Iron-depleting nutritional immunity controls extracellular bacterial replication in Legionella pneumophila infections

Torres-Escobar, Ascención; Wilkins, Ashley; Juárez-Rodríguez, María D.; Circu, Magdalena; Latimer, Brian; Dragoi, Ana-Maria; Ivanov, Stanimir S.

doi:10.1038/s41467-024-52184-x

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Published: 08 September 2024

Iron-depleting nutritional immunity controls extracellular bacterial replication in Legionella pneumophila infections

Ascención Torres-Escobar¹,
Ashley Wilkins¹^nAff4,
María D. Juárez-Rodríguez²,
Magdalena Circu¹,
Brian Latimer³,
Ana-Maria Dragoi ORCID: orcid.org/0000-0001-9841-6802^2,3 &
…
Stanimir S. Ivanov ORCID: orcid.org/0000-0002-3294-7595¹

Nature Communications volume 15, Article number: 7848 (2024) Cite this article

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Abstract

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.

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Introduction

Legionella species are environmental Gram-negative γ-proteobacteria that parasitize and replicate within free-living unicellular eukaryotes^1,2. 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 treatment^4,5. Outbreaks from a single clinical isolate frequently manifest in both the mild and the severe disease forms^{6,7,8,9,10,11,12,13}, 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’ disease^4,5, however the mechanistic links remain unresolved.

In human infections and animal models of disease, alveolar macrophages are the primary cellular niche for bacterial replication^{14,15,16,17,18}. 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 replication^19,20,21,22. 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 cell^23,24. 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 models^26,27; thus, mutants that cannot grow intracellularly (such as T4SS− strains) are avirulent in both animal and tissue culture models of infection^28,29,30,31. 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 infection^{27,29,31,32,33,34}. 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 models^{35,36,37,38,39}. 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 pathogens^{41,42,43,44,45,46} where a microorganism cycles between two phenotypically distinct forms each with a unique gene expression profile^{47,48,49,50,51}. 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 replication^{6,40,52,53,54,55,56}. IFNγ-stimulated primary human alveolar and monocyte-derived macrophages limit Lp intracellular replication in vitro^52,53 through a variety of cell autonomous defense mechanisms in part impinging on LCV rupture and host cell death^{57,58,59,60,61,62}. IFNγ secretion is required for legionellosis resolution in animal models, emphasizing the importance of IFNγ effector functions in Lp pathogenesis^55,56. 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 immunity^64,65,66. 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 consumption^{68,69,70,71,72}. 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 protein^73,74. During inflammation FPN1 is downregulated, which favors intracellular iron accumulation and a decrease in extracellular iron bioavailability^69,75. 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 Lp^76,77. 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 approaches^79,80 (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 intracellularly^25,32. 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.**

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 infections^25,32.

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 complex^82,83, whereas the DotB is the energy-generating ATPase that facilitates translocation of effector proteins^84,85. 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.**

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 degradation^25,32 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 cultures^87,88. 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 membrane^88,90,91. 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.**

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 replication^36,39. 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.**

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 replication^92,93. 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 cysteine^94,95. 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.**

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 replication^96,97. Macrophages regulate extracellular iron levels, both locally and systemically, via dedicated uptake and secretion transport mechanisms^98,99,100. 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.**

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 replication^52,53,103. Primary human alveolar and monocyte-derived macrophages primed with IFNγ restrict Lp intracellular replication in vitro^52,53, 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-smokers^{104,105,106,107,108,109}—a phenomenon that is exacerbated with age¹¹⁰. Unlike iron, the amount of alveolar Transferrin remains unchanged in smokers^106,107,108 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 IRF1^111,112 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.**

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 notion^76,77, 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.**

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 Transferrin^113,114,115. 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’ disease^4,5. Notably, bronchoalveolar lavage (BAL) fluid samples from smokers as compared to non-smokers are highly enriched in iron, without a concomitant increase in Transferrin^{104,105,106,107,108,109}. 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 restored^76,77. However, this assertion is incongruent with more recent data showing IFN stimulation results in (i) death of infected macrophages due to LCV rupture^57,58,60 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 models^4,5,119. 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 administration^120,121,122. 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 disease^{6,7,8,9,10,11,12,13}. 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 P_tac 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 P_tac 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 dotB_Lj. 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 Salt^R, whereas T4SS+ strains were Salt^S. 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.

