes Lp rapidly remodels the early phagosome into a unique endoplasmic reticulum (ER)-like organelle, which within 4 h post uptake matures to a vacuolar niche that supports robust bacterial replication19–22. The critical virulence factor for intracellular replication conserved in all Legionellaceae is a type IVb secretion system (T4SS), referred to as the Dot/Icm apparatus, which translocates ~300 bacterial proteins into the cytosol of the infected cell23,24. Mutant strains lacking functional T4SS are transported to and degraded within lysosomes of infected macrophages25. Although axenic growth conditions have been developed, Legionella does not replicate extracellularly during infections in tissue cultures and in vivo murine models26,27; thus, mutants that cannot grow intracellularly (such as T4SS− strains) are avirulent in both animal and tissue culture models of infection28–31. Convincing experimental data from numerous labs over the years demonstrate that T4SS– bacteria are gradually eliminated when cultured with either human or murine macrophages or unicellular amoebae, whereas the wild-type strain grows several orders of magnitude—consistent with the idea of obligate intracellular lifestyle during infection27,29,31–34. Moreover, Legionella is fastidious due to auxotrophy for several proteogenic amino acids, including cysteine, which are acquired intracellularly from the host cell and partially explain the inability of the bacteria to replicate extracellularly in cellular infection models35–39. Since the foundational work by Dr. Horowitz 40 years ago, the paradigm in the field is that Legionella under infection conditions replicates strictly intracellularly40. Indeed, Legionella has biphasic developmental program typical of obligate vacuolar bacterial pathogens41–46 where a microorganism cycles between two phenotypically distinct forms each with a unique gene expression profile47–51. A motile highly infectious transmissive form, which upregulates the T4SS, is the predominant extracellular form detected during infection and in late exponential/early stationary phase in axenic growth cultures51. The transition into a non-motile poorly infectious replicative form, which is highly similar to the bacterial population during exponential growth in axenic cultures, occurs intracellularly within the vacuolar niche51. Thus, in terms of its lifecycle during infection Legionella resembles obligate vacuolar bacterial pathogens.

An inflammatory response coordinated by multiple innate immune cell types results in IFNγ production, which resolves legionellosis by reprograming macrophages into a phenotypic state that restrict intracellular bacterial replication6,40,52–56. IFNγ-stimulated primary human alveolar and monocyte-derived macrophages limit Lp intracellular replication in vitro52,53 through a variety of cell autonomous defense mechanisms in part impinging on LCV rupture and host cell death57–62. IFNγ secretion is required for legionellosis resolution in animal models, emphasizing the importance of IFNγ effector functions in Lp pathogenesis55,56. Yet, leukocytes from recovered Legionnaires’ disease patients secreted IFNγ when stimulated with Lp similarly to leukocytes isolated from the control cohort indicating that differences in IFNγ responses alone cannot account for disease progression63. Thus, it is poorly understood what mechanistic determinants allow progression towards severe legionellosis—an infection that is normally well-controlled by innate immunity via IFNγ production.

Host defense mechanisms that sequester various trace metals and other micronutrients from invading pathogens, thus limiting pathogen replication and disease severity, are collectively known as nutritional immunity64–66. Iron is essential for life and therefore is under strict nutritional immunity protection where high affinity iron-binding carriers, such as Transferrin and Lactoferrin, limit bioavailable iron in the extracellular milieu67. Imbalance in systemic iron homeostasis—a complex tightly regulated process—increases susceptibility to bacterial infections especially under systemic iron-overload brought by saturation of host iron-carrier as a result of hereditary genetic disorders or high dietary iron consumption68–72. Macrophages control bioavailable iron locally and systemically by regulated uptake of iron-carrier proteins via cell-surface receptors and secretion through Ferroportin (FPN1)—the only known iron exporter protein73,74. During inflammation FPN1 is downregulated, which favors intracellular iron accumulation and a decrease in extracellular iron bioavailability69,75. Nutritional immunity in legionellosis is poorly understood; nevertheless, under iron overload conditions IFNγ-primed macrophages fail to restrict Lp growth ex vivo suggesting that compromised nutritional immunity impacts cell autonomous responses to Lp76,77. The molecular mechanism driving this phenotype remains unresolved.

In this work, we demonstrate that human macrophages can support Lp extracellular replication, which can be suppressed by Transferrin through sequestration of micronutrients. Accordingly, iron-overload is sufficient to incapacitate Transferrin-dependent nutritional immunity and restore Lp replication even when IFNγ-mediated cell immunity is functional. Thus, we provide insight in the cooperation between nutritional and cell mediated immunity for the restriction of Lp growth in macrophage infections.

Results

Legionella mutant strains lacking a functional Dot/Icm secretion apparatus replicate in the presence of human macrophages under serum-free conditions

We measured the intracellular growth kinetics of different Lp strains in human macrophage infections to investigate the connection between serum derived nutrients and bacterial replication. To avoid a pyroptotic cell death response triggered by cytosolic delivery of flagellin78, which can convolute data interpretation in infection-based growth assays, we used a flagellin clean deletion mutant. We engineered two distinct L. pneumophila Philadelphia-1 derived strains—Lp01 and JR32—to bioluminescence by inserting the LuxR operon (luxC luxD luxA luxB luxE) from Photorhabdus luminescens on the bacterial chromosome under the constitutive IcmR promoter using two different genetic approaches79,80 (Supplementary Fig. 1a). For both strains the bioluminescence output increased exponentially during logarithmic growth in axenic cultures and peaked as the bacteria entered stationary phase (Supplementary Fig. 1b, c)79,80. The Lp growth kinetics with human U937 macrophages peaked at 48hrs for both JR32 and Lp01 and the curves were comparable regardless of serum availability (Fig. 1A, B). As negative controls in these assays, we used dotA clean deletion strains on the respective backgrounds, which lack functional Dot/Icm apparatus and have been extensively used in the Legionella field as negative growth controls because dotA mutants cannot replicate intracellularly25,32. In the presence of serum, the bioluminescence signal in macrophage infections with the dotA mutants decreased sharply and remained below the level produced by the inoculum, consistent with the demonstrated inability of dot/icm deletion strains to replicate intracellularly (Fig. 1A, B). Unexpectedly, in infections under serum-free (SF) conditions dotA strains appear to replicate similarly in magnitude to the parental Dot/Icm+ strains, albeit with delayed kinetics (Fig. 1A, B). Area under the curve analysis from at least 9 biological replicates demonstrates the growth restriction-capacity of serum specifically towards the dotA mutant (Fig. 1C). The unexpected growth of the dotA mutant in SF infections was confirmed by an alternative Colony Forming Units (CFUs) growth assay, in which bacteria were collected at distinct times post infection and subsequently were serially diluted, plated and enumerated (Fig. 1D). When serum was present, Lp01 ∆dotA CFUs decreased 100-fold over the course of the infection, whereas in the absence of serum 100-fold increase in CFUs occurred for the same time period (Fig. 1D). Unlike Dot/Icm- strains, Dot/Icm+ strains fail to grow on CYE plates supplemented with 150 mM NaCl due to ion imbalance81. Therefore, to confirm that dotA bacteria did not regain Dot/Icm functionality in SF infections, eight recovered colonies from every infection condition at each time point were randomly selected and replica plated under high- and low-salt conditions. The results clearly demonstrate that CFUs recovered from Lp01 ∆dotA infections remained salt resistant regardless of serum supplementation, confirming the Dot/Icm- genotype (Fig. 1D).Fig. 1 Effects of serum supplementation on Lp growth in human macrophage infections.

A–D Growth kinetics of DotA+ and DotA– Lp in U937 MFs cell culture infections (MOI = 5). Bacterial growth was measured at discrete time-points via bioluminescence output (A–C) or recovered CFUs (D) for mutant strains on the JR32 (A, C) and the Lp01 (B–D) backgrounds as indicated. C Area under the curve (AUC) analysis of Lp growth kinetics in the presence vs. absence of hiFBS for each infection condition. Each data point represents the AUC calculated from the growth curve in a single biological replicate as shown in A and B. Statistical significance was determined by two-tailed non-parametric Wilcoxon matched-pairs T-test and the respective p-values are indicated; n number of biological replicates. D In the CFU growth assays for every timepoint eight of the recovered colonies were selected at random and replica plated on CYA agar with or without 150 mM NaCl. E Axenic growth curves of the indicated strains in AYE liquid cultures in the presence/absence of 10% (v/v) hiFBS were generated from OD600 measurements every 10 min over the indicated time period. Each time point represents an average of a technical triplicate. F Growth kinetics of JR32 ∆flaA dotA in U937 MFs cell culture infections (MOI = 5) supplemented or not with different amounts of heat-inactivated human (hS1) or heat-inactivated bovine serum (bS4). A, B, D–F Averages from three technical replicates +/−StDev are shown. At least three biological replicates were completed for each experiment. Source data are provided as a Source Data file.

