scholarly journals Sequence Elements Upstream of the Core Promoter Are Necessary for Full Transcription of the Capsule Gene Operon in Streptococcus pneumoniae Strain D39

2015 ◽  
Vol 83 (5) ◽  
pp. 1957-1972 ◽  
Author(s):  
Zhensong Wen ◽  
Odeniel Sertil ◽  
Yongxin Cheng ◽  
Shanshan Zhang ◽  
Xue Liu ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Capsular Polysaccharide ◽  
Core Promoter ◽  
Virulent Strain ◽  
Bacterial Pathogen ◽  
Content Type ◽  
The Core ◽  
Sequence Elements ◽  
Almost All

Streptococcus pneumoniaeis a major bacterial pathogen in humans. Its polysaccharide capsule is a key virulence factor that promotes bacterial evasion of human phagocytic killing. WhileS. pneumoniaeproduces at least 94 antigenically different types of capsule, the genes for biosynthesis of almost all capsular types are arranged in the same locus. The transcription of the capsular polysaccharide (cps) locus is not well understood. This study determined the transcriptional features of thecpslocus in the type 2 virulent strain D39. The initial analysis revealed that thecpsgenes are cotranscribed from a major transcription start site at the −25 nucleotide (G) upstream ofcps2A, the first gene in the locus. Using unmarked chromosomal truncations and a luciferase-based transcriptional reporter, we showed that the full transcription of thecpsgenes not only depends on the core promoter immediately upstream ofcps2A, but also requires additional elements upstream of the core promoter, particularly a 59-bp sequence immediately upstream of the core promoter. Unmarked deletions of these promoter elements in the D39 genome also led to significant reduction in CPS production and virulence in mice. Lastly, commoncpsgene (cps2ABCD) mutants did not show significant abnormality incpstranscription, although they produced significantly less CPS, indicating that the CpsABCD proteins are involved in the encapsulation ofS. pneumoniaein a posttranscriptional manner. This study has yielded important information on the transcriptional characteristics of thecpslocus inS. pneumoniae.

scholarly journals The Core Promoter of the Capsule Operon of Streptococcus pneumoniae Is Necessary for Colonization and Invasive Disease

2013 ◽  
Vol 82 (2) ◽  
pp. 694-705 ◽  
Author(s):  
Mara G. Shainheit ◽  
Matthew Mulé ◽  
Andrew Camilli
Keyword(s):  
Transcriptional Regulation ◽  
Streptococcus Pneumoniae ◽  
Capsular Polysaccharide ◽  
Core Promoter ◽  
Invasive Disease ◽  
Fine Tuning ◽  
Anatomical Site ◽  
Content Type ◽  
The Core

ABSTRACTStreptococcus pneumoniaeis a commensal of the human nasopharynx but can cause invasive diseases, including otitis media, pneumonia, sepsis, and meningitis. The capsular polysaccharide (capsule) is a critical virulence factor required for both asymptomatic colonization and invasive disease, yet the expression level is different in each anatomical site. During colonization, reduced levels of capsule promote binding to the host epithelium and biofilm formation, while during systemic infection, increased capsule is required to evade opsonophagocytosis. How this regulation of capsule expression occurs is incompletely understood. To investigate the contribution of transcriptional regulation on capsule level in the serotype 4 strain TIGR4, we constructed two mutants harboring a constitutive promoter that was either comparably weaker (Pcat) or stronger (PtRNAGlu) than the wild-type (WT) capsule promoter, Pcps. Mild reductions incpsAandcpsEtranscript levels in the Pcatpromoter mutant resulted in a 2-fold reduction in total amounts of capsule and in avirulence in murine models of lung and blood infection. Additionally, the PtRNAGlumutant revealed that, despite expressing enhanced levels ofcpsAandcpsEand possessing levels of capsule comparable to those of WT TIGR4, it was still significantly attenuated in all testedin vivoniches. Further analysis using chimeric promoter mutants revealed that the WT −10 and −35 boxes are required for optimal nasopharyngeal colonization and virulence. These data support the hypothesis that dynamic transcriptional regulation of the capsule operon is required and that the core promoter region plays a central role in fine-tuning levels of capsule to promote colonization and invasive disease.

