Neisseria gonorrhoeae Evades Calprotectin-Mediated Nutritional Immunity and Survives Neutrophil Extracellular Traps by Production of TdfH

Neisseria gonorrhoeaesuccessfully overcomes host strategies to limit essential nutrients, termed nutritional immunity, by production of TonB-dependent transporters (TdTs)—outer membrane proteins that facilitate nutrient transport in an energy-dependent manner. Four gonococcal TdTs facilitate utilization of iron or iron chelates from host-derived proteins, including transferrin (TbpA), lactoferrin (LbpA), and hemoglobin (HpuB), in addition to xenosiderophores from other bacteria (FetA). The roles of the remaining four uncharacterized TdTs (TdfF, TdfG, TdfH, and TdfJ) remain elusive. Regulatory data demonstrating that production of gonococcal TdfH and TdfJ are unresponsive to or upregulated under iron-replete conditions led us to evaluate the role of these TdTs in the acquisition of nutrients other than iron. In this study, we found that production of gonococcal TdfH is both Zn and Zur repressed. We also found that TdfH confers resistance to calprotectin, an immune effector protein highly produced in neutrophils that has antimicrobial activity due to its ability to sequester Zn and Mn. We found that TdfH directly binds calprotectin, which enables gonococcal Zn accumulation in a TdfH-dependent manner and enhances bacterial survival after exposure to neutrophil extracellular traps (NETs). These studies highlight Zn sequestration by calprotectin as a key functional arm of NET-mediated killing of gonococci. We demonstrate for the first time thatN. gonorrhoeaeexploits this host strategy in a novel defense mechanism, in which TdfH production hijacks and directly utilizes the host protein calprotectin as a zinc source and thereby evades nutritional immunity.

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Neutrophil Extracellular Traps Exhibit Antibacterial Activity against Burkholderia pseudomallei and Are Influenced by Bacterial and Host Factors

Infection and Immunity ◽

10.1128/iai.00806-12 ◽

2012 ◽

Vol 80 (11) ◽

pp. 3921-3929 ◽

Cited By ~ 51

Author(s):

Donporn Riyapa ◽

Surachat Buddhisa ◽

Sunee Korbsrisate ◽

Jon Cuccui ◽

Brendan W. Wren ◽

...

Keyword(s):

Burkholderia Pseudomallei ◽

Capsular Polysaccharide ◽

Neutrophil Extracellular Traps ◽

Polymorphonuclear Neutrophils ◽

Dependent Manner ◽

Predisposing Factor ◽

Bacterial Killing ◽

Content Type ◽

Extracellular Traps ◽

Type Iii Protein Secretion

ABSTRACTBurkholderia pseudomalleiis the causative pathogen of melioidosis, of which a major predisposing factor is diabetes mellitus. Polymorphonuclear neutrophils (PMNs) kill microbes extracellularly by the release of neutrophil extracellular traps (NETs). PMNs play a key role in the control of melioidosis, but the involvement of NETs in killing ofB. pseudomalleiremains obscure. Here, we showed that bactericidal NETs were released from human PMNs in response toB. pseudomalleiin a dose- and time-dependent manner.B. pseudomallei-induced NET formation required NADPH oxidase activation but not phosphatidylinositol-3 kinase, mitogen-activated protein kinases, or Src family kinase signaling pathways.B. pseudomalleimutants defective in the virulence-associated Bsa type III protein secretion system (T3SS) or capsular polysaccharide I (CPS-I) induced elevated levels of NETs. NET induction by such mutants was associated with increased bacterial killing, phagocytosis, and oxidative burst by PMNs. Taken together the data imply that T3SS and the capsule may play a role in evading the induction of NETs. Importantly, PMNs from diabetic subjects released NETs at a lower level than PMNs from healthy subjects. Modulation of NET formation may therefore be associated with the pathogenesis and control of melioidosis.