Because the growth restriction phenotype was specific for the dotA mutants, we investigated whether dotA deletion sensitized Lp to a yet unidentified bacteriocidal component of the heat-inactivated FBS (hiFBS) used in the previous infection assays, which would explain the selective growth-restriction phenotype. However, the dotA mutant growth rate in axenic cultures was not reduced upon addition of hiFBS (Fig. 1E), which is inconsistent with the presence of an anti-microbial activity in the serum. Sera from human and bovine origin blocked dotA replication in cellular infections in a dose-dependent manner (Fig. 1F). A panel of 12 commercially sourced sera used in macrophage infections (Supplementary Fig. 2a) and axenic growth assays (Supplementary Fig. 2b) revealed that while sera blocked dotA replication with various potency in cellular infections, Lp growth in axenic cultures was not affected. Thus, we conclude that serum-mediated growth restriction by a yet unidentified mechanism is required for the previously reported inability of the dotA mutant to growth in macrophage infections25,32.

The dotA growth in macrophage infections was puzzling because this strain, similar to other dot/icm deletion mutants, is readily cleared via lysosomal degradation after internalization by the host cell. Thus, we investigated whether the growth phenotype is common to other mutant strains lacking a functional Dot/Icm system. To this end, we engineered single gene knockout strains lacking either dotB or dotH—two genes essential for a functional Dot/Icm apparatus. DotH participates in the formation of the outer membrane cap and the periplasmic ring of the Dot/Icm T4SS core complex82,83, whereas the DotB is the energy-generating ATPase that facilitates translocation of effector proteins84,85. The growth kinetics of dotB and dotH mutant strains in macrophage infections phenocopied the data from the dotA deletion strains (Fig. 2A) and all dot/icm deficient strains grew under high salt concentrations (Fig. 2B). Deletion of dotB in Legionella jordanis (Supplementary Fig. 1a)—another Legionella species that encodes a Dot/Icm system and replicates intracellularly86—produced a strain capable of replication in macrophage infections under SF conditions (Supplementary Fig. 3a). Similar to the Lp dotA deletion strains, the Lj dotB mutant grew in axenic cultures regardless of serum supplementation (Supplementary Fig. 3b). Thus, we conclude that serum from bovine and human origins restrict the replication of dot/icm mutants from L. pneumophila and L. jordanis specifically in macrophage infections but not in axenic growth cultures.Fig. 2 Serum withdrawal restores the growth of several Lp01 mutants with distinct intracellular growth defects.

A Growth kinetics of different Lp01 Dot/Icm mutant strains with U937 macrophages under SF conditions (MOI = 5). B Lp01 Dot/Icm mutants grow in the presence of 150 mM NaCl. C Lp mutant strains (T4SS-, mavN and sdhA) exhibit intracellular growth-restriction phenotype due to distinct mechanisms—lysosomal degradation, iron starvation and vacuolar rupture. (D-E) Growth kinetics of the ∆sdhA (D) and mavN::KanR (E) mutants with U937 macrophages in the presence or absence of serum (MOI = 5). Averages from three technical replicates +/−StDev are shown. Statistical significance was determined by one-way ANOVA and the respective p-values are indicated. A–E At least three biological replicates were completed for each experiment. Source data are provided as a Source Data file.

Serum restricts L. pneumophila extracellular replication in macrophage and monocyte infections

In light of the well-documented inability of (i) dot/icm mutants to avoid lysosomal degradation25,32 and (ii) Lp to replicate extracellularly during infection40, we sought to determine which of the two postulates is affected by serum availability during infection. We took advantage of two other Lp knockout strains—sdhA and mavN—which fail to replicate specifically in macrophage infections because of distinct intracellular growth defects (Fig. 2C) but otherwise grow normally in axenic cultures87,88. SdhA is a Dot/Icm effector protein involved in the early stages of LCV remodeling89 and sdhA deletion results in rapid destabilization of the Lp-occupied phagosome, which triggers a pyroptotic host-cell death response trapping the bacteria within the macrophage corpse thus restricting bacterial replication87. Conversely, MavN is multi-transmembrane domain iron transporter translocated by the Dot/Icm apparatus that delivers ferrous iron across the LCV membrane88,90,91. The LCVs occupied by mavN deletion mutants are remodeled and traffic normally, however bacterial replication is arrested due to iron starvation88. Indeed, mutants lacking sdhA (Fig. 2D) or mavN (Fig. 2E) did not replicate in macrophage infections when serum was present; however, both strains grew with kinetics similar to the dot/icm deletion strains under SF conditions (Fig. 2A). Taken together, these data from infections with Lp mutants incapable of intracellular growth points to extracellular replication as a putative mechanism facilitating bacterial growth under SF conditions. This notion challenges the prevailing paradigm that Lp cannot grow outside of the host cell during infection40.

Next, we set out to test directly whether Lp can replicate extracellularly during infection. To this end, we focused on monocyte infections because Lp entry in monocytes requires opsonization and FcR-mediated phagocytosis, which allows us to dictate bacteria location during infection. Using microscopy, we confirmed that U937 macrophages phagocytosed both opsonized and non-opsonized Lp efficiently, whereas U937 monocytes strictly internalized opsonized bacteria (Fig. 3A). Importantly, serum supplementation did not alter the Lp internalization determinants for either cell line (Fig. 3A). In monocyte infections, opsonized Lp initiated bacterial replication quickly and achieved maximal growth at ~36 hpi (Fig. 3B) with kinetics similar to intracellular growth in macrophages (Fig. 2A). Non-opsonized Lp also replicated in monocytes infections but with an obvious delay where growth was initiated at ~ 48hpi and peaked at ~ 96hpi (Fig. 3B). The growth kinetics of non-opsonized Lp in monocyte infections (Fig. 3B) and dot/icm mutants in both macrophage and monocyte infections (Figs. 2A and 3B) were comparable, which would be predicted if growth determinants in all circumstances were similar. Importantly, non-opsonized Lp replicated in monocyte infections with kinetics similar to the sdhA and mavN mutants in serum-dependent manner (Fig. 3C). Together these data are consistent with extracellular replication taking place in the absence of serum supplementation during infection. To test this idea with an alternative approach, we performed antibiotic protection assay using different spectinomycin concentrations to distinguish intracellular from extracellular replication. Spectinomycin is bacteriostatic and should impact extracellular replication at lower concentrations because host cell membranes limit drug diffusion. Indeed, spectinomycin at 30 µg/ml completely blocked the replication of the dotA mutant in macrophage infections, which we posit to take place extracellularly, whereas the intracellular growth of the parental strain was only slightly reduced (Supplementary Fig. 4a). Notably, both strains were equally sensitive to spectinomycin in axenic cultures (Supplementary Fig. 4b, c) indicating that the distinct infection growth kinetics likely result from the inhibition of extracellular replication. In agreement, low dose spectinomycin (20 µg/ml) completely blocked the growth of non-opsonized Lp in monocytes infections—condition in which the bacteria remain extracellular (Supplementary Fig. 4d).Fig. 3 Monocytes and macrophages support extracellular Lp growth upon serum withdrawal.

A Internalization of opsonized and non-opsonized Lp01 ∆flaA GFP+ by U937 cells at 6hpi (MOI = 5). Representative z-projection micrographs from fixed cells strained by the inside/out method show both intracellular (single positive) and surface-associated (double positive) bacteria on monocytes (left panel) and macrophages (right panel). Graphed data represents averages of internalized bacteria for each condition from three technical replicates +/−StDev (n > 150). Lp were opsonized with a polyclonal rabbit a-Lp and were stained with a chicken a-Lp IgY antibody. B Growth kinetics of the indicated Lp stains in cultures with U937 monocytes under SF conditions (MOI = 5). Some bacteria were opsonized with a polyclonal rabbit a-Lp IgG for 30 min prior to infection. C Growth kinetics of the indicated Lp strains with U937 monocytes (MOI = 5) in the absence (top panel) or presence of hiFBS (bottom panel). D Representative micrographs from time-lapse live-cell microscopy showing growth kinetics of Lp01 ∆flaA GFP+ with U937 monocytes (left panels) and U937 macrophages (right panels) (MOI = 5). Source data is included in Supplementary Movies 1 and 2 (macrophages and monocytes respectively). Arrows denote individual LCVs in each frame. E, F Loss of direct bacterium-host cell contact alters Lp growth kinetics in macrophage but not monocyte cultures under SF conditions (MOI = 5). E JR32 ∆flaA growth kinetics when cultured with U937 monocytes together or when separated by a 0.4 µm membrane are comparable. F Loss-of-contact significantly delays initiation of JR32 ∆flaA replication in macrophage infections. G Lp01 ∆flaA growth kinetics with U937 monocytes (MOI = 5) in the presence of the actin-polymerization inhibitor cytochalasin D (5 µM CytoD) or the secretory transport pathway inhibitor Brefeldin A (1 μM BFA), which were added 60 min prior to infection. Statistical significance was determined by one-way ANOVA and the respective p-values are indicated. B, C, E–G Averages from three technical replicates +/−StDev are shown. Two (A, D) or at least three (B, C, E–G) biological replicates were completed for each experiment. Source data are provided as a Source Data file.

