RESUMO
Metachromatic leukodystrophy (MLD) is an autosomal recessively inherited sulfatide storage disease caused by deficient activity of the lysosomal enzyme arylsulfatase A (ASA). Genetic analysis of the ARSA gene is important in MLD diagnosis and screening of family members. In addition, more information on genotype prevalence will help interpreting MLD population differences between countries. In this study, we identified 31 different ARSA variants in the patient cohort (n = 67) of the Dutch expertise center for MLD. The most frequently found variant, c.1283C > T, p.(Pro428Leu), was present in 43 (64%) patients and resulted in a high prevalence of the juvenile MLD type (58%) in The Netherlands. Furthermore, we observed in five out of six patients with a non-Caucasian ethnic background previously unreported pathogenic ARSA variants. In total, we report ten novel variants including four missense, two nonsense, and two frameshift variants and one in-frame indel, which were all predicted to be disease causing in silico. In addition, one silent variant was found, c.1200C > T, that most likely resulted in erroneous exonic splicing, including partial skipping of exon 7. The c.1200C > T variant was inherited in cis with the pseudodeficiency allele c.1055A > G, p.(Asn352Ser) + ∗96A > G. With this study we provide a genetic base of the unique MLD phenotype distribution in The Netherlands. In addition, our study demonstrated the importance of genetic analysis in MLD diagnosis and the increased likelihood of unreported, pathogenic ARSA variants in patients with non-Caucasian ethnic backgrounds.
Assuntos
Cerebrosídeo Sulfatase/genética , Leucodistrofia Metacromática/genética , Adolescente , Alelos , Criança , Pré-Escolar , Códon sem Sentido , Éxons , Feminino , Mutação da Fase de Leitura , Estudos de Associação Genética , Predisposição Genética para Doença , Genótipo , Humanos , Lactente , Leucodistrofia Metacromática/etnologia , Masculino , Mutação , Mutação de Sentido Incorreto , Países Baixos , Fenótipo , Estudos Retrospectivos , Adulto JovemRESUMO
Deficiency of succinate semialdehyde dehydrogenase (SSADH; aldehyde dehydrogenase 5a1 (ALDH5A1), OMIM 271980, 610045), the second enzyme of GABA degradation, represents a rare autosomal-recessively inherited disorder which manifests metabolically as gamma-hydroxybutyric aciduria. The neurological phenotype includes intellectual disability, autism spectrum, epilepsy and sleep and behavior disturbances. Approximately 70 variants have been reported in the ALDH5A1 gene, half of them being missense variants. In this study, 34 missense variants, of which 22 novel, were evaluated by in silico analyses using PolyPhen2 and SIFT prediction tools. Subsequently, the effect of these variants on SSADH activity was studied by transient overexpression in HEK293 cells. These studies showed severe enzymatic activity impairment for 27 out of 34 alleles, normal activity for one allele and a broad range of residual activities (25 to 74%) for six alleles. To better evaluate the alleles that showed residual activity above 25%, we generated an SSADH-deficient HEK293-Flp-In cell line using CRISPR-Cas9, in which these alleles were stably expressed. This model proved essential in the classification as deficient for one out of the seven studied alleles. For 8 out of 34 addressed alleles, there were discrepant results among the used prediction tools, and/or in correlating the results of the prediction tools with the functional data. In case of diagnostic urgency of missense alleles, we propose the use of the transient transfection model for confirmation of their effect on the SSADH catalytic function, since this model resulted in fast and robust functional characterization for the majority of the tested variants. In selected cases, stable transfections can be considered and may prove valuable.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/patologia , Deficiências do Desenvolvimento/patologia , Mutação de Sentido Incorreto , Succinato-Semialdeído Desidrogenase/deficiência , Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Simulação por Computador , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/metabolismo , Células HEK293 , Humanos , Succinato-Semialdeído Desidrogenase/genética , Succinato-Semialdeído Desidrogenase/metabolismoRESUMO