Data availability

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

References

Boamah, D. K., Zhou, G., Ensminger, A. W. & O’Connor, T. J. From Many Hosts, One Accidental Pathogen: The Diverse Protozoan Hosts of Legionella. Front. Cell Infect. Microbiol. 7, 477 (2017).
Article PubMed PubMed Central Google Scholar
Gomez-Valero, L. & Buchrieser, C. Genome dynamics in Legionella: the basis of versatility and adaptation to intracellular replication. Cold Spring Harb. Perspect. Med. 3, https://doi.org/10.1101/cshperspect.a009993 (2013).
Collier, S. A. et al. Estimate of Burden and Direct Healthcare Cost of Infectious Waterborne Disease in the United States. Emerg. Infect. Dis. 27, 140–149 (2021).
Article PubMed PubMed Central Google Scholar
Cunha, B. A., Burillo, A. & Bouza, E. Legionnaires’ disease. Lancet 387, 376–385 (2016).
Article PubMed Google Scholar
Phin, N. et al. Epidemiology and clinical management of Legionnaires’ disease. Lancet Infect. Dis. 14, 1011–1021 (2014).
Article PubMed Google Scholar
Ambrose, J. et al. Large outbreak of Legionnaires’ disease and Pontiac fever at a military base. Epidemiol. Infect. 142, 2336–2346 (2014).
Article CAS PubMed Google Scholar
Benin, A. L. et al. An outbreak of travel-associated Legionnaires disease and Pontiac fever: the need for enhanced surveillance of travel-associated legionellosis in the United States. J. Infect. Dis. 185, 237–243 (2002).
Article PubMed Google Scholar
Burnsed, L. J. et al. A large, travel-associated outbreak of legionellosis among hotel guests: utility of the urine antigen assay in confirming Pontiac fever. Clin. Infect. Dis. 44, 222–228 (2007).
Article PubMed Google Scholar
Euser, S. M., Pelgrim, M. & den Boer, J. W. Legionnaires’ disease and Pontiac fever after using a private outdoor whirlpool spa. Scand. J. Infect. Dis. 42, 910–916 (2010).
Article PubMed Google Scholar
Girod, J. C. et al. Pneumonic and nonpneumonic forms of legionellosis. The result of a common-source exposure to Legionella pneumophila. Arch. Intern. Med. 142, 545–547 (1982).
Article CAS PubMed Google Scholar
Hunt, D. A. et al. An outbreak of Legionnaires’ disease in Gloucester. Epidemiol. Infect. 107, 133–141 (1991).
Article CAS PubMed PubMed Central Google Scholar
Smith, S. S. et al. Legionellosis Outbreak Associated With a Hotel Fountain. Open Forum Infect. Dis. 2, ofv164 (2015).
Article PubMed PubMed Central Google Scholar
Thomas, D. L., Mundy, L. M. & Tucker, P. C. Hot tub legionellosis. Legionnaires’ disease and Pontiac fever after a point-source exposure to Legionella pneumophila. Arch. Intern. Med. 153, 2597–2599 (1993).
Article CAS PubMed Google Scholar
Baskerville, A., Dowsett, A. B., Fitzgeorge, R. B., Hambleton, P. & Broster, M. Ultrastructure of pulmonary alveoli and macrophages in experimental Legionnaires’ disease. J. Pathol. 140, 77–90 (1983).
Article CAS PubMed Google Scholar
Chandler, F. W., Blackmon, J. A., Hicklin, M. D., Cole, R. M. & Callaway, C. S. Ultrastructure of the agent of Legionnaires’ disease in the human lung. Am. J. Clin. Pathol. 71, 43–50 (1979).
Article CAS PubMed Google Scholar
Davis, G. S., Winn, W. C. Jr., Gump, D. W. & Beaty, H. N. The kinetics of early inflammatory events during experimental pneumonia due to Legionella pneumophila in guinea pigs. J. Infect. Dis. 148, 823–835 (1983).
Article CAS PubMed Google Scholar
Nash, T. W., Libby, D. M. & Horwitz, M. A. Interaction between the legionnaires’ disease bacterium (Legionella pneumophila) and human alveolar macrophages. Influence of antibody, lymphokines, and hydrocortisone. J. Clin. Invest. 74, 771–782 (1984).
Article CAS PubMed PubMed Central Google Scholar
Winn, W. C. Jr. & Myerowitz, R. L. The pathology of the Legionella pneumonias. A review of 74 cases and the literature. Hum. Pathol. 12, 401–422 (1981).
Article PubMed Google Scholar
Horwitz, M. A. Formation of a novel phagosome by the Legionnaires’ disease bacterium (Legionella pneumophila) in human monocytes. J. Exp. Med. 158, 1319–1331 (1983).
Article CAS PubMed Google Scholar
Ivanov, S. S. & Roy, C. R. Modulation of ubiquitin dynamics and suppression of DALIS formation by the Legionella pneumophila Dot/Icm system. Cell Microbiol. 11, 261–278 (2009).
Article CAS PubMed Google Scholar
Swanson, M. S. & Isberg, R. R. Association of Legionella pneumophila with the macrophage endoplasmic reticulum. Infect. Immun. 63, 3609–3620 (1995).
Article CAS PubMed PubMed Central Google Scholar