Because the previous data is indicative of Lp extracellular replication in monocyte infections, we sought to visualize this process using live-cell imaging. To this end, monocytes or macrophages were cultured with GFP-expressing Lp in the absence of serum and were imaged every 4 h over a period of several days spanning multiple rounds of infection (Fig. 3D and Supplementary Movies 1 and 2). As expected, Lp replication was spatially confined in the host cell resulting in the biogenesis of prototypical GFP+ spherical organelles that ultimately rupture releasing bacteria in the milieu (Fig. 3D and Supplementary Movie 1). Conversely, individual bacteria with unrestricted spatial lateral mobility were observed in monocyte cultures (Fig. 3D and Supplementary Movie 2), which indicates that Lp is not confined within the eukaryotic cell and is consistent with the uptake defect for non-opsonized Lp (Fig. 3A). Little bacterial replication was evident prior to ~70hpi but the bacteria population increased afterwards dramatically within a short period of time (<8 h) (Fig. 3D and Supplementary Movie 2). Once bacterial replication started, the Lp GFP signal intensity increased uniformly throughout the imaging field in monocyte cultures, unlike the spatially localized signal produced in macrophage infections (Fig. 3D and Supplementary Movies 1 and 2). Thus, the live-cell imaging data clearly demonstrate the distinct properties of bacterial replication with host cells produced under conditions allowing or preventing bacterial uptake and strongly support the notion of Lp extracellular replication in specific infection settings.

Next, we investigated if Lp growth with monocytes required direct contact between the two cell types. Accordingly, infections were performed in a chamber assay, where bacteria are separated from the eukaryotic cells by a 0.4 µm pore-seized membrane which allows only fluid phase exchange to occur between the two compartments. In this setting, Lp grew with similar kinetics regardless of whether direct contact with monocytes was allowed (Fig. 3E) confirming that the bacteria replicates extracellularly under those conditions. In the same setting, Lp growth was delayed when direct contact with macrophages was blocked by a physical membranous barrier (Fig. 3F) and phenocopied the contact-independent growth curve of the of the dotA mutant strain, which would be expected if both strains grew extracellularly. Furthermore, inhibition of actin polymerization with cytochalasin D resulting in a phagocytosis blockade did not interfere with Lp growth with monocytes (Fig. 3G). Based on the collective data we conclude that (i) Lp can replicate extracellularly in a contact-independent manner when cultured with monocytes or macrophages and (ii) serum presence restricts Lp extracellular growth.

Macrophages and monocytes produce nutrients required for Lp extracellular replication

Lp is a fastidious bacterium with an amino acid-centric metabolism and is also an auxotroph for several amino acids which are acquired from the host cell during intracellular replication36,39. Therefore, we sought to determine the nutrients source facilitating Lp extracellular growth. SF-RPMI by itself did not support bacterial growth (Fig. 4A), which would be expected because Lp cannot transport cystine across the outer membrane92. Supplementation of RPMI with both cysteine and iron—another Lp growth factor93—did not trigger bacterial replication either (Fig. 4A), indicating that the supplemented RPMI also lacks the capacity to support Lp growth. Because Lp can grow extracellularly in RPMI when macrophages or monocytes are present, one can posit that host cells likely produce one or more nutrients need by Lp for growth. Indeed, Lp grew in the absence of host cells when the bacteria were cultured in 0.2 µm filtered conditioned medium (SF-RPMI), which was collected from 4 days-old macrophage cultures (Fig. 4A). Conditioned medium from unstimulated and E.coli LPS-stimulated macrophages supported Lp replication indistinguishably (Fig. 4A). Thus, we conclude that host cells are required and provide the necessary growth factors for Lp extracellular replication.Fig. 4 Nutrients secreted by live macrophages are sufficient and required for Lp extracellular replication.

A Lp01∆flaA growth kinetics in the absence of host cells. Bacteria (5 × 105) were cultured in SF RPMI media supplemented with 3.3 mM cysteine and 330 µM Fe(NO3)3 or in 4 days RPMI conditioned media (CM) from resting or LPS-stimulated U937 macrophages (MFs). CMs were passaged through a 0.2 µm membrane filter prior to bacteria addition. Statistical significance was determined by one-way ANOVA and the respective p-values are indicated. B Changes in U937 monocyte viability inferred from LDH released in the culture supernatant under the indicated conditions in serum free RPMI media (MOI = 5). Data is normalized to LDH activity released from monocytes lysed with 1% Triton X-100. C Growth kinetics of the indicated Lp strains in the presence of live (non-sonicated) or physically ruptured (sonicated) U937 monocytes cultured in SF RPMI media (MOI = 5). D Bacterial replication of the indicated strains in nutrient poor amino acids-free PBSG medium (PBS, 7.5 mM glucose, 900 µM CaCl2, 500 µM MgCl2) media in the presence/absence of U937 macrophages (MOI = 5). A–D Averages from three technical replicates +/−StDev are shown. Representative of three biological replicates for each experiment is shown. Source data are provided as a Source Data file.

One possibility is that Lp grows extracellularly via necrotrophy fueled by nutrients released from dying macrophages/monocytes, which would explain the kinetic delay prior to initiation of extracellular replication as host cell viability gradually decreases (Fig. 4B). To investigate this scenario, Lp was cultured with monocytes that were sonicated in SF-RPMI prior to infection, which liberates intracellular host-derived metabolites for Lp consumption immediately (Fig. 4C). However, sonicated monocytes did not support the growth of either T4SS+ or T4SS- strains whereas the equivalent number of live (non-sonicated) monocytes did, demonstrating that at least some of the nutrients utilized by Lp during extracellular replication are produced by live host cells. To determine if macrophages can be the sole nutrients source for growth factors during Lp extracellular replication, akin to nutrients acquisition during intracellular replication, the growth assays were carried out in amino acids-free PBSG medium (PBS + 7.5 mM glucose). Under those conditions, the dotA deletion mutant grew only in the presence of macrophages (Fig. 4D); thus, demonstrating that Lp acquires nutrients and other growth factors needed for extracellular growth from the host cells. Such a model would predict that nutrients concentrations sufficient for the initiation of bacterial replication would be reached faster in a smaller cell culture volume. Indeed, initiation of extracellular replication but not maximal growth positively correlated with the cell culture volume in monocytic infections, where bacterial replication started and peaked earlier when lower cell culture volumes were used during infection (Supplementary Fig. 5a, b). Altogether, we conclude that (i) macrophages/monocytes produce and release in the extracellular environment all nutrients necessary to support Lp replication and (ii) live host cells are required for at least the initiation of extracellular replication. In agreement, inhibition of the secretory pathway in monocytes with Brefeldin A (BFA) interfered with Lp extracellular replication (Fig. 3G). The exact nature of the host-derived nutrients and how they are sourced remains to be elucidated.

Serum restrict Lp extracellular replication by scavenging bioavailable iron

It is apparent from our data that the practice of serum supplementation in cellular infection models restricted Lp extracellular replication, thus supporting the obligate intracellular growth paradigm in the field. To gain insight in the serum-mediated growth-restriction of Lp extracellular replication we focused on nutrients limitation as a plausible mechanism because heat-inactivated serum lacked microbiocidal activity towards Lp (Supplementary Fig. 2b). Iron and cysteine are well-documented nutrient determinants of optimal Lp replication92,93. Macrophages modulate the concentration of extracellular cysteine by direct export through the ASC neutral amino acid antiporters and by secretion of Thioredoxin, which reduces extracellular cystine to cysteine94,95. Cysteine supplementation could not rescue Lp extracellular growth in macrophage infections when serum was present (Fig. 5A) indicating cysteine utilization is not limiting bacterial growth under those conditions. However, ferric iron supplementation fully restored Lp extracellular growth in the presence of either bovine (Fig. 5B–D) or human (Fig. 5E) serum in a dose-dependent manner (Supplementary Fig. 6a) where the growth kinetics in the absence of serum were indistinguishable from the ‘serum + Fe3+’ condition (Fig. 5B–E). Supplementation of either ferrous or ferric iron abolished the serum-dependent restriction of Lp extracellular replication and triggered earlier bacterial replication under SF conditions (Supplementary Fig. 6b), implicating iron availability in the extracellular milieu as a key growth determinant. The addition of other divalent metals (Zn2+, Mn2+ and Ni2+) did not counteract the serum restriction of Lp extracellular growth underlying the need specifically for iron (Fig. 5F). Conversely, iron supplementation minimally impacted Lp growth intracellularly (Supplementary Fig. 6b), indicating bioavailable iron concentration inside macrophages is sufficient for optimal intracellular replication during infection. Importantly, primary human monocyte-derived macrophages (hMDMs) similar to U937 macrophages supported extracellular replication but only in the presence of exogenous iron when serum was present (Fig. 5E). Regardless of serum or iron supplementation Lp extracellular growth was independent of flagellin (Supplementary Fig. 6c).Fig. 5 Iron overload overcomes serum restriction of Legionella extracellular growth.

Ferric iron but not cysteine rescues JR32 ∆flaA dotA growth with U937 macrophages (A–E) and primary human monocyte-derived macrophages (hMDMs) (E) in the presence of serum. Culture media supplementation with 33 µM Fe(NO3)3 (Fe3+) (B–E) but not 3.3 mM cysteine (Cys) (A) restores JR32 ∆flaA dotA growth in the presence of heat-inactivated FBS (hiFBS). D Fold difference in JR32 ∆flaA dotA bioluminescence output at 116 hpi normalized to the PRMI+hiFBS infection condition. E Culture media supplementation with the indicated Fe(NO3)3 (Fe3+) amounts rescues JR32 ∆flaA dotA growth in the presence of heat-inactivated human serum (hiHS). Note that hMDMs viability was rapidly lost in SF conditions and thus is not included in the analysis. F Non-iron divalent metal salts cannot restore JR32 ∆flaA dotA replication in the presence of hiFBS. G In the presence of serum, iron supplementation (33 µM Fe(NO3)3) is sufficient to restore the growth of Lp mutants (sdhA, mavN, dotH and dotB) that cannot replicate intracellularly. H Ferric iron supplementation affects specifically Lp extracellular growth. Growth kinetics of the indicated Lp strains with U937 macrophages under Iron limiting (hiFBS) vs Iron overload (hiFBS+33 µM Fe(NO3)3) conditions are shown. Bacterial internalization was blocked by the addition of 5 µM cytochalasin D (CytoD) at 60 min prior to infection. A–H Averages from three technical replicates +/−StDev are shown, where the infection dose was MOI = 5. Representative of three biological replicates for each experiment is shown. Statistical significance was determined by one-way ANOVA (A–E, G) and the respective p-values are indicated. Source data are provided as a Source Data file.