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3′-Nucleotidase/Nuclease Activity Allows Leishmania Parasites To Escape Killing by Neutrophil Extracellular Traps

Infection and Immunity ◽

10.1128/iai.01232-13 ◽

2014 ◽

Vol 82 (4) ◽

pp. 1732-1740 ◽

Cited By ~ 66

Author(s):

Anderson B. Guimarães-Costa ◽

Thiago S. DeSouza-Vieira ◽

Rafael Paletta-Silva ◽

Anita Leocádio Freitas-Mesquita ◽

José Roberto Meyer-Fernandes ◽

...

Keyword(s):

Leishmania Infantum ◽

Nuclease Activity ◽

Neutrophil Extracellular Traps ◽

Human Neutrophils ◽

Dependent Manner ◽

Content Type ◽

Extracellular Traps ◽

Leishmania Parasites ◽

High Phosphate

ABSTRACTLeishmaniasis is a widespread neglected tropical disease caused by parasites of theLeishmaniagenus. These parasites express the enzyme 3′-nucleotidase/nuclease (3′NT/NU), which has been described to be involved in parasite nutrition and infection. Bacteria that express nucleases escape the toxic effects of neutrophil extracellular traps (NETs). Hence, we investigated the role of 3′NT/NU inLeishmaniasurvival of NET-mediated killing. Promastigotes ofLeishmania infantumwere cultured in high-phosphate (HP) or low-phosphate (LP) medium to modulate nuclease activity. We compared the survival of the two different groups ofLeishmaniaduring interaction with human neutrophils, assessing the role of neutrophil extracellular traps. As previously reported, we detected higher nuclease activity in parasites cultured in LP medium. Both LP and HP promastigotes were capable of inducing the release of neutrophil extracellular traps from human neutrophils in a dose- and time-dependent manner. LP parasites had 2.4 times more survival than HP promastigotes. NET disruption was prevented by the treatment of the parasites with ammonium tetrathiomolybdate (TTM), a 3′NT/NU inhibitor. Inhibition of 3′NT/NU by 3′-AMP, 5′-GMP, or TTM decreased promastigote survival upon interaction with neutrophils. Our results show thatLeishmania infantuminduces NET release and that promastigotes can escape NET-mediated killing by 3′-nucleotidase/nuclease activity, thus ascribing a new function to this enzyme.

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Secreted Phosphatase and Deoxyribonuclease Are Required by Pseudomonas aeruginosa To Defend against Neutrophil Extracellular Traps

Infection and Immunity ◽

10.1128/iai.00403-18 ◽

2018 ◽

Vol 86 (9) ◽

Cited By ~ 8

Author(s):

Mike Wilton ◽

Tyler W. R. Halverson ◽

Laetitia Charron-Mazenod ◽

Michael D. Parkins ◽

Shawn Lewenza

Keyword(s):

Pseudomonas Aeruginosa ◽

Antimicrobial Activity ◽

Alkaline Phosphatase ◽

Defense Mechanism ◽

Neutrophil Extracellular Traps ◽

Immune Defense ◽

Extracellular Dna ◽

Protective Function ◽

Content Type ◽

Extracellular Traps

ABSTRACT Neutrophil extracellular traps (NETs) are produced by neutrophils as an innate immune defense mechanism to trap and kill microbial pathogens. NETs are comprised of ejected chromatin that forms a lattice structure enmeshed with numerous antimicrobial proteins. In addition to forming the structural backbone of NETs, extracellular DNA (eDNA) has membrane-disrupting antimicrobial activity that contributes to NET killing. Many pathogens produce secreted extracellular DNases to evade the antimicrobial activity of NETs. Pseudomonas aeruginosa encodes an operon of two secreted enzymes, a predicted alkaline phosphatase and a DNase. The DNase (eddB) degrades eDNA to use as a nutrient source. Here we report that both eDNA and NETs are potent inducers of this DNase-phosphatase operon. Furthermore, the secreted DNase contributes to degrading NET DNA and defends P. aeruginosa against NET-mediated killing. We demonstrate that EddA has both alkaline phosphatase and phosphodiesterase (PDase) activities and also protects against the antimicrobial activity of NETs. Although the phosphatase does not cause DNA degradation similar to that of the DNase, its protective function is likely a result of removing the cation-chelating phosphates from the eDNA phosphodiester backbone. Therefore, both the DNase and PDase contribute to defense against NET killing of P. aeruginosa, highlighting the role of DNA-manipulating enzymes in targeting the eDNA in neutrophil extracellular traps.