Tilney, L. G., Harb, O. S., Connelly, P. S., Robinson, C. G. & Roy, C. R. How the parasitic bacterium Legionella pneumophila modifies its phagosome and transforms it into rough ER: implications for conversion of plasma membrane to the ER membrane. J. Cell Sci. 114, 4637–4650 (2001).
Article CAS PubMed Google Scholar
Cornejo, E., Schlaermann, P. & Mukherjee, S. How to rewire the host cell: A home improvement guide for intracellular bacteria. J. Cell Biol. 216, 3931–3948 (2017).
Article CAS PubMed PubMed Central Google Scholar
Personnic, N., Barlocher, K., Finsel, I. & Hilbi, H. Subversion of Retrograde Trafficking by Translocated Pathogen Effectors. Trends Microbiol. 24, 450–462 (2016).
Article CAS PubMed Google Scholar
Roy, C. R., Berger, K. H. & Isberg, R. R. Legionella pneumophila DotA protein is required for early phagosome trafficking decisions that occur within minutes of bacterial uptake. Mol. Microbiol. 28, 663–674 (1998).
Article CAS PubMed Google Scholar
Elliott, J. A. & Winn, W. C. Jr. Treatment of alveolar macrophages with cytochalasin D inhibits uptake and subsequent growth of Legionella pneumophila. Infect. Immun. 51, 31–36 (1986).
Article CAS PubMed PubMed Central Google Scholar
Horwitz, M. A. & Silverstein, S. C. Legionnaires’ disease bacterium (Legionella pneumophila) multiples intracellularly in human monocytes. J. Clin. Invest. 66, 441–450 (1980).
Article CAS PubMed PubMed Central Google Scholar
Andrews, H. L., Vogel, J. P. & Isberg, R. R. Identification of linked Legionella pneumophila genes essential for intracellular growth and evasion of the endocytic pathway. Infect. Immun. 66, 950–958 (1998).
Article CAS PubMed PubMed Central Google Scholar
Berger, K. H. & Isberg, R. R. Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila. Mol. Microbiol. 7, 7–19 (1993).
Article CAS PubMed Google Scholar
Brand, B. C., Sadosky, A. B. & Shuman, H. A. The Legionella pneumophila icm locus: a set of genes required for intracellular multiplication in human macrophages. Mol. Microbiol. 14, 797–808 (1994).
Article CAS PubMed Google Scholar
Marra, A., Blander, S. J., Horwitz, M. A. & Shuman, H. A. Identification of a Legionella pneumophila locus required for intracellular multiplication in human macrophages. Proc. Natl Acad. Sci. USA 89, 9607–9611 (1992).
Article ADS CAS PubMed PubMed Central Google Scholar
Berger, K. H., Merriam, J. J. & Isberg, R. R. Altered intracellular targeting properties associated with mutations in the Legionella pneumophila dotA gene. Mol. Microbiol. 14, 809–822 (1994).
Article CAS PubMed Google Scholar
Segal, G. & Shuman, H. A. Legionella pneumophila utilizes the same genes to multiply within Acanthamoeba castellanii and human macrophages. Infect. Immun. 67, 2117–2124 (1999).
Article CAS PubMed PubMed Central Google Scholar
Solomon, J. M., Rupper, A., Cardelli, J. A. & Isberg, R. R. Intracellular growth of Legionella pneumophila in Dictyostelium discoideum, a system for genetic analysis of host-pathogen interactions. Infect. Immun. 68, 2939–2947 (2000).
Article CAS PubMed PubMed Central Google Scholar
Akinsulore, A., Owojuyigbe, A. M., Faponle, A. F. & Fatoye, F. O. Assessment of Preoperative and Postoperative Anxiety among Elective Major Surgery Patients in a Tertiary Hospital in Nigeria. Middle East J. Anaesthesiol. 23, 235–240 (2015).
PubMed Google Scholar
Eisenreich, W. & Heuner, K. The life stage-specific pathometabolism of Legionella pneumophila. FEBS Lett. 590, 3868–3886 (2016).
Article CAS PubMed Google Scholar
George, J. R., Pine, L., Reeves, M. W. & Harrell, W. K. Amino acid requirements of Legionella pneumophila. J. Clin. Microbiol. 11, 286–291 (1980).
Article CAS PubMed PubMed Central Google Scholar
Pine, L., George, J. R., Reeves, M. W. & Harrell, W. K. Development of a chemically defined liquid medium for growth of Legionella pneumophila. J. Clin. Microbiol. 9, 615–626 (1979).
Article CAS PubMed PubMed Central Google Scholar
Tesh, M. J. & Miller, R. D. Amino acid requirements for Legionella pneumophila growth. J. Clin. Microbiol. 13, 865–869 (1981).
Article CAS PubMed PubMed Central Google Scholar
Horwitz, M. A. Cell-mediated immunity in Legionnaires’ disease. J. Clin. Invest. 71, 1686–1697 (1983).
Article CAS PubMed PubMed Central Google Scholar
Coleman, S. A., Fischer, E. R., Howe, D., Mead, D. J. & Heinzen, R. A. Temporal analysis of Coxiella burnetii morphological differentiation. J. Bacteriol. 186, 7344–7352 (2004).
Article CAS PubMed PubMed Central Google Scholar
Elwell, C., Mirrashidi, K. & Engel, J. Chlamydia cell biology and pathogenesis. Nat. Rev. Microbiol. 14, 385–400 (2016).
Article CAS PubMed PubMed Central Google Scholar
Gitsels, A., Sanders, N. & Vanrompay, D. Chlamydial Infection From Outside to Inside. Front. Microbiol. 10, 2329 (2019).