Iron-binding carrier proteins, such as Transferrin and Lactoferrin, are major serum constituents performing nutritional immunity functions by sequestering and limiting availability of divalent metals to bacterial pathogens67. The iron-sequestration capacity of serum is a function of the saturation state of iron-binding proteins, thus iron supplementation can overload nutritional immunity defenses and restore iron availability for bacterial replication96,97. Macrophages regulate extracellular iron levels, both locally and systemically, via dedicated uptake and secretion transport mechanisms98–100. Without serum, the sole source of iron in our cell culture infection model remains the macrophage, which likely export sufficient amounts of iron in the extracellular milieu for Lp to growth. Therefore, we tested directly whether Lp extracellular replication specifically is dependent upon serum iron overload. To this end, the growth kinetics of different Lp mutant strains that cannot replicate intracellularly—dotB, dotH, mavN and sdhA—were measured under iron-limiting (i.e. ‘serum only’) or iron-overload (i.e. ‘serum+Fe3+’) conditions (Fig. 5G). For all strains, Lp extracellular growth was restored under iron-overload but not iron-limiting conditions (Fig. 5G). Notably, bacterial growth kinetics in iron-overload conditions either phenocopied (dotH) or were accelerated (sdhA, mavN and dotH) as compared to bacterial replication in the absence of serum (Fig. 5G). These data implicate serum iron-binding capacity as a critical regulator of Lp extracellular replication as demonstrated by strains that fail to replicate intracellularly. To assess serum capacity to inhibit T4SS+ Lp extracellular growth promoted by macrophages, we blocked phagocytosis with the actin-polymerization inhibitor cytochalasin D (cytoD) and measured bacterial replication under iron-overload and iron-limiting setting. Robust T4SS+ Lp replication was evident when iron-saturated serum and cytoD were present suggesting that macrophages can support extracellular replication of Dot/Icm+ Lp providing sufficient iron is available (Fig. 5H). Conversely, extracellular T4SS+ Lp replication was restricted under iron-limiting conditions (i.e. ‘+ serum and cyto D’), confirming the capacity of serum to restrict Lp extracellular replication specifically by limiting bioavailable iron released from macrophages (Fig. 5H). Similarly, the dotA deletion strain, which strictly replicates extracellularly, grew only under iron-overload conditions regardless of cytoD presence (Fig. 5H).

Because iron was critical for Lp extracellular growth in the presence of serum, we investigated if iron is required of Lp growth under SF conditions by treating cells with deferoxamine (DFO)—a non-toxic iron chelator that is clinically approved and effective for treatment of iron-overload pathological conditions101. Treatment with 25 µM DFO restricted Lp extracellular replication in monocyte infections as measured by a bioluminescence growth assay (Fig. 6A) and live-cell imaging (Fig. 6B). Importantly, addition of iron salt restored Lp growth in the presence of DFO demonstrating that the restriction of bacteria replication by DFO is due to iron-sequestration (Fig. 6A). Because iron is insufficient to trigger extracellular replication in SF conditions in the absence of macrophages (Fig. 4A), we conclude that iron is required but not sufficient for Lp extracellular replication during infection. Thus, as long as extracellular iron concentration does not exceed the sequestration capacity of nutritional immunity, macrophage/monocyte ability to support Lp extracellular replication is neutralized.Fig. 6 Sequestration of host-derived iron by nutritional immunity proteins restricts Lp extracellular replication.

A The iron chelator Deferoxamine (DFO) blocks Lp extracellular growth in an iron-dependent manner. Lp growth kinetics with U937 monocytes (MOI = 5), where cells were treated with 25 µM DFO or 33 µM Fe(NO3)3 individually or together. Growth of opsonized and non-opsonized bacteria are shown. B Representative micrographs from time-lapse live-cell microscopy showing growth of non-opsonized Lp01 ∆flaA GFP+ and Lp01 ∆flaA dotA GFP+ with U937 monocytes in the presence of hiFBS or 25 µM DFO (MOI = 5). Bar = 100 µm (left panels). Quantitative analysis of the changes in GFP signal integrated intensity (GCU x µm2) over time. C Supplementation of purified apo-Transferrin (Tfn) blocks Lp extracellular replication in a dose- and iron-dependent manner (MOI = 5). Graph shows peak Lp01 ∆dotA growth (highest Biolux output/Biolux at T0) with U937 macrophages when Tfn was added at the indicated concentrations in the presence or absence of 330 µM Fe(NO3)3. (D) Lp growth kinetics with U937 macrophages at MOI = 5 in SF RPMI under the indicated growth conditions (Tfn = 62.5 µg/ml purified apo-Transferrin; Fe3 + = 33 µM Fe(NO3)3). E Maximum growth of JR32 ∆flaA in U937 macrophage infections (MOI = 5) under functional (+hiFBS or +Tfn) or compromised (+hiFBS/Fe3+ or +Tfn/Fe3+) nutritional immunity; Tfn = 62.5 µg/ml purified apo-Transferrin, hiFBS = 10%v/v, Fe3 + = 33 µM Fe(NO3)3. F Apo-Transferrin does not reduce Lp growth (OD600) or bioluminescence output (Biolux RLUs) in axenic liquid AYE culture media. Fe3+ = 330 µM Fe(NO3)3. G Lactoferrin blocks Lp extracellular growth in a dose-dependent manner. JR32 ∆flaA dotA growth kinetics with U937 macrophages (MOI = 5) under SF conditions in the presence of purified human apo-Lactoferrin (Lfn). Fe3+ = 66 µM Fe(NO3)3. A–G Averages from three technical replicates +StDev are shown. Representative of two biological replicates for each experiment is shown. Statistical significance was determined by two-tailed paired T-test (C, E) or one-way ANOVA (A, D, G) and the respective p-values are indicated. Source data are provided as a Source Data file.

Host iron-binding proteins sustain nutritional immunity defenses against Lp

Transferrin and Lactoferrin are the major nutritional immunity iron-binding proteins in human airway secretions102. Transferrin is a beta globulin with two high affinity ferric iron binding sites and the relative proportions of apo-, mono-, and diferric forms of Transferrin determines the percentage of Transferrin iron saturation in serum as well as its capacity to sequester additional iron molecules—a critical parameter for nutritional immunity. Because serum blocked Lp extracellular replication in an iron-dependent manner, we hypothesized that iron-binding carriers could mediate Lp restriction through iron-starvation. Therefore, the capacity of apo-Transferrin to restrict Lp extracellular growth in macrophage infections was investigated with the dotA mutant. The growth kinetics of the dotA mutant strain in macrophage cultures measured in the presence of different amounts of purified bovine apo-Transferrin (from 250.0 µg/ml to 7.8 µg/ml) clearly showed a dose-dependent restriction of Lp growth (Fig. 6C) that was reversed upon addition of ferric nitrate implying that Lp growth restriction is dependent on the iron-binding capacity of Transferrin (Fig. 6C). At 125 µg/ml apo-Transferrin was microbiostatic against the dotA mutant in macrophage infections but also lowered the apex of the growth curve of the T4SS+ parental strain by ~50% without affecting the slope (Fig. 6D, E). Iron supplementation reversed the growth defect brought by apo-Transferrin under infection conditions. Importantly, apo-Transferrin did not reduce Lp viability nor bioluminescence output in axenic growth cultures (Fig. 6F). Lactoferrin also interfered with Lp extracellular replication in a manner that was abrogated by ferric iron (Fig. 6G). Thus, we conclude that iron-sequestration by host iron-binding proteins block Lp extracellular replication by absorbing iron released from macrophages during infection.