scholarly journals Infection with Conditionally Virulent Streptococcus pneumoniae ΔpabStrains Induces Antibody to Conserved Protein Antigens but Does Not Protect against Systemic Infection with Heterologous Strains

2011 ◽  
Vol 79 (12) ◽  
pp. 4965-4976 ◽  
Author(s):  
Suneeta Chimalapati ◽  
Jonathan Cohen ◽  
Emilie Camberlein ◽  
Claire Durmort ◽  
Helen Baxendale ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Capsular Polysaccharide ◽  
Bacterial Pathogen ◽  
Systemic Infection ◽  
Complete Medium ◽  
Protein Antigens ◽  
Live Attenuated Vaccine ◽  
Content Type ◽  
Immunological Responses ◽  
Heterologous Challenge

ABSTRACTAvirulent strains of a bacterial pathogen could be useful tools for investigating immunological responses to infection and potentially effective vaccines. We have therefore constructed an auxotrophic TIGR4 Δpabstrain ofStreptococcus pneumoniaeby deleting thepabBgene Sp_0665. The TIGR4 Δpabstrain grew well in complete medium but was unable to grow in serum unless it was supplemented withpara-aminobenzoic acid (PABA). The TIGR4 Δpabstrain was markedly attenuated in virulence in mouse models ofS. pneumoniaenasopharyngeal colonization, pneumonia, and sepsis. Supplementing mouse drinking water with PABA largely restored the virulence of TIGR4 Δpab. An additional Δpabstrain constructed in the D39 capsular serotype 2 background was also avirulent in a sepsis model. Systemic inoculation of mice with TIGR4 Δpabinduced antibody responses toS. pneumoniaeprotein antigens, including PpmA, PsaA, pneumolysin, and CbpD, but not capsular polysaccharide. Flow cytometry demonstrated that IgG in sera from TIGR4 Δpab-vaccinated mice bound to the surface of TIGR4 and D39 bacteria but not to a capsular serotype 3 strain, strain 0100993. Mice vaccinated with the TIGR4 Δpabor D39 Δpabstrain by intraperitoneal inoculation were protected from developing septicemia when challenged with the homologousS. pneumoniaestrain. Vaccination with the TIGR4 Δpabstrain provided only weak or no protection against heterologous challenge with the D39 or 0100993 strain but did strongly protect against a TIGR4 capsular-switch strain expressing a serotype 2 capsule. The failure of cross-protection after systemic vaccination with Δpabbacteria suggests that parenteral administration of a live attenuated vaccine is not an attractive approach for preventingS. pneumoniaeinfection.

scholarly journals Antibodies to Streptococcus pneumoniae Capsular Polysaccharide Enhance Pneumococcal Quorum Sensing

mBio ◽  
2011 ◽  
Vol 2 (5) ◽  
Author(s):  
Masahide Yano ◽  
Shruti Gohil ◽  
J. Robert Coleman ◽  
Catherine Manix ◽  
Liise-anne Pirofski
Keyword(s):  
Monoclonal Antibodies ◽  
Streptococcus Pneumoniae ◽  
Quorum Sensing ◽  
Direct Effect ◽  
Pneumococcal Disease ◽  
Effector Cell ◽  
Capsular Polysaccharide ◽  
Genetic Exchange ◽  
Content Type ◽  
Specific Antibodies