Article PubMed PubMed Central Google Scholar
Heinzen, R. A., Hackstadt, T. & Samuel, J. E. Developmental biology of Coxiella burnettii. Trends Microbiol. 7, 149–154 (1999).
Article CAS PubMed Google Scholar
Troese, M. J. & Carlyon, J. A. Anaplasma phagocytophilum dense-cored organisms mediate cellular adherence through recognition of human P-selectin glycoprotein ligand 1. Infect. Immun. 77, 4018–4027 (2009).
Article CAS PubMed PubMed Central Google Scholar
Zhang, J. Z., Popov, V. L., Gao, S., Walker, D. H. & Yu, X. J. The developmental cycle of Ehrlichia chaffeensis in vertebrate cells. Cell Microbiol. 9, 610–618 (2007).
Article CAS PubMed Google Scholar
Bachman, M. A. & Swanson, M. S. Genetic evidence that Legionella pneumophila RpoS modulates expression of the transmission phenotype in both the exponential phase and the stationary phase. Infect. Immun. 72, 2468–2476 (2004).
Article CAS PubMed PubMed Central Google Scholar
Byrne, B. & Swanson, M. S. Expression of Legionella pneumophila virulence traits in response to growth conditions. Infect. Immun. 66, 3029–3034 (1998).
Article CAS PubMed PubMed Central Google Scholar
Garduno, R. A., Garduno, E., Hiltz, M. & Hoffman, P. S. Intracellular growth of Legionella pneumophila gives rise to a differentiated form dissimilar to stationary-phase forms. Infect. Immun. 70, 6273–6283 (2002).
Article CAS PubMed PubMed Central Google Scholar
Hammer, B. K., Tateda, E. S. & Swanson, M. S. A two-component regulator induces the transmission phenotype of stationary-phase Legionella pneumophila. Mol. Microbiol. 44, 107–118 (2002).
Article CAS PubMed Google Scholar
Molofsky, A. B. & Swanson, M. S. Differentiate to thrive: lessons from the Legionella pneumophila life cycle. Mol. Microbiol. 53, 29–40 (2004).
Article CAS PubMed Google Scholar
Bhardwaj, N., Nash, T. W. & Horwitz, M. A. Interferon-gamma-activated human monocytes inhibit the intracellular multiplication of Legionella pneumophila. J. Immunol. 137, 2662–2669 (1986).
Article CAS PubMed Google Scholar
Nash, T. W., Libby, D. M. & Horwitz, M. A. IFN-gamma-activated human alveolar macrophages inhibit the intracellular multiplication of Legionella pneumophila. J. Immunol. 140, 3978–3981 (1988).
Article CAS PubMed Google Scholar
Price, J. V. et al. IRG1 and Inducible Nitric Oxide Synthase Act Redundantly with Other Interferon-Gamma-Induced Factors To Restrict Intracellular Replication of Legionella pneumophila. mBio 10, https://doi.org/10.1128/mBio.02629-19 (2019).
Shinozawa, Y. et al. Role of interferon-gamma in inflammatory responses in murine respiratory infection with Legionella pneumophila. J. Med. Microbiol. 51, 225–230 (2002).
Article CAS PubMed Google Scholar
Sporri, R., Joller, N., Albers, U., Hilbi, H. & Oxenius, A. MyD88-dependent IFN-gamma production by NK cells is key for control of Legionella pneumophila infection. J. Immunol. 176, 6162–6171 (2006).
Article PubMed Google Scholar
Bass, A. R. et al. Human GBP1 facilitates the rupture of the Legionella-containing vacuole and inflammasome activation. mBio 14, e0170723 (2023).
Article PubMed Google Scholar
Feeley, E. M. et al. Galectin-3 directs antimicrobial guanylate binding proteins to vacuoles furnished with bacterial secretion systems. Proc. Natl Acad. Sci. USA 114, E1698–E1706 (2017).
Article CAS PubMed PubMed Central Google Scholar
Lippmann, J. et al. Dissection of a type I interferon pathway in controlling bacterial intracellular infection in mice. Cell Microbiol. 13, 1668–1682 (2011).
Article CAS PubMed PubMed Central Google Scholar
Liu, B. C. et al. Constitutive Interferon Maintains GBP Expression Required for Release of Bacterial Components Upstream of Pyroptosis and Anti-DNA Responses. Cell Rep. 24, 155–168.e155 (2018).
Article CAS PubMed PubMed Central Google Scholar
Naujoks, J. et al. IFNs Modify the Proteome of Legionella-Containing Vacuoles and Restrict Infection Via IRG1-Derived Itaconic Acid. PLoS Pathog. 12, e1005408 (2016).
Article PubMed PubMed Central Google Scholar
Pilla, D. M. et al. Guanylate binding proteins promote caspase-11-dependent pyroptosis in response to cytoplasmic LPS. Proc. Natl Acad. Sci. USA 111, 6046–6051 (2014).
Article ADS CAS PubMed PubMed Central Google Scholar
Lettinga, K. D. et al. Reduced interferon-gamma release in patients recovered from Legionnaires’ disease. Thorax 58, 63–67 (2003).
Article CAS PubMed PubMed Central Google Scholar
Murdoch, C. C. & Skaar, E. P. Nutritional immunity: the battle for nutrient metals at the host-pathogen interface. Nat. Rev. Microbiol. 20, 657–670 (2022).