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Neisseria gonorrhoeae Elicits Extracellular Traps in Primary Neutrophil Culture While Suppressing the Oxidative Burst

mBio ◽

10.1128/mbio.02452-14 ◽

2015 ◽

Vol 6 (1) ◽

Cited By ~ 14

Author(s):

Carl W. Gunderson ◽

H. Steven Seifert

Keyword(s):

Neisseria Gonorrhoeae ◽

Oxidative Burst ◽

Bacterial Species ◽

Immune Surveillance ◽

Large Body ◽

Surface Antigens ◽

Neutrophil Extracellular Traps ◽

Content Type ◽

Extracellular Traps ◽

Variable Surface

ABSTRACT Neisseria gonorrhoeae(the gonococcus) causes gonorrhea and is uniquely adapted to survive within the human reproductive tract. Gonococci evade host immune surveillance in part by varying their pili and opacity-associated proteins. These variable surface antigens influence interactions with host epithelial and immune cells. A potent polymorphonuclear leukocyte (PMN) response is a hallmark of symptomatic gonococcal infection, with vast numbers of PMNs recruited to the site of infection. A large body of literature describes gonococcus-PMN interactions, but the factors driving the outcome of infection are not fully understood. Gonococci have been described to both induce and suppress the PMN oxidative burst, but we determined that gonococci differentially affect induction of the PMN oxidative burst depending on the multiplicity of infection (MOI). Infecting PMN at an MOI of <20 gonococci elicits an oxidative burst, while an MOI of >20 suppresses the burst. Oxidative burst in response to gonococci is enhanced by, but does not require, expression of pili or opacity proteins. Neutrophil extracellular traps (NETs) were observed in gonococcus-infected PMNs, a process which requires an oxidative burst, yet gonococci induced NETs under suppressing conditions. The NETs were unable to kill gonococci despite killing the common vaginal bacteriumLactobacillus crispatus. Thus, gonococci influence PMN biology to promote their own survival by suppressing the oxidative burst of PMNs and stimulating the formation of NETs, which do not effectively kill gonococci, illustrating how N. gonorrhoeae has evolved to modulate PMN responses to promote infection.IMPORTANCE Neisseria gonorrhoeae, the gonococcus, is the only causative agent of gonorrhea and is exclusively found within the human host. Gonococci stochastically vary the composition of antigens on their surface to evade immune surveillance. We used gonococcal mutants which stably express different surface antigens to dissect interactions between gonococci and primary human polymorphonuclear leukocytes (PMNs). We found that gonococci, depending on the number of bacteria present, either induce or suppress the oxidative burst of PMNs regardless of other stimuli. Gonococci also cause PMNs to release DNA, forming neutrophil extracellular traps (NETs) independently of the oxidative burst. The NETs were unable to kill gonococci but were able to kill commensal bacteria, suggesting that NET production can help gonococci outcompete other bacterial species. We propose that gonococci have evolved to manipulate PMN responses to promote their own survival during infection.

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Immunomodulatory Role of Clarithromycin in Acinetobacter baumannii Infection via Formation of Neutrophil Extracellular Traps

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02063-15 ◽

2015 ◽

Vol 60 (2) ◽

pp. 1040-1048 ◽

Cited By ~ 26

Author(s):

Theocharis Konstantinidis ◽

Konstantinos Kambas ◽

Alexandros Mitsios ◽

Maria Panopoulou ◽

Victoria Tsironidou ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Ex Vivo ◽