Cooperation between cell intrinsic host defenses and nutritional immunity is required for restriction of Legionella growth

A robust pulmonary innate immune response orchestrated by TNF and IFNγ is critical for limiting Lp replication and resolving the infection by reprograming macrophages into a phenotypic state that is not permissive for intracellular bacterial replication52,53,103. Primary human alveolar and monocyte-derived macrophages primed with IFNγ restrict Lp intracellular replication in vitro52,53, emphasizing the importance IFNγ effector functions in Lp pathogenesis. Yet, the molecular determinants that allow a mild self-resolving legionellosis that is well-controlled by innate immunity and IFNγ to progress into Legionnaires’ disease—a severe atypical pneumonia—are unclear. Could extracellular replication driven by a pulmonary iron-overload potentially allow Lp to overcome innate immunity tilting the balance towards severe disease? It is well established that aging and history of cigarette smoking are major risk factors for severe legionellosis based on extensive epidemiological evidence5. Smokers accumulate significantly higher concentration of iron in the alveolar compartment vs. non-smokers104–109—a phenomenon that is exacerbated with age110. Unlike iron, the amount of alveolar Transferrin remains unchanged in smokers106–108 indicating lowered pulmonary nutritional immune capacity in smokers may lead to iron-overload conditions sufficient to support Lp extracellular replication, which in turn would allow evasion of cell autonomous innate immune defenses, explosive bacterial growth and disease exacerbation. Such a scenario would be dependent on an intrinsic capacity of IFNγ stimulated macrophages to support Lp extracellular replication, especially under conditions of compromised nutritional immunity. Therefore, the capacity of macrophages to support Lp extracellular growth when primed with IFNγ alone or with IFNγ and E.coli LPS was investigated in the presence or absence of serum. First, we confirmed that IFNγ and IFNγ + LPS treatments elicited the appropriate transcriptional response in macrophages by demonstrating the induction of the interferon-stimulated genes IDO1 and IRF1111,112 as well as the LPS regulated gene IL6 (Fig. 7A). Next, the capacity of IFNγ-primed macrophages to restrict Lp intracellular replication was investigated via live-cell imaging. Macrophages primed with IFNγ in the presence of serum for 16hrs were infected with T4SS+ Lp expressing GFP in the presence of IFNγ under SF conditions and subsequently imaged for 24 h (Fig. 7B). LCVs in non-primed macrophages increased in size unlike the LCVs in IFNγ-stimulated cells (Fig. 7B) indicating that serum withdrawal did not interfere with IFNγ-dependent restriction of Lp intracellular replication.Fig. 7 IFNγ and nutritional immunity cooperate to restrict Lp growth in macrophage infections.

A qPCR analysis of IFNγ-inducible and inflammatory genes expression in U937 macrophages treated for 16hrs as indicated. For each gene expression data from each treatment is relative to respective GAPDH amount and is represented as fold change from unstimulated macrophages. Averages from five biological replicates ±SEM are shown. B Quantitative analysis of LCVs expansion from time-lapse microscopy of U937 macrophages infected with Lp01 ∆flaA GFP+ for 24 h at MOI = 5 that have been either primed with IFNγ (2 µg/ml) for 16 h or left unstimulated (UN). Serum was present during priming and was removed prior to the infection. Representative micrographs in which the LCVs are highlighted by a red border are shown in the left panel, whereas the change in LCV size (n > 200) is graphed in the right panel. Lp growth restriction by macrophages requires both serum and IFNγ priming. Growth kinetics of JR32 ∆flaA (C) and JR32 ∆flaA dotA (D) with U937 macrophages (MOI = 5) primed with either IFNγ (2 µg/ml) alone or IFNγ (2 µg/ml) +E.coli LPS (100 ng/ml) for 16 h prior to infection in the presence of hiFBS (left panels). Area-under-the-curve analysis from four independent biological replicates are shown in the right panels. Treatments were maintained during the infections. In some conditions, the serum-containing RPMI was replaced with SF RPMI just before the inoculum was added. E Blockade of extracellular replication is required for the IFNγ-dependent restriction of Lp growth with human macrophages. Growth kinetics of JR32 ∆flaA in SF RPMI with unstimulated (UN) or IFNγ-primed U937 macrophages (MOI = 5) in the absence/presence of 20 µg/ml Spectinomycin (Sp) or 125 ng/ml purified apo-Transferrin (Tfn). IFNγ stimulation was maintained during the infections. A–C left panel, D left panel, E, F Averages from three technical replicates + StDev are shown. C right panel, D right panel each data point denotes one biological replicate. Representative of three (B, E, F) or four (C, D) biological replicates for each experiment is shown. Statistical significance was determined by two-tailed non-parametric Wilcoxon matched-pairs T-test (C, D) or one-way ANOVA (E, F) the respective p-values are indicated. n number of biological replicates. Source data are provided as a Source Data file.

The growth of T4SS+ Lp with macrophages primed by either IFNγ or LPS + IFNγ was restricted in the presence of serum (Fig. 7C). However, when infections were carried out under SF conditions, T4SS+ Lp growth with IFNγ-primed macrophages was restored (Fig. 7C). Under the same conditions, the dotA mutant, which can only replicate extracellularly, grew with similar kinetics regardless of IFNγ priming, but strictly in the absence of serum (Fig. 7D). Thus, IFNγ-primed macrophages can support robust Lp extracellular replication. Considering that Lp intracellular growth is restricted by IFNγ in SF conditions (Fig. 7B), these data support a scenario where extracellular replication expands the bacterial population under conditions where IFNγ is present and nutritional immunity is absent. To test this scenario experimentally we utilized two distinct approaches to block Lp extracellular replication. The first was based on a low-dose spectinomycin antibiotic protection assay at concentrations that preferentially interfered with extracellular but not intracellular Lp replication (Supplementary Fig. 4) and the second was based on the capacity of apo-Transferrin to provide nutritional immunity and restrict Lp extracellular growth (Fig. 6D). In SF conditions, addition of either apo-Transferrin or low-dose spectinomycin was sufficient to restore the replication blockade imposed by IFNγ-primed macrophages (Fig. 7E), demonstrating that extracellular replication overcomes IFNγ restriction. Similarly, when nutritional immunity was compromised by iron-overload (serum + Fe3+) IFNγ-primed macrophages no longer restricted Lp growth (Fig. 7F). Previously published data with primary human cells demonstrating that iron supplementation overcomes IFNγ-mediated restriction of Lp replication also supports this notion76,77, although the authors assumed that intracellular growth was restored. Notably, IFNγ- and LPS-priming significantly decreased expression of iron uptake and export regulators in macrophages indicating that iron release might potentially occur via a non-canonical mechanism independent of Ferroportin-1 (Supplementary Fig. 7). Taken together, our data demonstrates that efficient host defenses to Lp require the joint action of IFNγ-driven cell mediated immunity and iron-sequestration by nutritional immunity proteins for the simultaneous blockade of intracellular and extracellular bacterial replication (see model in Fig. 8).Fig. 8 Proposed model for the role of nutritional immunity in Lp pathogenesis.

Initially, several rounds of intracellular replication within a membrane-bound organelle inside alveolar macrophages amplify the Lp population triggering inflammation that drives the subsequent infiltration of innate immune cells leading to localized IFNγ production by NK or NK T-cells. We propose that Transferrin and IFNγ form a two-pronged defense barrier to limit Lp infection where nutritional immunity limits extracellular replication when Transferrin saturation is low and IFNγ restricts intracellular replication. When Transferrin saturation is high because of a chronic pulmonary iron-overload state, nutritional immunity is compromised, which renders the extracellular milieu permissive for Lp growth as growth-sustaining nutrients are released by tissue-resident and infiltrating monocytes/macrophages. Under conditions of compromised nutritional immunity one critical barrier to infection is removed and Lp growth can continue despite IFNγ secretion from innate immune cells because majority of clinical Lp strains express the lag-1 gene which renders them resistant to complement killing and neutrophils internalization.

Discussion

In the last four decades the idea that Legionella cannot replicate extracellularly under infection conditions has been a cornerstone of Legionella biology. This paradigm has shaped our view of Legionella pathogenicity in a way that parallels are drawn to obligate intracellular bacterial pathogens, where a transmissive form exists outside of the host which transforms into a replicative form within the intracellular niche, replicates and switches back to transmissive form prior to egress51. Our data challenges this longstanding notion by demonstrating that Lp readily replicates extracellularly during infection in the absence of or upon disruption of nutritional immunity. Serum utilization in tissue culture models of infections for decades has masked this phenomenon because of iron-dependent nutritional blockade. Several lines of evidence from our data demonstrate robust Lp extracellularly replication occurred when nutritional immunity is absent or is compromised due to saturation of iron-absorption capacity: (i) mutant strains with distinct intracellular growth defects now grew during infection; (ii) bacterial growth was observed even when host cells could not internalize bacteria; (iii) bacterial replication occurred despite a physical barrier separated Lp from the host cells; (iv) bacteria replicated in macrophage-conditioned medium in the absence of host cells altogether. We demonstrated that human immortalized (U937 monocytes and macrophages) as well as primary (hMDMs) cells support extracellular replication of L. pneumophila and L. jordanis when nutritional immunity is compromised. Thus, extracellular replication under infection conditions could be feature common to multiple pathogenic Legionellaceae species.

Notably, host cells were required for extracellular replication as macrophages and monocytes promoted bacterial growth likely by providing necessary nutrients missing from the cell culture media, which is evident by the conditioned medium experiments. We identified iron as one such milieu micronutrient that is required but is not sufficient for extracellular replication. Iron sequestration caused by serum-derived or purified iron-binding proteins imposed a blockade on extracellular replication that was abolished specifically under iron saturation conditions. Under SF conditions, the iron fueling Lp extracellular replication could only come from host cells either by Ferroportin mediated secretion or more likely by passive release from dying host cells because Ferroportin transcripts decreased by ~80% in response to LPS in U937 macrophages (Supplementary Fig. 7). One iron-acquisition strategy evolved by human adapted bacterial pathogens to bypass nutritional immunity is the production of high-affinity bacterial receptors for Transferrin113–115. However, apo-Transferrin interfered with Lp extracellular growth under infection conditions in a dose-dependent manner demonstrating that Transferrin blocks access to iron released from macrophages indicating that Legionella cannot source iron from holo-Transferrin directly and likely its siderophores have lower Fe3+ affinity compared to host iron-binding proteins.