Article CAS PubMed PubMed Central Google Scholar
Weinberg, E. D. Iron and susceptibility to infectious disease. Science 184, 952–956 (1974).
Article ADS CAS PubMed Google Scholar
Weinberg, E. D. Nutritional immunity. Host’s attempt to withold iron from microbial invaders. JAMA 231, 39–41 (1975).
Article CAS PubMed Google Scholar
Neves, J., Haider, T., Gassmann, M. & Muckenthaler, M. U. Iron Homeostasis in the Lungs-A Balance between Health and Disease. Pharmaceuticals 12, https://doi.org/10.3390/ph12010005 (2019)
Gangaidzo, I. T. et al. Association of pulmonary tuberculosis with increased dietary iron. J. Infect. Dis. 184, 936–939 (2001).
Article CAS PubMed Google Scholar
Ganz, T. Iron in innate immunity: starve the invaders. Curr. Opin. Immunol. 21, 63–67 (2009).
Article CAS PubMed PubMed Central Google Scholar
Khan, F. A., Fisher, M. A. & Khakoo, R. A. Association of hemochromatosis with infectious diseases: expanding spectrum. Int. J. Infect. Dis. 11, 482–487 (2007).
Article CAS PubMed Google Scholar
Sazawal, S. et al. Effects of routine prophylactic supplementation with iron and folic acid on admission to hospital and mortality in preschool children in a high malaria transmission setting: community-based, randomised, placebo-controlled trial. Lancet 367, 133–143 (2006).
Article CAS PubMed Google Scholar
Wander, K., Shell-Duncan, B. & Brindle, E. Lower incidence of respiratory infections among iron-deficient children in Kilimanjaro, Tanzania. Evol. Med. Public Health 2017, 109–119 (2017).
Article PubMed PubMed Central Google Scholar
Donovan, A. et al. Positional cloning of zebrafish ferroportin1 identifies a conserved vertebrate iron exporter. Nature 403, 776–781 (2000).
Article ADS CAS PubMed Google Scholar
Donovan, A. et al. The iron exporter ferroportin/Slc40a1 is essential for iron homeostasis. Cell Metab. 1, 191–200 (2005).
Article CAS PubMed Google Scholar
Cassat, J. E. & Skaar, E. P. Iron in infection and immunity. Cell Host Microbe 13, 509–519 (2013).
Article CAS PubMed PubMed Central Google Scholar
Byrd, T. F. & Horwitz, M. A. Interferon gamma-activated human monocytes downregulate transferrin receptors and inhibit the intracellular multiplication of Legionella pneumophila by limiting the availability of iron. J. Clin. Invest 83, 1457–1465 (1989).
Article CAS PubMed PubMed Central Google Scholar
Byrd, T. F. & Horwitz, M. A. Lactoferrin inhibits or promotes Legionella pneumophila intracellular multiplication in nonactivated and interferon gamma-activated human monocytes depending upon its degree of iron saturation. Iron-lactoferrin and nonphysiologic iron chelates reverse monocyte activation against Legionella pneumophila. J. Clin. Invest. 88, 1103–1112 (1991).
Article CAS PubMed PubMed Central Google Scholar
Ren, T., Zamboni, D. S., Roy, C. R., Dietrich, W. F. & Vance, R. E. Flagellin-deficient Legionella mutants evade caspase-1- and Naip5-mediated macrophage immunity. PLoS Pathog. 2, e18 (2006).
Article PubMed PubMed Central Google Scholar
Ondari, E., Wilkins, A., Latimer, B., Dragoi, A. M. & Ivanov, S. S. Cellular cholesterol licenses Legionella pneumophila intracellular replication in macrophages. Micro Cell 10, 1–17 (2023).
Article CAS Google Scholar
Wilkins, A. A. et al. The intracellular growth of the vacuolar pathogen Legionella pneumophila is dependent on the acyl chain composition of host membranes. Front. Bacteriol. 3, https://doi.org/10.3389/fbrio.2024.1322138 (2024)
Vogel, J. P., Roy, C. & Isberg, R. R. Use of salt to isolate Legionella pneumophila mutants unable to replicate in macrophages. Ann. N. Y. Acad. Sci. 797, 271–272 (1996).
Article ADS CAS PubMed Google Scholar
Kubori, T. et al. Native structure of a type IV secretion system core complex essential for Legionella pathogenesis. Proc. Natl Acad. Sci. USA 111, 11804–11809 (2014).
Article ADS CAS PubMed PubMed Central Google Scholar
Vincent, C. D. et al. Identification of the core transmembrane complex of the Legionella Dot/Icm type IV secretion system. Mol. Microbiol. 62, 1278–1291 (2006).
Article CAS PubMed Google Scholar
Chetrit, D., Hu, B., Christie, P. J., Roy, C. R. & Liu, J. A unique cytoplasmic ATPase complex defines the Legionella pneumophila type IV secretion channel. Nat. Microbiol. 3, 678–686 (2018).
Article CAS PubMed PubMed Central Google Scholar
Sexton, J. A. et al. The Legionella pneumophila PilT homologue DotB exhibits ATPase activity that is critical for intracellular growth. J. Bacteriol. 186, 1658–1666 (2004).
Article CAS PubMed PubMed Central Google Scholar