ABSTRACTThe use of pneumococcal capsular polysaccharide (PPS)-based vaccines has resulted in a substantial reduction in invasive pneumococcal disease. However, much remains to be learned about vaccine-mediated immunity, as seven-valent PPS-protein conjugate vaccine use in children has been associated with nonvaccine serotype replacement and 23-valent vaccine use in adults has not prevented pneumococcal pneumonia. In this report, we demonstrate that certain PPS-specific monoclonal antibodies (MAbs) enhance the transformation frequency of two differentStreptococcus pneumoniaeserotypes. This phenomenon was mediated by PPS-specific MAbs that agglutinate but do not promote opsonic effector cell killing of the homologous serotypeinvitro. Compared to the autoinducer, competence-stimulating peptide (CSP) alone, transcriptional profiling of pneumococcal gene expression after incubation with CSP and one such MAb to the PPS of serotype 3 revealed changes in the expression of competence (com)-related and bacteriocin-like peptide (blp) genes involved in pneumococcal quorum sensing. This MAb was also found to induce a nearly 2-fold increase in CSP2-mediated bacterial killing or fratricide. These observations reveal a novel, direct effect of PPS-binding MAbs on pneumococcal biology that has important implications for antibody immunity to pneumococcus in the pneumococcal vaccine era. Taken together, our data suggest heretofore unsuspected mechanisms by which PPS-specific antibodies could affect genetic exchange and bacterial viability in the absence of host cells.IMPORTANCECurrent thought holds that pneumococcal capsular polysaccharide (PPS)-binding antibodies protect against pneumococcus by inducing effector cell opsonic killing of the homologous serotype. While such antibodies are an important part of how pneumococcal vaccines protect against pneumococcal disease, PPS-specific antibodies that do not exhibit this activity but are highly protective against pneumococcus in mice have been identified. This article examines the effect of nonopsonic PPS-specific monoclonal antibodies (MAbs) on the biology ofStreptococcus pneumoniae. The results showed that in the presence of a competence-stimulating peptide (CSP), such MAbs increase the frequency of pneumococcal transformation. Further studies with one such MAb showed that it altered the expression of genes involved in quorum sensing and increased competence-induced killing or fratricide. These findings reveal a novel, previously unsuspected mechanism by which certain PPS-specific antibodies exert a direct effect on pneumococcal biology that has broad implications for bacterial clearance, genetic exchange, and antibody immunity to pneumococcus.

scholarly journals Identification of Pneumococcal Factors Affecting Pneumococcal Shedding Shows that thedltLocus Promotes Inflammation and Transmission

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
M. Ammar Zafar ◽  
Alexandria J. Hammond ◽  
Shigeto Hamaguchi ◽  
Weisheng Wu ◽  
Masamitsu Kono ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Respiratory Tract ◽  
Capsular Polysaccharide ◽  
Inflammatory Responses ◽  
Transposon Mutagenesis ◽  
Respiratory Pathogen ◽  
Invasive Infection ◽  
Upper Respiratory Tract ◽  
Content Type ◽  
Upper Respiratory

ABSTRACTHost-to-host transmission is a necessary but poorly understood aspect of microbial pathogenesis. Herein, we screened a genomic library of mutants of the leading respiratory pathogenStreptococcus pneumoniaegenerated by mariner transposon mutagenesis (Tn-Seq) to identify genes contributing to its exit or shedding from the upper respiratory tract (URT), the limiting step in the organism’s transmission in an infant mouse model. Our analysis focused on genes affecting the bacterial surface that directly impact interactions with the host. Among the multiple factors identified was thedltlocus, which addsd-alanine onto lipoteichoic acids (LTA) and thereby increases Toll-like receptor 2-mediated inflammation and resistance to antimicrobial peptides. The more robust proinflammatory response in the presence ofd-alanylation promotes secretions that facilitate pneumococcal shedding and allows for transmission. Expression of thedltlocus is controlled by the CiaRH system, which senses cell wall stress in response to antimicrobial activity, including in response to lysozyme, the most abundant antimicrobial along the URT mucosa. Accordingly, in alysM−/−host, there was no longer an effect of thedltlocus on pneumococcal shedding. Thus, our findings demonstrate how a pathogen senses the URT milieu and then modifies its surface characteristics to take advantage of the host response for transit to another host.IMPORTANCEStreptococcus pneumoniae(the pneumococcus) is a common cause of respiratory tract and invasive infection. The overall effectiveness of immunization with the organism’s capsular polysaccharide depends on its ability to block colonization of the upper respiratory tract and thereby prevent host-to-host transmission. Because of the limited coverage of current pneumococcal vaccines, we carried out an unbiasedin vivotransposon mutagenesis screen to identify pneumococcal factors other than its capsular polysaccharide that affect transmission. One such candidate was expressed by thedltlocus, previously shown to addd-alanine onto the pneumococcal lipoteichoic acid present on the bacterial cell surface. This modification protects against host antimicrobials and augments host inflammatory responses. The latter increases secretions and bacterial shedding from the upper respiratory tract to allow for transmission. Thus, this study provides insight into a mechanism employed by the pneumococcus to successfully transit from one host to another.

