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

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.

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A Cross-Reactive Protein Vaccine Combined with PCV-13 Prevents Streptococcus pneumoniae- and Haemophilus influenzae-Mediated Acute Otitis Media

Infection and Immunity ◽

10.1128/iai.00253-19 ◽

2019 ◽

Vol 87 (10) ◽

Cited By ~ 2

Author(s):

Hannah M. Rowe ◽

Beth Mann ◽

Amy Iverson ◽

Aaron Poole ◽

Elaine Tuomanen ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Haemophilus Influenzae ◽

Otitis Media ◽

Capsular Polysaccharide ◽

Vaccine Coverage ◽

Acute Otitis Media ◽

Protein Vaccine ◽

Content Type ◽

Fusion Construct ◽

Reactive Protein

ABSTRACT Acute otitis media is one of the most common childhood infections worldwide. Currently licensed vaccines against the common otopathogen Streptococcus pneumoniae target the bacterial capsular polysaccharide and confer no protection against nonencapsulated strains or capsular types outside vaccine coverage. Mucosal infections such as acute otitis media remain prevalent, even those caused by vaccine-covered serotypes. Here, we report that a protein-based vaccine, a fusion construct of epitopes of CbpA to pneumolysin toxoid, confers effective protection against pneumococcal acute otitis media for non-PCV-13 serotypes and enhances protection for PCV-13 serotypes when coadministered with PCV-13. Having cross-reactive epitopes, the fusion protein also induces potent antibody responses against nontypeable Haemophilus influenzae and S. pneumoniae, engendering protection against acute otitis media caused by emerging unencapsulated otopathogens. These data suggest that augmenting capsule-based vaccination with conserved, cross-reactive protein-based vaccines broadens and enhances protection against acute otitis media.

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Antibodies to Streptococcus pneumoniae Capsular Polysaccharide Enhance Pneumococcal Quorum Sensing

mBio ◽

10.1128/mbio.00176-11 ◽

2011 ◽

Vol 2 (5) ◽

Cited By ~ 22

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.

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Influenza-Induced Inflammation Drives Pneumococcal Otitis Media

Infection and Immunity ◽

10.1128/iai.01278-12 ◽

2013 ◽

Vol 81 (3) ◽

pp. 645-652 ◽

Cited By ~ 43

Author(s):

Kirsty R. Short ◽

Patrick C. Reading ◽

Lorena E. Brown ◽

John Pedersen ◽

Brad Gilbertson ◽

...

Keyword(s):

Otitis Media ◽

Middle Ear ◽

Influenza A ◽

Pneumococcal Disease ◽

Influenza Viruses ◽

Dependent Manner ◽

Proinflammatory Response ◽

Content Type ◽

Infant Mouse ◽

Human Middle Ear

ABSTRACTInfluenza A virus (IAV) predisposes individuals to secondary infections with the bacteriumStreptococcus pneumoniae(the pneumococcus). Infections may manifest as pneumonia, sepsis, meningitis, or otitis media (OM). It remains controversial as to whether secondary pneumococcal disease is due to the induction of an aberrant immune response or IAV-induced immunosuppression. Moreover, as the majority of studies have been performed in the context of pneumococcal pneumonia, it remains unclear how far these findings can be extrapolated to other pneumococcal disease phenotypes such as OM. Here, we used an infant mouse model, human middle ear epithelial cells, and a series of reverse-engineered influenza viruses to investigate how IAV promotes bacterial OM. Our data suggest that the influenza virus HA facilitates disease by inducing a proinflammatory response in the middle ear cavity in a replication-dependent manner. Importantly, our findings suggest that it is the inflammatory response to IAV infection that mediates pneumococcal replication. This study thus provides the first evidence that inflammation drives pneumococcal replication in the middle ear cavity, which may have important implications for the treatment of pneumococcal OM.