Izu, K. et al. Grouping of 20 reference strains of Legionella species by the growth ability within mouse and guinea pig macrophages. FEMS Immunol. Med. Microbiol. 26, 61–68 (1999).
Article CAS PubMed Google Scholar
Creasey, E. A. & Isberg, R. R. The protein SdhA maintains the integrity of the Legionella-containing vacuole. Proc. Natl Acad. Sci. USA 109, 3481–3486 (2012).
Article ADS CAS PubMed PubMed Central Google Scholar
Isaac, D. T., Laguna, R. K., Valtz, N. & Isberg, R. R. MavN is a Legionella pneumophila vacuole-associated protein required for efficient iron acquisition during intracellular growth. Proc. Natl Acad. Sci. USA 112, E5208–E5217 (2015).
Article ADS CAS PubMed PubMed Central Google Scholar
Choi, W. Y. et al. SdhA blocks disruption of the Legionella-containing vacuole by hijacking the OCRL phosphatase. Cell Rep. 37, 109894 (2021).
Article CAS PubMed PubMed Central Google Scholar
Abeyrathna, S. S. et al. IroT/MavN Is a Legionella Transmembrane Fe(II) Transporter: Metal Selectivity and Translocation Kinetics Revealed by in Vitro Real-Time Transport. Biochemistry 58, 4337–4342 (2019).
Article CAS PubMed Google Scholar
Christenson, E. T. et al. The iron-regulated vacuolar Legionella pneumophila MavN protein is a transition-metal transporter. Proc. Natl Acad. Sci. USA 116, 17775–17785 (2019).
Article ADS CAS PubMed PubMed Central Google Scholar
Ewann, F. & Hoffman, P. S. Cysteine metabolism in Legionella pneumophila: characterization of an L-cystine-utilizing mutant. Appl. Environ. Microbiol. 72, 3993–4000 (2006).
Article ADS CAS PubMed PubMed Central Google Scholar
Reeves, M. W., Pine, L., Hutner, S. H., George, J. R. & Harrell, W. K. Metal requirements of Legionella pneumophila. J. Clin. Microbiol. 13, 688–695 (1981).
Article CAS PubMed PubMed Central Google Scholar
Castellani, P., Angelini, G., Delfino, L., Matucci, A. & Rubartelli, A. The thiol redox state of lymphoid organs is modified by immunization: role of different immune cell populations. Eur. J. Immunol. 38, 2419–2425 (2008).
Article CAS PubMed Google Scholar
Gmunder, H., Eck, H. P., Benninghoff, B., Roth, S. & Droge, W. Macrophages regulate intracellular glutathione levels of lymphocytes. Evidence for an immunoregulatory role of cysteine. Cell Immunol. 129, 32–46 (1990).
Article CAS PubMed Google Scholar
Bullen, J. J., Rogers, H. J., Spalding, P. B. & Ward, C. G. Natural resistance, iron and infection: a challenge for clinical medicine. J. Med. Microbiol. 55, 251–258 (2006).
Article CAS PubMed Google Scholar
Hunter, R. L., Bennett, B., Towns, M. & Vogler, W. R. Transferrin in disease II: defects in the regulation of transferrin saturation with iron contribute to susceptibility to infection. Am. J. Clin. Pathol. 81, 748–753 (1984).
Article CAS PubMed Google Scholar
Ganz, T. & Nemeth, E. Iron homeostasis in host defence and inflammation. Nat. Rev. Immunol. 15, 500–510 (2015).
Article CAS PubMed PubMed Central Google Scholar
Nairz, M., Theurl, I., Swirski, F. K. & Weiss, G. Pumping iron”-how macrophages handle iron at the systemic, microenvironmental, and cellular levels. Pflug. Arch. 469, 397–418 (2017).
Article CAS Google Scholar
Winn, N. C., Volk, K. M. & Hasty, A. H. Regulation of tissue iron homeostasis: the macrophage “ferrostat”. JCI Insight 5, https://doi.org/10.1172/jci.insight.132964 (2020).
Parker, J. B. et al. Chelating the valley of death: Deferoxamine’s path from bench to wound clinic. Front. Med. 10, 1015711 (2023).
Article Google Scholar
Szabo, S., Barbu, Z., Lakatos, L., Laszlo, I. & Szabo, A. Local production of proteins in normal human bronchial secretion. Respiration 39, 172–178 (1980).
Article CAS PubMed Google Scholar
Horwitz, M. A. & Silverstein, S. C. Activated human monocytes inhibit the intracellular multiplication of Legionnaires’ disease bacteria. J. Exp. Med. 154, 1618–1635 (1981).
Article CAS PubMed PubMed Central Google Scholar
Corhay, J. L. et al. Iron content in human alveolar macrophages. Eur. Respir. J. 5, 804–809 (1992).
Article CAS PubMed Google Scholar
Ghio, A. J. et al. Particulate matter in cigarette smoke alters iron homeostasis to produce a biological effect. Am. J. Respir. Crit. Care Med. 178, 1130–1138 (2008).
Article CAS PubMed Google Scholar
McGowan, S. E. & Henley, S. A. Iron and ferritin contents and distribution in human alveolar macrophages. J. Lab. Clin. Med. 111, 611–617 (1988).
CAS PubMed Google Scholar
Thompson, A. B., Bohling, T., Heires, A., Linder, J. & Rennard, S. I. Lower respiratory tract iron burden is increased in association with cigarette smoking. J. Lab Clin. Med. 117, 493–499 (1991).