scholarly journals Two DHH Subfamily 1 Proteins Contribute to Pneumococcal Virulence and Confer Protection against Pneumococcal Disease

2011 ◽  
Vol 79 (9) ◽  
pp. 3697-3710 ◽  
Author(s):  
L. E. Cron ◽  
K. Stol ◽  
P. Burghout ◽  
S. van Selm ◽  
E. R. Simonetti ◽  
...  
Keyword(s):  
Otitis Media ◽  
Pneumococcal Disease ◽  
Capsular Polysaccharide ◽  
Pneumococcal Infection ◽  
Bacterial Pathogen ◽  
Protein Vaccine ◽  
Double Knockout ◽  
Content Type ◽  
Knockout Mutants ◽  
Different Strains

ABSTRACTStreptococcus pneumoniaeis an important human bacterial pathogen, causing such infections as pneumonia, meningitis, septicemia, and otitis media. Current capsular polysaccharide-based conjugate vaccines protect against a fraction of the over 90 serotypes known, whereas vaccines based on conserved pneumococcal proteins are considered promising broad-range alternatives. The pneumococcal genome encodes two conserved proteins of an as yet unknown function, SP1298 and SP2205, classified as DHH (Asp-His-His) subfamily 1 proteins. Here we examined their contribution to pneumococcal pathogenesis using single and double knockout mutants in three different strains: D39, TIGR4, and BHN100. Mutants lacking both SP1298 and SP2205 were severely impaired in adherence to human epithelial Detroit 562 cells. Importantly, the attenuated phenotypes were restored upon genetic complementation of the deleted genes. Single and mixed mouse models of colonization, otitis media, pneumonia, and bacteremia showed that bacterial loads in the nasopharynx, middle ears, lungs, and blood of mice infected with the mutants were significantly reduced from those of wild-type-infected mice, with an apparent additive effect upon deletion of both genes. Minor strain-specific phenotypes were observed, i.e., deletion of SP1298 affected host-cell adherence in BHN100 only, and deletion of SP2205 significantly attenuated virulence in lungs and blood in D39 and BHN100 but not TIGR4. Finally, subcutaneous vaccination with a combination of both DHH subfamily 1 proteins conferred protection to nasopharynx, lungs, and blood of mice infected with TIGR4. We conclude that SP1298 and SP2205 play a significant role at several stages of pneumococcal infection, and importantly, these proteins are potential candidates for a multicomponent protein vaccine.

Structural studies of the capsular polysaccharide from streptococcus pneumoniae type 2, a reinvestigation

1988 ◽  
Vol 182 (1) ◽  
pp. 111-117 ◽  
Author(s):  
Per-Erik Jansson ◽  
Bengt Lindberg ◽  
Mats Andersson ◽  
Ulf Lindquist ◽  
Jørgen Henrichsen
Keyword(s):  
Streptococcus Pneumoniae ◽  
Capsular Polysaccharide ◽  
Structural Studies

scholarly journals Streptococcus pneumoniae DNA Initiates Type I Interferon Signaling in the Respiratory Tract

mBio ◽  
2011 ◽  
Vol 2 (3) ◽  
Author(s):  
Dane Parker ◽  
Francis J. Martin ◽  
Grace Soong ◽  
Bryan S. Harfenist ◽  
Jorge L. Aguilar ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Type I Interferon ◽  
Bacterial Pathogen ◽  
Central Component ◽  
Type I ◽  
Respiratory Pathogens ◽  
Airway Epithelial ◽  
Type I Ifn ◽  
Mucosal Epithelium ◽  
Content Type