Neutrophil Extracellular Traps ◽

Macrolide Antibiotics ◽

Dependent Manner ◽

Biofilm Inhibition ◽

Content Type ◽

Extracellular Traps

ABSTRACTMacrolide antibiotics have been shown to act as immunomodulatory molecules in various immune cells. However, their effect on neutrophils has not been extensively investigated. In this study, we investigated the role of macrolide antibiotics in the generation of neutrophil extracellular traps (NETs). By assessingex vivoandin vivoNET formation, we demonstrated that clarithromycin is able to induce NET generation bothin vitroandin vivo. Clarithromycin utilizes autophagy in order to form NETs, and these NETs are decorated with antimicrobial peptide LL-37. Clarithromycin-induced NETs are able to inhibitAcinetobacter baumanniigrowth and biofilm formation in an LL-37-dependent manner. Additionally, LL-37 antimicrobial function depends on NET scaffold integrity. Collectively, these data expand the knowledge on the immunomodulatory role of macrolide antibiotics via the generation of LL-37-bearing NETs, which demonstrate LL-37-dependent antimicrobial activity and biofilm inhibition againstA. baumannii.

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Molecular Insight into TdfH-Mediated Zinc Piracy from Human Calprotectin by Neisseria gonorrhoeae

mBio ◽

10.1128/mbio.00949-20 ◽

2020 ◽

Vol 11 (3) ◽

Cited By ~ 1

Author(s):

Michael T. Kammerman ◽

Aloke Bera ◽

Runrun Wu ◽

Simone A. Harrison ◽

C. Noel Maxwell ◽

...

Keyword(s):

Neisseria Gonorrhoeae ◽

Binding Sites ◽

Building Blocks ◽

Titration Calorimetry ◽

Site Preference ◽

Dependent Manner ◽

Bacterial Survival ◽

Transmitted Infection ◽

Content Type ◽

High Affinity

ABSTRACT Neisseria gonorrhoeae, responsible for the sexually transmitted infection gonorrhea, is an obligate human pathogen exquisitely adapted for survival on mucosal surfaces of humans. This host-pathogen relationship has resulted in evolution by N. gonorrhoeae of pathways that enable the use of host metalloproteins as required nutrients through the deployment of outer membrane-bound TonB-dependent transporters (TdTs). Recently, a TdT called TdfH was implicated in binding to calprotectin (CP) and in removal of the bound zinc (Zn), enabling gonococcal growth. TdfH is highly conserved among the pathogenic Neisseria species, making it a potentially promising candidate for inclusion into a gonococcal vaccine. Currently, the nature and specificity of the TdfH-CP interaction have not been determined. In this study, we found that TdfH specifically interacted with human calprotectin (hCP) and that growth of the gonococcus was supported in a TdfH-dependent manner only when hCP was available as a sole zinc source and not when mouse CP was provided. The binding interactions between TdfH and hCP were assessed using isothermal titration calorimetry where we observed a multistate model having both high-affinity and low-affinity sites of interaction. hCP has two Zn binding sites, and gonococcal growth assays using hCP mutants deficient in one or both of the Zn binding sites revealed that TdfH exhibited a site preference during Zn piracy and utilization. This report provides the first insights into the molecular mechanism of Zn piracy by neisserial TdfH and further highlights the obligate human nature of N. gonorrhoeae and the high-affinity interactions occurring between TdTs and their human ligands during pathogenesis. IMPORTANCE The dramatic rise in antimicrobial resistance among Neisseria gonorrhoeae isolates over the last few decades, paired with dwindling treatment options and the lack of a protective vaccine, has prompted increased interest in identifying new bacterial targets for the treatment and, ideally, prevention of gonococcal disease. TonB-dependent transporters are a conserved set of proteins that serve crucial functions for bacterial survival within the host. In this study, binding between the gonococcal transporter, TdfH, and calprotectin was determined to be of high affinity and host restricted. The current study identified a preferential TdfH interaction at the calprotectin dimer interface. An antigonococcal therapeutic could potentially block this site on calprotectin, interrupting Zn uptake by N. gonorrhoeae and thereby prohibiting continued bacterial growth. We describe protein-protein interactions between TdfH and calprotectin, and our findings provide the building blocks for future therapeutic or prophylactic targets.