Because legionellosis is caused by an environmental bacterium which co-evolved with unicellular amoebae and not a mammalian host, it has been difficult to explain how Lp escapes IFNγ-driven cell mediated immune defenses that efficiently block intracellular replication to cause severe disease. History of smoking as well as aging are the main risk factors for developing Legionnaires’ disease4,5. Notably, bronchoalveolar lavage (BAL) fluid samples from smokers as compared to non-smokers are highly enriched in iron, without a concomitant increase in Transferrin104–109. Similarly, aging has been shown to coincide with progressive accumulation of pulmonary iron110, thus under both circumstances access to bioavailable iron is increased due to a state of lowered nutritional immunity. Indeed, BAL samples from smokers support higher growth of Staphylococcus aureus and Pseudomonas aeruginosa in an iron-dependent and Lactoferrin-dependent manner as compared to non-smokers116. Moreover, a significant protective effect specifically against respiratory infections has been observed in the context of mild-to-moderate iron deficiency without an increase in cell-mediated immune responses72. Could extracellular replication facilitated by iron overload potentially explain progression towards severe legionellosis? We demonstrated that iron-enriched milieu permits Lp replication outside of the host cell and bypass killing by IFNγ-primed macrophages. Previously, exogenous iron has been shown to rescue Lp growth with IFNγ-primed macrophages and the assumption was made that intracellular growth was restored76,77. However, this assertion is incongruent with more recent data showing IFN stimulation results in (i) death of infected macrophages due to LCV rupture57,58,60 caused by the Guanylate binding protein 1 (GBP1) GTPase57; (ii) downregulation of iron uptake via the Transferrin Receptor76; (iii) an increase in iron export117. Our work reconciles these observations by demonstrating that extracellular replication in iron-enriched milieus accounts for restoration of Lp growth with IFNγ-primed macrophages.

Another important aspect of our work is the discovery that outside of the host cell Lp can transition into a replicative form under infection conditions by sourcing nutrients for extracellular growth from macrophages and monocytes. Lp cannot grow on glucose as the sole carbon source because of amino acid auxotrophy38, yet our data revealed robust bacterial growth strictly in the presence of host cells in nutrient depleted medium lacking amino acids. Under such conditions, nutrients and growth factors can only be sourced from host cells. Even though heat-killed microorganisms (gram-negative bacteria as well as amoeba) have been shown to support limited Lp necrotrophic growth118, in our assays sonicated monocytes did not support bacterial growth whereas live monocytes and macrophage-conditioned medium did. Thus, live monocytes/macrophages are necessary at least for initiation of extracellular bacterial replication during infection likely through production of critical nutrient(s) or a growth-initiating sensory cue; although, a potential role for necrotrophy cannot be excluded in the later growth stages. The sensory cues that trigger and the nutrients that fuel extracellular replication represent an important aspect of Lp pathogenesis that warrants further research effort.

Our data argues for an updated pathogenesis paradigm taking into account nutritional immunity and extracellular bacterial replication. We propose the following model (Fig. 8). Upon inhalation and deposit in the lower respiratory tract, Lp likely undergoes several rounds of intracellular replication within alveolar macrophages triggering an inflammatory response, subsequent infiltration of innate immune cells, and localized IFNγ production by NK or NK T-cells. Once majority of bystander macrophages are primed by IFNγ, Lp intracellular growth would be restricted and the infection resolved providing nutritional immunity suppresses extracellular replication. This scenario is consistent with the self-resolving features of mild legionellosis in human infections (aka Pontiac fever) and low-dose infections in murine models4,5,119. Because of the prolonged lag phage Lp extracellular replication likely coincides with IFNγ production and we argue that this is a critical junction in pathogenesis and disease progression. A defect in nutritional immunity caused by Transferrin saturation from a chronic pulmonary iron overload (as in patients with history of smoking) would establish an alveolar milieu permissive for extracellular replication further fueled by nutrients released by tissue-resident and infiltrating monocytes/macrophages. Under those conditions, extracellular replication can sustain bacterial growth despite IFNγ production further aggravating inflammation, eliciting the severe symptomatology associated with Legionnaires’ disease and causing pulmonary failure without a timely antibiotic administration120–122. Such a model places an emphasis on extracellular survival and replication as important determinants of disease progression, assertion strongly supported by the dominance of clinical strains with the capacity to evade host defenses targeting extracellular bacteria. Majority of serogroup-1 clinical isolates unlike environmental isolates (80% vs 30%) express the lag-1 gene encoding a LPS O-acetyltransferase, which confers resistance to complement killing and neutrophil internalization123. Because ‘clinical’ strains are frequently isolated during outbreaks from symptomatic infections, the lag-1 gene predominance in clinical isolates links extracellular antimicrobial defenses (i.e killing by complement and neutrophils) with clinical symptomatology and disease severity. Epidemiological data also argues for host-intrinsic rather than bacteria-intrinsic drivers of disease severity because outbreaks caused by a single clinical isolate frequently produces both mild and severe disease6–13. Thus, extracellular replication as well as the defense mechanisms that counteract it are emerging as important previously underappreciated aspects of Legionella pathogenesis. Our work highlights the critical interdependency between nutritional and cell mediate immunity for generation of robust defense against bacterial infections and provides a reasonable mechanistic link between the well-documented deficiency in pulmonary nutritional immunity of smokers and severe legionellosis.

Methods

Bacterial strains, plasmids, and media

The L. pneumophila strains used in this study were derived from the L. pneumophila serogroup 1, strain Lp01 or strain JR3229 and have a clean deletion of the flaA gene to avoid NLRC4-mediated pyroptotic cell death response triggered by flagellin. Importantly, strains lacking a functional T4SS as a result of inactivation of dotA, dotB or dotH do not trigger pyroptosis regardless of flagellin expression.

The bacterial strains, primers and plasmids used in this study are listed in Supplementary Tables 1, 2 and 3 respectively. Legionella strains were cultured either on charcoal yeast extract (CYE) plates [1% yeast extract, 1%N-(2-acetamido)−2-aminoethanesulphonic acid (ACES; pH6.9), 3.3mM l-cysteine, 0.33 mM Fe(NO3)3, 1.5% bacto-agar, 0.2% activated charcoal] or in complete ACES-buffered yeast extract (AYE) broth (10 mg/ml ACES: pH6.9, 10 mg/ml yeast extract, 400 mg/l L-cysteine, 135 mg/l Fe(NO3)3) supplemented with 100 µg/ml streptomycin124. As needed, the medium were supplemented with the following: 25 μg/ml kanamycin (Kam), 100 μg/ml streptomycin, 10 μg/ml chloramphenicol, 150 mM sodium chloride, 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG), or different concentrations of spectinomycin.

Bacterial culture conditions for inoculum preparation

for all infection experiments, Legionella strains obtained from dense patches grown on CYE plates for two days were resuspended to OD600 of 0.5 U in 1 ml AYE, placed in 15-ml glass culture tubes, and cultivated aerobically 24–26 h with continuous shaking (175 rpm) at 37 °C until early stationary phase was reached (OD600 range of 2.0 to 3.0 U). The L. jordanis reporter strains with the chromosomally encoded LuxR operon under the control of Ptac received 0.5 mM IPTG at 8 h post seeding in the AYE broth. The AYE broth cultures of L. pneumophila GFP+ strains harboring a plasmid carrying the gfp under the control of the Ptac contained 10 μg/ml chloramphenicol and were supplemented with 0.1 mM IPTG between 18 to 20 h post seeding in AYE broth.

Generation of bioluminescent Legionella strains

all three bioluminescence producing regulons (see Supplementary Fig. 1a) were based on the luxR operon (luxCDABE) from Photorhabdus luminescens. The L. pneumophila JR32 strains used in the study encoded the constitutive Lp icmRp-luxR regulon, which has been previously described in detail79. The L. jordanis strains encoded the IPTG-inducible Lj pTAC-luxR regulon, which has been previously described in detail125. The L. pneumophila Lp01 strains used in the study encoded a new constitutive Lp icmR-luxR locus regulon. The Lp icmR-luxR locus is a synthetic regulon in which the luxR operon from P. luminescens is inserted immediately downstream of the Lp icmR gene in the context of the native icmR locus. For cloning, the synthetic icmR locus (1943 nt) was obtained as a gBlock (IDT). It contained the Lp chromosome region spanning icmT, icmS, icmR, and icmQ genes and included four unique restriction enzyme sites: (i) BamHI—localized upstream of the icmT gene; (ii) SacI—localized downstream of the icmQ gene, as well as (iii) BglII and (iv) NotI localized in the intergenic region between the icmR and icmQ genes. The gBlock was digested with BamHI/SacI and cloned into similarly digested suicide plasmid pSR47s. Next, the 5,854nt luxR operon (luxCDABE) fragment was released from pXen13 with BamHI and NotI, gel-purified and inserted between icmR and icmQ to yield pSR47s-icmR-luxR locus. The synthetic icmR regulon encoded by pSR47s-icmR-luxR locus was swapped with the native regulon in Lp01 strains by allelic exchange via double homologous recombination and was confirmed via bioluminescence emission; kanamycin sensitivity and sucrose resistance126.