ABSTRACTThe mucosal epithelium is the initial target for respiratory pathogens of all types. While type I interferon (IFN) signaling is traditionally associated with antiviral immunity, we demonstrate that the extracellular bacterial pathogenStreptococcus pneumoniaeactivates the type I IFN cascade in airway epithelial and dendritic cells. This response is dependent upon the pore-forming toxin pneumolysin. Pneumococcal DNA activates IFN-β expression through a DAI/STING/TBK1/IRF3 cascade.Tlr4−/−,Myd88−/−,Trif−/−, andNod2−/−mutant mice had no impairment of type I IFN signaling. Induction of type I IFN signaling contributes to the eradication of pneumococcal carriage, as IFN-α/β receptor null mice had significantly increased nasal colonization withS. pneumoniaecompared with that of wild-type mice. These studies suggest that the type I IFN cascade is a central component of the mucosal response to airway bacterial pathogens and is responsive to bacterial pathogen-associated molecular patterns that are capable of accessing intracellular receptors.IMPORTANCEThe bacteriumStreptococcus pneumoniaeis a leading cause of bacterial pneumonia, leading to upwards of one million deaths a year worldwide and significant economic burden. Although it is known that antibody is critical for efficient phagocytosis, it is not known how this pathogen is sensed by the mucosal epithelium. We demonstrate that this extracellular pathogen activates mucosal signaling typically activated by viral pathogens via the pneumolysin pore to activate intracellular receptors and the type I interferon (IFN) cascade. Mice lacking the receptor to type I IFNs have a reduced ability to clearS. pneumoniae, suggesting that the type I IFN cascade is central to the mucosal clearance of this important pathogen.

scholarly journals Monoclonal antibodies against protease-sensitive pneumococcal antigens can protect mice from fatal infection with Streptococcus pneumoniae.

1984 ◽  
Vol 160 (2) ◽  
pp. 386-397 ◽  
Author(s):  
L S McDaniel ◽  
G Scott ◽  
J F Kearney ◽  
D E Briles
Keyword(s):  
Cell Wall ◽  
Monoclonal Antibodies ◽  
Streptococcus Pneumoniae ◽  
Capsular Polysaccharide ◽  
Minimum Amount ◽  
Binding Studies ◽  
Fatal Infection ◽  
Rough Strain ◽  
Type 3

Monoclonal antibodies were raised against surface determinants of Streptococcus pneumoniae by hyperimmunizing X-linked immunodeficient (xid) CBA/N mice with the heat-killed rough strain R36A. 17 hybridomas produced antibody that bound intact R36A and did not cross-react with phosphocholine, an antigen common in the cell wall of all S. pneumoniae. The antibody produced by at least two of these hybridomas, Xi64 (IgM) and Xi126 (IgG2b), could protect mice from a lethal intravenous challenge of type 3 S. pneumoniae strains WU2 and A66 and of the type 2 strain D39. The minimum amount of antibody required to protect xid mice from 100 WU2 was 4.5 micrograms/mouse for Xi64 and 2.6 micrograms/mouse for Xi126,. Free phosphocholine, C-polysaccharide, and type 3 capsular polysaccharide all failed to inhibit the binding of Xi64 or Xi126 to R36A. These antibodies appeared to bind surface polypeptides, since treatment of R36A with either pepsin or trypsin, or of R36A lysate with trypsin, effectively eliminated the ability of Xi64 and Xi126 to bind antigens in these preparations. Binding studies indicated that these two antibodies recognized different epitopes that were expressed on several but not all serotypes of pneumococci.

scholarly journals Novel Role for the Streptococcus pneumoniae Toxin Pneumolysin in the Assembly of Biofilms

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Joshua R. Shak ◽  
Herbert P. Ludewick ◽  
Kristen E. Howery ◽  
Fuminori Sakai ◽  
Hong Yi ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Hemolytic Activity ◽  
Biofilm Formation ◽  
Early Time ◽  
Wild Type ◽  
Knockout Mutant ◽  
Content Type ◽  
Biofilm Development ◽  
Almost All