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Loss of Outer Membrane Protein C in Escherichia coli Contributes to Both Antibiotic Resistance and Escaping Antibody-Dependent Bactericidal Activity

Infection and Immunity ◽

10.1128/iai.06395-11 ◽

2012 ◽

Vol 80 (5) ◽

pp. 1815-1822 ◽

Cited By ~ 52

Author(s):

Yi-Fang Liu ◽

Jing-Jou Yan ◽

Huan-Yao Lei ◽

Ching-Hao Teng ◽

Ming-Cheng Wang ◽

...

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Outer Membrane Proteins ◽

Bactericidal Effect ◽

Bacterial Virulence ◽

Classical Pathway ◽

Dependent Manner ◽

Bacterial Survival ◽

Content Type ◽

E Coli

ABSTRACTOuter membrane proteins (OMPs) serve as the permeability channels for nutrients, toxins, and antibiotics. InEscherichia coli, OmpA has been shown to be involved in bacterial virulence, and OmpC is related to multidrug resistance. However, it is unclear whether OmpC also has a role in the virulence ofE. coli. The aims of this study were to characterize the role of OmpC in antimicrobial resistance and bacterial virulence inE. coli. TheompCdeletion mutant showed significantly decreased susceptibility to carbapenems and cefepime. To investigate the survival ofE. coliexposed to the innate immune system, a human blood bactericidal assay showed that theompCmutant increased survival in blood and serum but not in complement-inactivated serum. These effects were also demonstrated in the natural selection of OmpC mutants. Also, C1q interacted withE. colithrough a complex of antibodies bound to OmpC as a major target. Bacterial survival was increased in the wild-type strain in a dose-dependent manner by adding free recombinant OmpC protein or anti-C1q antibody to human serum. These results demonstrated that the interaction of OmpC-specific antibody and C1q was the key step in initiating the antibody-dependent classical pathway for the clearance of OmpC-expressingE. coli. Anti-OmpC antibody was detected in human sera, indicating that OmpC is an immunogen. These data indicate that the loss of OmpC inE. coliis resistant to not only antibiotics, but also the serum bactericidal effect, which is mediated from the C1q and anti-OmpC antibody-dependent classical pathway.

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Disruption of Glycolysis by Nutritional Immunity Activates a Two-Component System That Coordinates a Metabolic and Antihost Response by Staphylococcus aureus

mBio ◽

10.1128/mbio.01321-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 4

Author(s):

Paola K. Párraga Solórzano ◽

Jiangwei Yao ◽

Charles O. Rock ◽

Thomas E. Kehl-Fie

Keyword(s):

Staphylococcus Aureus ◽

Metabolic Flux ◽

Bacterial Species ◽

Critical Role ◽

Dependent Manner ◽

Two Component System ◽

Component System ◽

Content Type ◽

Nutritional Immunity ◽

Two Component

ABSTRACT During infection, bacteria use two-component signal transduction systems to sense and adapt to the dynamic host environment. Despite critically contributing to infection, the activating signals of most of these regulators remain unknown. This also applies to the Staphylococcus aureus ArlRS two-component system, which contributes to virulence by coordinating the production of toxins, adhesins, and a metabolic response that enables the bacterium to overcome host-imposed manganese starvation. Restricting the availability of essential transition metals, a strategy known as nutritional immunity, constitutes a critical defense against infection. In this work, expression analysis revealed that manganese starvation imposed by the immune effector calprotectin or by the absence of glycolytic substrates activates ArlRS. Manganese starvation imposed by calprotectin also activated the ArlRS system even when glycolytic substrates were present. A combination of metabolomics, mutational analysis, and metabolic feeding experiments revealed that ArlRS is activated by alterations in metabolic flux occurring in the latter half of the glycolytic pathway. Moreover, calprotectin was found to induce expression of staphylococcal leukocidins in an ArlRS-dependent manner. These studies indicated that ArlRS is a metabolic sensor that allows S. aureus to integrate multiple environmental stresses that alter glycolytic flux to coordinate an antihost response and to adapt to manganese starvation. They also established that the latter half of glycolysis represents a checkpoint to monitor metabolic state in S. aureus. Altogether, these findings contribute to understanding how invading pathogens, such as S. aureus, adapt to the host during infection and suggest the existence of similar mechanisms in other bacterial species. IMPORTANCE Two-component regulatory systems enable bacteria to adapt to changes in their environment during infection by altering gene expression and coordinating antihost responses. Despite the critical role of two-component systems in bacterial survival and pathogenesis, the activating signals for most of these regulators remain unidentified. This is exemplified by ArlRS, a Staphylococcus aureus global regulator that contributes to virulence and to resisting host-mediated restriction of essential nutrients, such as manganese. In this report, we demonstrate that manganese starvation and the absence of glycolytic substrates activate ArlRS. Further investigations revealed that ArlRS is activated when the latter half of glycolysis is disrupted, suggesting that S. aureus monitors flux through the second half of this pathway. Host-imposed manganese starvation also induced the expression of pore-forming toxins in an ArlRS-dependent manner. Cumulatively, this work reveals that ArlRS acts as a sensor that links nutritional status, cellular metabolism, and virulence regulation.