Inactivation of Lp genes via insertion mutagenesis

the single gene knockout strains lacking mavN, dotB or dotH were produced by intragenic insertion of the pSR47 plasmid on the Lp chromosome via homologous recombination. To this end, intragenic regions for mavN (137→ 1,131nt), dotB (151→ 937nt) and dotH (121→ 830nt) were PCR amplified from genomic DNA and cloned in pSR47 using primers listed in Supplementary Table 2. The respective plasmids pSR47-mavN, pSR47-dotB and pSR47-dotH were introduced in Lp via tri-parental mating. Insertion mutants were isolated by kanamycin resistance and were genotyped by colony PCR to confirm correct insertion using the T3 promoter primer (5’-CAATTAACCCTCACTAAAGG-3’) together with the respective screening primer for each gene listed in Supplementary Table 3.

Inactivation of Lp genes via clean deletion (CD) mutagenesis

the L. jordanis ΔdotB allele was generated by fusing ~1 kb regions upstream and downstream of the dotB gene. To this end the upstream region was PCR amplified from genomic DNA using the primers UpF_Lj_dotB and UpR_Lj_dotB. The downstream region was similarly produced with the primers DownF_Lj_dotB and DownR_Lj_dotB. The resulting fragments were linked by an EcoRI site and cloned into the BamHI/SacI sites of the gene replacement vector pSR47s creating pSR47s-CD dotBLj. The pSR47s-CD was electroporated (voltage-1800, capacitance- 25µF and resistance- 200ohms) in L. jordanis for the generation of the ΔdotB strain by allelic exchange of dotB with ΔdotB via double homologous recombination. The allelic exchange was confirmed by genotyping clonal isolates with the primers SP1_Lj_dotB, SP2_Lj_dotB SP3_Lj_dotB in a single PCR reaction that produces either a fragment of ~300nt for dotB or a fragment of ~450nt for ΔdotB. The clean deletion Lp strains lacking flaA or sdhA were generated with the gene replacement plasmids pSR47s-CD1340 and pSR47s-CD0376 via allelic exchange as detailed previously127.

Reagents

Detailed reagents list is provided in Supplementary Table 4. The purified polyclonal α-Lp IgY chicken antibody was custom generated by Cocalico Biologicals against formalin-killed bacteria127. The purified polyclonal α-Lp IgG rabbit antibody (lot# Marsha) was a kind gift from Dr. Craig Roy (Yale University).

Bacterial axenic growth assays

For axenic growth assays bacteria were collected from dense patches grown on CYE plates for two days, diluted in AYE with an initial OD600 = 0.5 U and were loaded onto a clear bottom white wall 96-well plate (Corning, cat# 3610). All experimental conditions were setup in triplicates and final culture volume was 200 µl. The plate was loaded into Tecan Spark luminometer and incubated at 37 °C for 24 h. Every 10 min, the cultures were agitated for 180 sec via a double orbital rotation at 108 rpm and luminescence as well as OD600 data were collected. The bioluminescence output from each well was captured for 2 sec and quantified as a total relative light unit counts per second.

Monocyte and macrophage cell culture conditions

The U937 cell line was obtained directly from ATCC (ATCC, CRL1593.2) and was authenticated by ATCC. Human U937 monocytes were cultured in RPMI-1640 with L-glutamine (BI Biologics, cat #01-100-1 A), 10% v/v FBS and penicillin/streptomycin at a temperature of 37 °C in the presence of 5% CO2. For macrophage differentiation, U937 monocytes were treated with 10 ng/ml Phorbol 12-myristate 13-acetate (PMA)(Adipogen) for the first 24 h after which the cells were cultured for additional 48 h without PMA and antibiotics. Human PBMCs were purchased from Sigma (cat#HUMANPBMC-0002644) and were differentiated into macrophage by continuous culture in RPMI-1640 with L-glutamine (BI Biologics, cat #01-100-1 A), 20% v/v human serum (Sigma, cat# H4522) and penicillin/streptomycin at 37 °C in the presence of 5% CO2 for 8 to 10 days, where the culture media were replaced every 4 days.

Bioluminescence assay for Legionella replication with human monocytes and macrophages

For human U937 macrophages infections, U937 monocytes were seeded at a density of 1 × 105 cells per well in white-wall clear-bottom 96-well plates (Corning cat# 3610) and differentiated into macrophages for 72 h as indicated in “Monocyte and macrophage cell culture conditions”. For infection, U937 monocytes were seeded at the same cell density in 100 µl of SF-RPMI or PBSG (PBS supplemented with 7.5 mM Glucose, 0.9 mM CaCl2, and 0.7 mM MgCl2) per well for 3 h prior to infection. The culture media used in each assay is indicated in the figure legends. Inoculum and other reagent were added to a final volume of 200 µl/well. Primary human monocyte-derived macrophages (hMDMs) were seeded at 1 × 105 cells per well in RPMI-1640 supplemented with human serum (10% v/v) (Sigma, cat# H4522) for 16 h prior to infection. Because of rapid loss of viability, infections with hMDMs were only carried out in the presence of human serum. Infections of U937 monocytes and macrophages were carried out under SF conditions or in the presence of FBS (10% v/v) as indicated for each assay with synchronized liquid culture grown bacteria at MOI = 5. Unless indicated, the culture volume during infection was 200 µl/well. Plates were centrifuged for 5 min at 47 g after inoculum addition to enhance bacteria contact with the host cells. Plates were kept in a tissue culture incubator at 37 °C with 5% CO2 and periodically, the bioluminescence output from each well was acquired by a Tecan Spark luminometer (integration time = 2 s). The data is presented as fold change in bioluminescence from the T0 reading. All conditions in all assays were performed in technical triplicates.

Infections of IFNγ-primed U937 macrophages

cells were cultured with either IFNγ (2 µg/ml) or IFNγ + E.coli LPS (100 ng/ml) for 16 h in RPMI-1640 supplemented with hiFBS (10% v/v). At the time of infection the culture medium was replaced with RPMI +/−hiFBS. The IFNγ and IFNγ + E.coli LPS treatments were maintained for the duration of the infection. The infections were carried out as outlined above and bacterial growth was measured via bioluminescence output.

Infections with opsonized bacteria

as indicated in certain monocyte infections Lp was opsonized with a polyclonal rabbit anti-Lp IgG (1:1000 dilution) for 30 min prior to infection at room temperature in SF-RPMI. The infections were carried out as outlined above and bacterial growth was measured via bioluminescence output.

Infections of sonicated monocytes

U937 monocytes were suspended in SF-RPMI at 2 × 106 cells per 300 µl. One group of cells was ruptured via sonication in Bioruptor® Pico for 5 min (30 s ON, 30 s OFF sonication cycle) and another group was not sonicated. Volume equivalents containing 1 ×105 cells (~15 µl) were added in each well from a 96 well plate to which the inoculums of liquid grown bacteria were added directly. The infections were carried out as outlined above and bacterial growth was measured via bioluminescence output. The final cell culture volume during infection was 200 µl.

Contact-dependent bacterial replication assay

U937 monocytes or macrophages were seeded in 24-well plates at 2.5 × 105 cells/well. A transwell plate insert with a 0.4 µm pore size bottom PET membrane (VWR, Cat# 76313-906) was placed in each well and the inoculum (MOI = 5) was added either to the transwell insert for measurement of contact-independent bacterial replication or in the well to measure contact-dependent growth. The plate was centrifuged for 5 min at 47 g to bring the bacteria in contact with the host cell. Plates were kept in a tissue culture incubator at 37 °C with 5% CO2 and periodically, the bioluminescence output from each well was acquired with a Tecan Spark luminometer (integration time = 2 s). The data is presented as fold change in bioluminescence from the T0 reading. All conditions in all assays were performed in technical triplicates. Transwell membrane integrity in the contact-independent growth conditions was confirmed by plating serial dilutions of the contents from the lower chamber on CYE agar, and as expected viable CFU were not recovered.

Bioluminescence assay for Legionella growth in conditioned media

For preparation of macrophage conditioned medium U937 monocytes were differentiated in 6 wells plates at 2 × 106 cells/well and cultured with SF RPMI in the presence of absence of 100 ng/ml E.coli LPS for 4 days. The conditioned medium was collected, centrifuged (10,000 rpm, 5 min) and passed through a 0.2 µm pore filter. The collected media was either immediately used in bacterial growth assays or stored at −80 °C for later use. Bacterial growth assay was setup in 96-well white wall plates and monitored by bioluminescence output, where 5 × 106 bacteria were added to 200 µl conditioned medium in each well. In control conditions, bacteria were added directly to SF RPMI in the presence or absence of 3.3 mM cysteine +330 µM Fe (NO3)3. All, experimental conditions were carried out in technical triplicates.

Colony forming units assay for Legionella growth

Macrophages were seeded, infected, and treated as listed in “Bioluminescence assay for Legionella intracellular replication”. Plates were kept in a tissue culture incubator at 37 °C and 5% CO2 and periodically removed to collect bacteria. For bacterial recovery, cell culture medium from the well was moved into a 1.5 ml Eppendorf tube and replaced with 100 µl of sterile water for lysis of the infected macrophages via hypotonic shock and the plate was returned to the incubator for 10 mins. Next, the contents of the well were pipetted at least 20 times and were collected in the respective Eppendorf tube which already contained the cell culture media. Eppendorf tubes were vortexed for 30 sec and contents (~300 µl) were serially diluted five times for a total of six dilutions. 25 µl from each dilution was plated on a CYE agar plate and incubated until colonies were visible. Colonies from a single dilution were counted and CFUs were calculated for each condition. Eight randomly selected colonies from each experimental condition at every time point were replicate streaked on CYA plates in the presence or absence of 150 mM NaCl to confirm that all T4SS- strains used in the assay were SaltR, whereas T4SS+ strains were SaltS. All infection conditions for every experiment were performed in three technical replicates. The data is presented as fold change in recovered CFU over T0.