ABSTRACTStreptococcus pneumoniaeis an important commensal and pathogen responsible for almost a million deaths annually in children under five. The formation of biofilms byS. pneumoniaeis important in nasopharyngeal colonization, pneumonia, and otitis media. Pneumolysin (Ply) is a toxin that contributes significantly to the virulence ofS. pneumoniaeand is an important candidate as a serotype-independent vaccine target. Having previously demonstrated that aluxSknockout mutant was unable to form early biofilms and expressed lessplymRNA than the wild type, we conducted a study to investigate the role of Ply in biofilm formation. We found that Ply was expressed in early phases of biofilm development and localized to cellular aggregates as early as 4 h postinoculation.S. pneumoniae plyknockout mutants in D39 and TIGR4 backgrounds produced significantly less biofilm biomass than wild-type strains at early time points, both on polystyrene and on human respiratory epithelial cells, cultured under static or continuous-flow conditions. Ply’s role in biofilm formation appears to be independent of its hemolytic activity, asS. pneumoniaeserotype 1 strains, which produce a nonhemolytic variant of Ply, were still able to form biofilms. Transmission electron microscopy of biofilms grown on A549 lung cells using immunogold demonstrated that Ply was located both on the surfaces of pneumococcal cells and in the extracellular biofilm matrix. Altogether, our studies demonstrate a novel role for pneumolysin in the assembly ofS. pneumoniaebiofilms that is likely important during both carriage and disease and therefore significant for pneumolysin-targeting vaccines under development.IMPORTANCEThe bacteriumStreptococcus pneumoniae(commonly known as the pneumococcus) is commonly carried in the human nasopharynx and can spread to other body sites to cause disease. In the nasopharynx, middle ear, and lungs, the pneumococcus forms multicellular surface-associated structures called biofilms. Pneumolysin is an important toxin produced by almost allS. pneumoniaestrains, extensively studied for its ability to cause damage to human tissue. In this paper, we demonstrate that pneumolysin has a previously unrecognized role in biofilm formation by showing that strains without pneumolysin are unable to form the same amount of biofilm on plastic and human cell substrates. Furthermore, we show that the role of pneumolysin in biofilm formation is separate from the hemolytic activity responsible for tissue damage during pneumococcal diseases. This novel role for pneumolysin suggests that pneumococcal vaccines directed against this protein should be investigated for their potential impact on biofilms formed during carriage and disease.

scholarly journals Conjugation of PspA4Pro with Capsular Streptococcus pneumoniae Polysaccharide Serotype 14 Does Not Reduce the Induction of Cross-Reactive Antibodies

2017 ◽  
Vol 24 (8) ◽  
Author(s):  
Míriam A. da Silva ◽  
Thiago R. Converso ◽  
Viviane M. Gonçalves ◽  
Luciana C. C. Leite ◽  
Martha M. Tanizaki ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Capsular Polysaccharide ◽  
Vaccine Coverage ◽  
Protein A ◽  
Surface Protein ◽  
Cross Reactivity ◽  
Carrier Protein ◽  
Content Type ◽  
Clade 1 ◽  
The Impact

ABSTRACT Current pneumococcal vaccines are composed of bacterial polysaccharides as antigens, plain or conjugated to carrier proteins. While efficacious against vaccine serotypes, epidemiologic data show an increasing incidence of infections caused by nonvaccine serotypes of Streptococcus pneumoniae. The use of pneumococcal surface protein A (PspA) as a carrier protein in a conjugate vaccine could help prevent serotype replacement by increasing vaccine coverage and reducing selective pressure of S. pneumoniae serotypes. PspA is present in all pneumococcal strains, is highly immunogenic, and is known to induce protective antibodies. Based on its sequence, PspA has been classified into three families and six clades. A PspA fragment derived from family 2, clade 4 (PspA4Pro), was shown to generate antibodies with a broad range of cross-reactivity, across clades and families. Here, PspA4Pro was modified and conjugated to capsular polysaccharide serotype 14 (PS14). We investigated the impact of conjugation on the immune response induced to PspA4Pro and PS14. Mice immunized with the PS14-mPspA4Pro conjugate produced higher titers of anti-PS14 antibodies than the animals that received coadministered antigens. The conjugate induced antibodies with opsonophagocytic activity against PS14-carrying strains, as well as against a panel of strains bearing PspAs from five clades (encompassing families 1 and 2) bearing a non-PS14 serotype. Furthermore, mice immunized with PS14-mPspA4Pro were protected against nasal colonization with a nonrelated S. pneumoniae strain bearing PspA from clade 1, serotype 6B. These results demonstrate that the cross-reactivity mediated by PspA4Pro is retained following conjugation, supporting the use of PspA4 as a carrier protein in order to enhance pneumococcal vaccine coverage and encourage its further investigation as a candidate in future vaccine designs.

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