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Ovine vital neutrophil extracellular traps bind and impairHaemonchus contortusL3 in a breed-dependent manner

Parasite Immunology ◽

10.1111/pim.12572 ◽

2018 ◽

Vol 40 (9) ◽

pp. e12572 ◽

Cited By ~ 3

Author(s):

Javier J. Garza ◽

Scott P. Greiner ◽

Scott A. Bowdridge

Keyword(s):

Neutrophil Extracellular Traps ◽

Dependent Manner ◽

Extracellular Traps

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ZupT Facilitates Clostridioides difficile Resistance to Host-Mediated Nutritional Immunity

mSphere ◽

10.1128/msphere.00061-20 ◽

2020 ◽

Vol 5 (2) ◽

Cited By ~ 3

Author(s):

Joseph P. Zackular ◽

Reece J. Knippel ◽

Christopher A. Lopez ◽

William N. Beavers ◽

C. Noel Maxwell ◽

...

Keyword(s):

Immune Response ◽

Therapeutic Potential ◽

Neutrophil Infiltration ◽

Toxin Production ◽

Host Protein ◽

Content Type ◽

Nutritional Immunity ◽

Clostridioides Difficile ◽

Therapeutic Development ◽

Dependent Growth

ABSTRACT Clostridioides difficile is a spore-forming bacterium that causes severe colitis and is a major public health threat. During infection, C. difficile toxin production results in damage to the epithelium and a hyperinflammatory response. The immune response to CDI leads to robust neutrophil infiltration at the sight of infection and the deployment of numerous antimicrobials. One of the most abundant host immune factors associated with CDI is calprotectin, a metal-chelating protein with potent antimicrobial activity. Calprotectin is essential to the innate immune response to C. difficile and increasing levels of calprotectin correlate with disease severity in both adults and children with CDI. The fact that C. difficile persists in the presence of high levels of calprotectin suggests that this organism may deploy strategies to compete with this potent antimicrobial factor for essential nutrient metals during infection. In this report, we demonstrate that a putative zinc (Zn) transporter, ZupT, is employed by C. difficile to survive calprotectin-mediated metal limitation. ZupT is highly expressed in the presence of calprotectin and is required to protect C. difficile against calprotectin-dependent growth inhibition. When competing against wild-type C. difficile, zupT mutants show a defect in colonization and persistence in a murine model of infection. Together these data demonstrate that C. difficile utilizes a metal import system to combat nutritional immunity during CDI and suggest that strategies targeting nutrient acquisition in C. difficile may have therapeutic potential. IMPORTANCE During infection, pathogenic organisms must acquire essential transition metals from the host environment. Through the process of nutritional immunity, the host employs numerous strategies to restrict these key nutrients from invading pathogens. In this study, we describe a mechanism by which the important human pathogen Clostridioides difficile resists transition-metal limitation by the host. We report that C. difficile utilizes a zinc transporter, ZupT, to compete with the host protein calprotectin for nutrient zinc. Inactivation of this transporter in C. difficile renders this important pathogen sensitive to host-mediated metal restriction and confers a fitness disadvantage during infection. Our study demonstrates that targeting nutrient metal transport proteins in C. difficile is a potential avenue for therapeutic development.

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