D-2-hydroxyglutaric aciduria Type I (D-2-HGA Type I), a neurometabolic disorder with a broad clinical spectrum, is caused by recessive variants in the D2HGDH gene encoding D-2-hydroxyglutarate dehydrogenase (D-2-HGDH). We and others detected 42 potentially pathogenic variants in D2HGDH of which 31 were missense. We developed functional studies to investigate the effect of missense variants on D-2-HGDH catalytic activity. Site-directed mutagenesis was used to introduce 31 missense variants in the pCMV5-D2HGDH expression vector. The wild type and missense variants were overexpressed in HEK293 cells. D-2-HGDH enzyme activity was evaluated based on the conversion of [2 H4 ]D-2-HG to [2 H4 ]2-ketoglutarate, which was subsequently converted into [2 H4 ]L-glutamate and the latter quantified by LC-MS/MS. Eighteen variants resulted in almost complete ablation of D-2-HGDH activity and thus, should be considered pathogenic. The remaining 13 variants manifested residual activities ranging between 17% and 94% of control enzymatic activity. Our functional assay evaluating the effect of novel D2HGDH variants will be beneficial for the classification of missense variants and determination of pathogenicity.
Assuntos
Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Encefalopatias Metabólicas Congênitas/genética , Mutação de Sentido Incorreto , Encefalopatias Metabólicas Congênitas/metabolismo , Cromatografia Líquida , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida , Espectrometria de Massas em Tandem , Anormalidades UrogenitaisRESUMO
Pyridoxine dependent epilepsy (PDE) is a treatable epileptic encephalopathy characterized by a positive response to pharmacologic doses of pyridoxine. Despite seizure control, at least 75% of individuals have intellectual disability and developmental delay. Current treatment paradigms have resulted in improved cognitive outcomes emphasizing the importance of an early diagnosis. As genetic testing is increasingly accepted as first tier testing for epileptic encephalopathies, we aimed to provide a comprehensive overview of ALDH7A1 mutations that cause PDE. The genotypes, ethnic origin and reported gender was collected from 185 subjects with a diagnosis of PDE. The population frequency for the variants in this report and the existing literature were reviewed in the Genome Aggregation Database (gnomAD). Novel variants identified in population databases were also evaluated through in silico prediction software and select variants were over-expressed in an E.coli-based expression system to measure α-aminoadipic semialdehyde dehydrogenase activity and production of α-aminoadipic acid. This study adds 47 novel variants to the literature resulting in a total of 165 reported pathogenic variants. Based on this report, in silico predictions, and general population data, we estimate an incidence of approximately 1:64,352 live births. This report provides a comprehensive overview of known ALDH7A1 mutations that cause PDE, and suggests that PDE may be more common than initially estimated. Due to the relative high frequency of the disease, the likelihood of under-diagnosis given the wide clinical spectrum and limited awareness among clinicians as well as the cognitive improvement noted with early treatment, newborn screening for PDE may be warranted.