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

Infection and Immunity ◽

10.1128/iai.05923-11 ◽

2011 ◽

Vol 79 (12) ◽

pp. 4965-4976 ◽

Cited By ~ 22

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.

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Cigarette Smoke Attenuates the Nasal Host Response to Streptococcus pneumoniae and Predisposes to Invasive Pneumococcal Disease in Mice

Infection and Immunity ◽

10.1128/iai.01504-15 ◽

2016 ◽

Vol 84 (5) ◽

pp. 1536-1547 ◽

Cited By ~ 14

Author(s):

Pamela Shen ◽

Mathieu C. Morissette ◽

Gilles Vanderstocken ◽

Yang Gao ◽

Muhammad Hassan ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Cigarette Smoke ◽

Invasive Pneumococcal Disease ◽

Bacterial Infections ◽

Pneumococcal Disease ◽

Pneumococcal Infection ◽

Cigarette Smoke Exposure ◽

Upper Respiratory Tract ◽

Content Type ◽

Pneumococcal Colonization

Streptococcus pneumoniaeis a leading cause of invasive bacterial infections, with nasal colonization an important first step in disease. While cigarette smoking is a strong risk factor for invasive pneumococcal disease, the underlying mechanisms remain unknown. This is partly due to a lack of clinically relevant animal models investigating nasal pneumococcal colonization in the context of cigarette smoke exposure. We present a model of nasal pneumococcal colonization in cigarette smoke-exposed mice and document, for the first time, that cigarette smoke predisposes to invasive pneumococcal infection and mortality in an animal model. Cigarette smoke increased the risk of bacteremia and meningitis without prior lung infection. Mechanistically, deficiency in interleukin 1α (IL-1α) or platelet-activating factor receptor (PAFR), an important host receptor thought to bind and facilitate pneumococcal invasiveness, did not rescue cigarette smoke-exposed mice from invasive pneumococcal disease. Importantly, we observed cigarette smoke to attenuate nasal inflammatory mediator expression, particularly that of neutrophil-recruiting chemokines, normally elicited by pneumococcal colonization. Smoking cessation during nasal pneumococcal colonization rescued nasal neutrophil recruitment and prevented invasive disease in mice. We propose that cigarette smoke predisposes to invasive pneumococcal disease by suppressing inflammatory processes of the upper respiratory tract. Given that smoking prevalence remains high worldwide, these findings are relevant to the continued efforts to reduce the invasive pneumococcal disease burden.

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

Infection and Immunity ◽

10.1128/iai.02944-14 ◽

2015 ◽

Vol 83 (5) ◽

pp. 1957-1972 ◽

Cited By ~ 15

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.

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Streptococcus pneumoniae Is Desiccation Tolerant and Infectious upon Rehydration

mBio ◽

10.1128/mbio.00092-11 ◽

2011 ◽

Vol 2 (3) ◽

Cited By ~ 36

Author(s):

Rebecca L. Walsh ◽

Andrew Camilli

Keyword(s):