Automated time-lapse live-cell imaging of Legionella growth with host cells

U937 monocytes or macrophages were seeded at 1 × 105 in 96-well black-wall clear-bottom plates (Corning, cat# 3904) in phenol red-free DMEM supplemented as indicated for each assay. All infections were carried out at MOI = 2.5 in 150 µl per well with AYE broth grown bacteria in the presence of 1 mM IPTG. In each experiment, all conditions were performed in technical triplicates. Plates were centrifuged (47 g, 5 min) and were loaded into the IncuCyte™ S3 microscope housing module. The IncuCyte™ S3 HD live-cell imaging platform (Sartorius) is a wide-filed microscope mounted inside a tissue culture incubator and is controlled by IncuCyte™ software. For each well, four single plane images in bright field and green channel (ExW 440–480 nm/EmW 504–544 nm) were automatically acquired with S Plan Fluor 20X/0.45 objective every four hours over several days. Images were analyzed with the IncuCyte™ Analysis software (Incucyte S3 GUI 2020.A). For quantitation of bacterial replication, GFP signal was used for the generation of a binary mask that defined total signal integrated intensity (GCU x µm2) in each image or for each GFP+ object when LCV expansion was measured. The IncuCyte™ S3 imaging analysis was performed in the Innovative North Louisiana Experimental Therapeutics program (INLET) core facility at LSU Health-Shreveport.

Gene expression analysis by quantitative real-time PCR

U937 macrophages were treated with 2 μg/ml IFNγ, 100 ng/ml E.coli LPS, or a combination of both for 16 h, then total RNA was extracted with the RNeasy Mini kit (Qiagen), treated with DNase I (NEB, cat# 50-814-118) and cDNA was prepared with the High Capacity cDNA reverse transcriptase kit (Thermo Fisher, cat# 4368814). Real-time qPCR amplification of the cDNA was completed with PowerUp SYBR Green master mix (Thermo Fisher, cat# A25741), following the manufacturer’s instructions. For each reaction, 1 μl cDNA (equivalent to 0.1−0.2 μg of total RNA) and 20 pmoles of each gene-specific primer set (listed in Supplementary Table 2) were combined in a 20 μl reaction volume. The LightCycler 96 Real-Time PCR System (Roche Diagnostics) was used for amplification with the following thermal cycle conditions: pre-amplification step at 95 °C for 10 min; amplification step of 40 cycles at 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 10 s; followed by a melting step at 95 °C for 10 s, 65 °C for 60 s, and 97 °C for 1 s. For each experiment, samples were analyzed in triplicates. GAPDH expression served as the internal reference for normalization to determine the relative expression levels of specific transcripts, and fold differences were calculated using the 2-ΔΔCt method.

Bacteria uptake assay

To adhere U937 monocytes onto glass cover slips placed inside a 24-well plate, 1 × 105 cells/well were cultured with Monocyte Attachment Medium (Sigma, cat# C-28051) for 2 h after which the cells attached, and the medium was replaced with RPMI. U937 macrophages were directly differentiated onto glass cover slips at the same cell density. Infections were carried out with AYE-grown Lp01 ∆flaA GFP+ that were either opsonized or not opsonized with a polyclonal rabbit α-Lp IgG for 60 min prior to infection. All experimental conditions were done in technical triplicates for each experiment. After the bacteria were added to each well at MOI = 5, plates were centrifuged (47 g, 5 min) and the infection was allowed to proceed for 6 h in the presence of 1 mM IPTG and in the presence/absence of hiFBS (10% v/v). Next, cells were washed with warm PBS (3X), the infection was stopped by adding 2% paraformaldehyde (PFA) for 60 min at ambient temperature. The surface-associated bacteria were immunolabeled without plasma membrane permeabilization with chicken α-Legionella IgY antibody (1:300) for 90 min in PBS containing goat serum (2% vol/vol) after which coverslips were washed with PBS (3X), fixed with 2% PFA (30 min) and permeabilized with 0.2% Triton X-100 (20 min). After washing with PBS (3X), the samples were stained with Phalloidin-iFluor 594 Conjugate (1:2000), goat α-chicken IgY-Alexa647 (Thermo Fisher, cat# A-21449) (1:500 dilution) and with Hoechst (1:2000 dilution) for 60 min in PBS containing goat serum (2% vol/vol). Coverslips were mounted with ProLong glass anti-fade mountant (ThermoFisher, cat# P36984) onto slides. Imaging was performed with inverted wide-field microscope (Nikon Eclipse Ti) controlled by NES Elements v4.3 imaging software (Nikon) using a 60X/1.40 oil objective (Nikon Plan Apo λ), LED illumination (Lumencor) and CoolSNAP MYO CCD camera. Image acquisition parameters—Hoechst (ExW 395 / EmW 455); GFP (ExW 470 / EmW 525), iFluor (ExW 555 / EmW 605) and Alexa647 (ExW 640 / EmW 705). The z-axis acquisition was set based on the out-of-focus boundaries and the distance between individual Z-slices was kept at 0.3 µm. Only linear image corrections in brightness or contrast were completed. For each condition, over 150 bacteria were imaged and scored as either intracellular (single positive—green only) or not-internalized (double positive—green/red).

Analysis of iron and Transferrin content in sera

The iron content of 12 commercially sourced sera from different manufacturers was determined by a ferrozine colorimetric assay. Ferrozine-based colorimetric assay. Briefly 100 ml of serum was mixed with 100 ml of 10 mM HCl and 100 ml of freshly prepared iron-releasing reagent (1.4 M HCl, and 4.5% (w/v) KMnO4 in H2O). Next the samples were incubated for 2 h at 60 °C, cooled to ambient temperature and 30 ml of the iron-detection reagent (6.6 mM ferrozine, 6.5 mM neocuproine, 2.5 M ammonium acetate, and 1 M ascorbic acid dissolved in water) was added. After 30 min incubation, 280 µl of each sample was moved to one well of 96-well plate and the absorbance was read at 550 nm on a Tecan Spark microplate reader. The iron content of each sample was calculated by comparing it absorbance to that of a range of concentration standards.

Immunoblot analysis for the Transferrin content in 2 µl volume from each serum was performed with a rabbit polyclonal α-Transferrin IgG (ProteinTech, cat# 17435-1-AP) (1:2000) after standard SDS-PAGE. The total protein content transferred on the nitrocellulose membrane for each sample was detected after incubation with Ponceau S staining solution (Thermo Scientific, cat# A40000279) prior to immunoblotting.

Monocyte cell viability assay

U937 monocytes were seeded in 96-well plates at 1 × 105 cells per well, in serum free RPMI medium at 3 h prior to infection with the indicated bacterial strains at MOI = 5. All conditions were performed in technical triplicates. Supernatants were collected from parallel infections every 24hrs from the start until 144 hpi and immediately frozen at 70 °C. Thawed supernatants (50 µl volume) were assayed for LDH activity using the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, cat# G1780) according to the manufacture’s protocol. In each experiment the dynamic range was determined from culture medium equivalent without monocytes (min) and from 1 × 105 monocytes cultured with serum free RPMI containing 1% Triton X-100 for 15 min (max). The data is normalized as percentage of maximum signal.

Statistical analysis

Calculations for statistical differences were completed with Prism v10.2.3 (GraphPad Software). The statistical tests applied for the different data sets are indicated in the figure legends and the resultant p-values are shown in the figures.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Supplementary information

Supplementary Information

Peer Review File

Description of Additional Supplementary Files

Supplementary Movie 1

Supplementary Movie 2

Reporting Summary

Source data

Source Data

Supplementary information

The online version contains supplementary material available at 10.1038/s41467-024-52184-x.

Acknowledgements

This work was supported by grants from following funding sources: NIAID (AI143839) to S.I., NIGMS (P20GM134974-5749) to A.M.D., Ike Muslow Predoctoral Fellowship from LSU Health Shreveport to A.A.W. We would like to thank the INLET High-Throughput Imaging Core at LSUHSC-Shreveport for technical assistance.

Author contributions

Conceptualization: S.I.; Methodology/Investigation: A.T.E., A.W., M.J.R., A.M.D., S.I.; Sample acquisition: A.T.E., A.W., M.J.R., M.C., B.L., S.I.; Visualization: A.T.E., S.I.; Funding acquisition: A.W., A.M.D., S.I.; Supervision: A.M.D., S.I.; Writing—original draft: A.T.E., A.M.D., S.I.; Writing—review & editing: all authors

Peer review

Peer review information

Nature Communications thanks the anonymous, reviewers for their contribution to the peer review of this work. A peer review file is available.

Data availability

The source data used in this study are available from Figshare at 10.6084/m9.figshare.26125729. Source data are provided with this paper.

Competing interests

The authors declare no competing interests.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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