CAS PubMed Google Scholar
Wesselius, L. J., Flowers, C. H. & Skikne, B. S. Alveolar macrophage content of isoferritins and transferrin. Comparison of nonsmokers and smokers with and without chronic airflow obstruction. Am. Rev. Respir. Dis. 145, 311–316 (1992).
Article CAS PubMed Google Scholar
Wesselius, L. J., Nelson, M. E. & Skikne, B. S. Increased release of ferritin and iron by iron-loaded alveolar macrophages in cigarette smokers. Am. J. Respir. Crit. Care Med. 150, 690–695 (1994).
Article CAS PubMed Google Scholar
Ghio, A. J., Pritchard, R. J., Dittrich, K. L. & Samet, J. M. Non-heme (Fe3+) in the lung increases with age in both humans and rats. J. Lab. Clin. Med. 129, 53–61 (1997).
Article CAS PubMed Google Scholar
Carlin, J. M., Borden, E. C., Sondel, P. M. & Byrne, G. I. Interferon-induced indoleamine 2,3-dioxygenase activity in human mononuclear phagocytes. J. Leukoc. Biol. 45, 29–34 (1989).
Article CAS PubMed Google Scholar
Der, S. D., Zhou, A., Williams, B. R. & Silverman, R. H. Identification of genes differentially regulated by interferon alpha, beta, or gamma using oligonucleotide arrays. Proc. Natl Acad. Sci. USA 95, 15623–15628 (1998).
Article ADS CAS PubMed PubMed Central Google Scholar
Loosmore, S. M. et al. Cloning and expression of the Haemophilus influenzae transferrin receptor genes. Mol. Microbiol. 19, 575–586 (1996).
Article CAS PubMed Google Scholar
Modun, B., Kendall, D. & Williams, P. Staphylococci express a receptor for human transferrin: identification of a 42-kilodalton cell wall transferrin-binding protein. Infect. Immun. 62, 3850–3858 (1994).
Article CAS PubMed PubMed Central Google Scholar
Noinaj, N. et al. Structural basis for iron piracy by pathogenic Neisseria. Nature 483, 53–58 (2012).
Article ADS CAS PubMed PubMed Central Google Scholar
Vargas Buonfiglio, L. G. et al. Airway surface liquid from smokers promotes bacterial growth and biofilm formation via iron-lactoferrin imbalance. Respir. Res. 19, 42 (2018).
Article PubMed PubMed Central Google Scholar
Abreu, R., Essler, L., Giri, P. & Quinn, F. Interferon-gamma promotes iron export in human macrophages to limit intracellular bacterial replication. PLoS One 15, e0240949 (2020).
Article CAS PubMed PubMed Central Google Scholar
Temmerman, R., Vervaeren, H., Noseda, B., Boon, N. & Verstraete, W. Necrotrophic growth of Legionella pneumophila. Appl. Environ. Microbiol. 72, 4323–4328 (2006).
Article ADS CAS PubMed PubMed Central Google Scholar
Archer, K. A. & Roy, C. R. MyD88-dependent responses involving toll-like receptor 2 are important for protection and clearance of Legionella pneumophila in a mouse model of Legionnaires’ disease. Infect. Immun. 74, 3325–3333 (2006).
Article CAS PubMed PubMed Central Google Scholar
Chidiac, C. et al. Factors associated with hospital mortality in community-acquired legionellosis in France. Eur. Respir. J. 39, 963–970 (2012).
Article CAS PubMed Google Scholar
Kostic, A. et al. The Different Clinical Courses of Legionnaires’ Disease in Newborns from the Same Maternity Hospital. Medicina 58, https://doi.org/10.3390/medicina58091150 (2022)
Levy, M. L. et al. Primary care summary of the British Thoracic Society Guidelines for the management of community acquired pneumonia in adults: 2009 update. Endorsed by the Royal College of General Practitioners and the Primary Care Respiratory Society UK. Prim. Care Respir. J. 19, 21–27 (2010).
Article PubMed PubMed Central Google Scholar
Wee, B. A. et al. Population analysis of Legionella pneumophila reveals a basis for resistance to complement-mediated killing. Nat. Commun. 12, 7165 (2021).
Article ADS CAS PubMed PubMed Central Google Scholar
Feeley, J. C. et al. Charcoal-yeast extract agar: primary isolation medium for Legionella pneumophila. J. Clin. Microbiol 10, 437–441 (1979).
Article CAS PubMed PubMed Central Google Scholar
Wilkins, A. A. et al. The intracellular growth of the vacuolar pathogen Legionella pneumophila is dependent on the acyl chain composition of host membranes. bioRxiv. https://doi.org/10.1101/2023.11.19.567753, (2023).
Merriam, J. J., Mathur, R., Maxfield-Boumil, R. & Isberg, R. R. Analysis of the Legionella pneumophila fliI gene: intracellular growth of a defined mutant defective for flagellum biosynthesis. Infect. Immun. 65, 2497–2501 (1997).
Article CAS PubMed PubMed Central Google Scholar
Abshire, C. F., Dragoi, A. M., Roy, C. R. & Ivanov, S. S. MTOR-Driven Metabolic Reprogramming Regulates Legionella pneumophila Intracellular Niche Homeostasis. PLoS Pathog. 12, e1006088 (2016).
Article PubMed PubMed Central Google Scholar