Streptococcus Pneumoniae ◽

Otitis Media ◽

Desiccation Tolerance ◽

Close Contact ◽

Pneumococcal Infection ◽

The United States ◽

Upper Respiratory Tract ◽

Term Survival ◽

Content Type

ABSTRACTStreptococcus pneumoniae(pneumococcus) is a frequent colonizer of the nasopharynx and one of the leading causative agents of otitis media, pneumonia, and meningitis. The current literature asserts thatS. pneumoniaeis transmitted person to person via respiratory droplets; however, environmental surfaces (fomites) have been linked to the spread of other respiratory pathogens. Desiccation tolerance has been to shown to be essential for long-term survival on dry surfaces. This study investigated the survival and infectivity ofS. pneumoniaefollowing desiccation under ambient conditions. We recovered viable bacteria after all desiccation periods tested, ranging from 1 h to 4 weeks. Experiments conducted under nutrient limitation indicate that desiccation is a condition separate from starvation. Desiccation of an acapsular mutant and 15 different clinical isolates shows thatS. pneumoniaedesiccation tolerance is independent of the polysaccharide capsule and is a species-wide phenomenon, respectively. Experiments demonstrating that nondesiccated and desiccatedS. pneumoniaestrains colonize the nasopharynx at comparable levels, combined with their ability to survive long-term desiccation, suggest that fomites may serve as alternate sources of pneumococcal infection.IMPORTANCEEven with the advent of multivalent capsular polysaccharide conjugate vaccines,S. pneumoniaecontinues to be a major cause of morbidity and mortality worldwide. Every year, there are approximately 7 million cases of pneumococcus-based otitis media in the United States alone, while pneumococcal invasive diseases are responsible for more than 1 million deaths globally. It is believed that the human upper respiratory tract is the sole niche ofS. pneumoniaeand, thus, that spread occurs via close contact with an infected individual. In this study, we characterized the desiccation tolerance ofS. pneumoniaeand found that it can survive for many weeks postdehydration and retain infectivity. Our results suggest that desiccation tolerance is an inherent trait of this genetically variable species and that fomites may be a source of transmission.

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Development of a Markerless Knockout Method for Actinobacillus succinogenes

Applied and Environmental Microbiology ◽

10.1128/aem.00492-14 ◽

2014 ◽

Vol 80 (10) ◽

pp. 3053-3061 ◽

Cited By ~ 11

Author(s):

Rajasi V. Joshi ◽

Bryan D. Schindler ◽

Nikolas R. McPherson ◽

Kanupriya Tiwari ◽

Claire Vieille

Keyword(s):

High Efficiency ◽

Direct Selection ◽

Selection Marker ◽

Actinobacillus Succinogenes ◽

Knockout Mutant ◽

Double Knockout ◽

Pyruvate Formate Lyase ◽

Content Type ◽

Knockout Mutants ◽

Encoding Genes

ABSTRACTActinobacillus succinogenesis one of the best natural succinate-producing organisms, but it still needs engineering to further increase succinate yield and productivity. In this study, we developed a markerless knockout method forA. succinogenesusing natural transformation or electroporation. TheEscherichia coliisocitrate dehydrogenase gene with flanking flippase recognition target sites was used as the positive selection marker, making use ofA. succinogenes's auxotrophy for glutamate to select for growth on isocitrate. TheSaccharomyces cerevisiaeflippase recombinase (Flp) was used to remove the selection marker, allowing its reuse. Finally, the plasmid expressingflpwas cured using acridine orange. We demonstrate that at least two consecutive deletions can be introduced into the same strain using this approach, that no more than a total of 1 kb of DNA is needed on each side of the selection cassette to protect from exonuclease activity during transformation, and that no more than 200 bp of homologous DNA is needed on each side for efficient recombination. We also demonstrate that electroporation can be used as an alternative transformation method to obtain knockout mutants and that an enriched defined medium can be used for direct selection of knockout mutants on agar plates with high efficiency. Single-knockout mutants of the fumarate reductase and of the pyruvate formate lyase-encoding genes were obtained using this knockout strategy. Double-knockout mutants were also obtained by deleting the citrate lyase-, β-galactosidase-, and aconitase-encoding genes in the pyruvate formate lyase knockout mutant strain.

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Discovery of Novel Pneumococcal Serotype 35D, a Natural WciG-Deficient Variant of Serotype 35B

Journal of Clinical Microbiology ◽

10.1128/jcm.00054-17 ◽

2017 ◽

Vol 55 (5) ◽

pp. 1416-1425 ◽

Cited By ~ 36

Author(s):

K. Aaron Geno ◽

Jamil S. Saad ◽

Moon H. Nahm

Keyword(s):

Pneumococcal Disease ◽

Genetic Basis ◽

Capsular Polysaccharide ◽

Vaccine Candidate ◽

Capsular Polysaccharides ◽

Conjugate Vaccines ◽

Content Type ◽

Magnetic Resonance Studies ◽

Pneumococcal Serotype ◽

Inactivating Mutations

ABSTRACTPneumococcus (Streptococcus pneumoniae) remains a significant cause of morbidity and mortality, especially among those at the extremes of age. Its capsular polysaccharide is essential for systemic virulence. Over 90 serologically distinct pneumococcal capsular polysaccharides (serotypes) are recognized, but they are unequal in prevalence. Because antibodies against the capsule are protective, polysaccharide conjugate vaccines, which are constructed against the most prevalent serotypes, have caused great reductions in pneumococcal disease caused by these serotypes. In response, however, the relative prevalences of serotypes have shifted. Certain previously rare serotypes, such as serotype 35B, are increasing in prevalence. Serotype 35B is thus a likely future vaccine candidate, but due to their previous rarity, serotype 35B strains have not been scrutinized for underlying heterogeneity. We studied putative serotype 35B clinical isolates to assess the uniformity of their serological reactions. While most isolates exhibited the accepted serology of serotype 35B, one isolate failed to bind to critical serotyping reagents. We determined that the genetic basis for this aberrant serology was the presence of inactivating mutations in theO-acetyltransferase genewciG. Complementation studies in awciGdeletion strain verified that the mutant WciG was nonfunctional, and the serology of the mutant could be restored through complementation with a construct encoding a functional WciG. Nuclear magnetic resonance studies confirmed that the capsule of the WciG-deficient isolate lackedO-acetylation but was otherwise identical to serotype 35B. As this isolate expresses a unique serology with unique biochemistry and a stable genetic basis, we named its novel capsule serotype 35D.

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Liposomal encapsulation of polysaccharides (LEPS) as an effective vaccine strategy to protect aged hosts against S. pneumoniae infection

10.1101/2021.10.18.464850 ◽

2021 ◽

Author(s):

Manmeet Bhalla ◽

Roozbeh Nayerhoda ◽

Essi Y. I Tchalla ◽

Alexsandra Abamonte ◽

Dongwon Park ◽

...

Keyword(s):

Pneumococcal Disease ◽

Complex Disease ◽

Capsular Polysaccharide ◽

Pneumococcal Infection ◽

The Elderly ◽

Effective Vaccine ◽

Pneumococcal Infections ◽

Vaccine Strategy ◽

Vaccine Platform ◽

Old Mice

Despite the availability of licensed vaccines, pneumococcal disease caused by the bacteria Streptococcus pneumoniae (pneumococcus), remains a serious infectious disease threat globally. Disease manifestations include pneumonia, bacteremia, and meningitis, resulting in over a million deaths annually. Pneumococcal disease disproportionally impacts elderly individuals ≥65 years old. Interventions are complicated through a combination of complex disease progression and 100 different bacterial capsular polysaccharide serotypes. This has made it challenging to develop a broad vaccine against S. pneumoniae, with current options utilizing capsular polysaccharides as the primary antigenic content. However, current vaccines are substantially less effective in protecting the elderly. We previously developed a Liposomal Encapsulation of Polysaccharides (LEPS) vaccine platform, designed around limitations of current pneumococcal vaccines, that allowed the non-covalent coupling of polysaccharide and protein antigen content and protected young hosts against pneumococcal infection in murine models. In this study, we modified the formulation to make it more economical and tested the novel LEPS vaccine in aged hosts. We found that in young mice (2-3 months), LEPS elicited comparable responses to the pneumococcal conjugate vaccine Prevnar-13. Further, LEPS immunization of old mice (20-22 months) induced comparable antibody levels and improved antibody function compared to Prevnar-13. Importantly, LEPS protected old mice against both invasive and lung localized pneumococcal infections. In summary, LEPS is an alternative and effective vaccine strategy that protects aged hosts against different manifestations of pneumococcal disease.

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