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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.

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Ashley Wilkins
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

Authors and Affiliations

Department of Microbiology and Immunology, Louisiana State University Health Sciences Center—Shreveport, Shreveport, LA, 71130, USA
Ascención Torres-Escobar, Ashley Wilkins, Magdalena Circu & Stanimir S. Ivanov
Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center—Shreveport, Shreveport, LA, 71130, USA
María D. Juárez-Rodríguez & Ana-Maria Dragoi
Innovative North Louisiana Experimental Therapeutics program (INLET), Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center—Shreveport, Shreveport, LA, 71130, USA
Brian Latimer & Ana-Maria Dragoi

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Ascención Torres-Escobar
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Ashley Wilkins
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María D. Juárez-Rodríguez
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Magdalena Circu
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Brian Latimer
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Ana-Maria Dragoi
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Stanimir S. Ivanov
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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

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Correspondence to Stanimir S. Ivanov.

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Torres-Escobar, A., Wilkins, A., Juárez-Rodríguez, M.D. et al. Iron-depleting nutritional immunity controls extracellular bacterial replication in Legionella pneumophila infections. Nat Commun 15, 7848 (2024). https://doi.org/10.1038/s41467-024-52184-x

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Received: 02 February 2024
Accepted: 29 August 2024
Published: 08 September 2024
DOI: https://doi.org/10.1038/s41467-024-52184-x

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