Assuntos
Aldeído Desidrogenase/genética , Epilepsia/genética , Ácido 2-Aminoadípico/metabolismo , Genótipo , Humanos , MutaçãoRESUMO
Combined D-2- and L-2-hydroxyglutaric aciduria (D/L-2-HGA) is a devastating neurometabolic disorder, usually lethal in the first years of life. Autosomal recessive mutations in the SLC25A1 gene, which encodes the mitochondrial citrate carrier (CIC), were previously detected in patients affected with combined D/L-2-HGA. We showed that transfection of deficient fibroblasts with wild-type SLC25A1 restored citrate efflux and decreased intracellular 2-hydroxyglutarate levels, confirming that deficient CIC is the cause of D/L-2-HGA. We developed and implemented a functional assay and applied it to all 17 missense variants detected in a total of 26 CIC-deficient patients, including eight novel cases, showing reduced activities of varying degrees. In addition, we analyzed the importance of residues affected by these missense variants using our existing scoring system. This allowed not only a clinical and biochemical overview of the D/L-2-HGA patients but also phenotype-genotype correlation studies.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Encefalopatias Metabólicas Congênitas/metabolismo , Ácido Cítrico/metabolismo , Glutaratos/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Transporte de Ânions/química , Proteínas de Transporte de Ânions/genética , Bioensaio/métodos , Encefalopatias Metabólicas Congênitas/genética , Células Cultivadas , Pré-Escolar , Análise Mutacional de DNA , Feminino , Fibroblastos , Predisposição Genética para Doença , Humanos , Lactente , Recém-Nascido , Masculino , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Modelos Moleculares , Mutação de Sentido Incorreto , Transportadores de Ânions Orgânicos , Fenótipo , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
The Krebs cycle is of fundamental importance for the generation of the energetic and molecular needs of both prokaryotic and eukaryotic cells. Both enantiomers of metabolite 2-hydroxyglutarate are directly linked to this pivotal biochemical pathway and are found elevated not only in several cancers, but also in different variants of the neurometabolic disease 2-hydroxyglutaric aciduria. Recently we showed that cancer-associated IDH2 germline mutations cause one variant of 2-hydroxyglutaric aciduria. Complementary to these findings, we now report recessive mutations in SLC25A1, the mitochondrial citrate carrier, in 12 out of 12 individuals with combined D-2- and L-2-hydroxyglutaric aciduria. Impaired mitochondrial citrate efflux, demonstrated by stable isotope labeling experiments and the absence of SLC25A1 in fibroblasts harboring certain mutations, suggest that SLC25A1 deficiency is pathogenic. Our results identify defects in SLC25A1 as a cause of combined D-2- and L-2-hydroxyglutaric aciduria.
Assuntos
Proteínas de Transporte de Ânions/genética , Encefalopatias Metabólicas Congênitas/etiologia , Ácido Cítrico/metabolismo , Genes Recessivos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Mutação/genética , Sequência de Aminoácidos , Biomarcadores/análise , Encefalopatias Metabólicas Congênitas/metabolismo , Encefalopatias Metabólicas Congênitas/patologia , Estudos de Casos e Controles , Células Cultivadas , Cromatografia Líquida , Exoma/genética , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Glutaratos/urina , Humanos , Masculino , Dados de Sequência Molecular , Transportadores de Ânions Orgânicos , Fenótipo , Estrutura Terciária de Proteína , Estudos Retrospectivos , Homologia de Sequência de Aminoácidos , Estereoisomerismo , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Aberrations in about 10-15% of X-chromosome genes account for intellectual disability (ID); with a prevalence of 1-3% (Gécz et al., 2009 [1]). The SLC6A8 gene, mapped to Xq28, encodes the creatine transporter (CTR1). Mutations in SLC6A8, and the ensuing decrease in brain creatine, lead to co-occurrence of speech/language delay, autism-like behaviors and epilepsy with ID. A splice variant of SLC6A8-SLC6A8C, containing intron 4 and exons 5-13, was identified. Herein, we report the identification of a novel variant - SLC6A8D, and functional relevance of these isoforms. METHODS: Via (quantitative) RT-PCR, uptake assays, and confocal microscopy, we investigated their expression and function vis-à-vis creatine transport. RESULTS: SLC6A8D is homologous to SLC6A8C except for a deletion of exon 9 (without occurrence of a frame shift). Both contain an open reading frame encoding a truncated protein but otherwise identical to CTR1. Like SLC6A8, both variants are predominantly expressed in tissues with high energy requirement. Our experiments reveal that these truncated isoforms do not transport creatine. However, in SLC6A8 (CTR1)-overexpressing cells, a subsequent infection (transduction) with viral constructs encoding either the SLC6A8C (CTR4) or SLC6A8D (CTR5) isoform resulted in a significant increase in creatine accumulation compared to CTR1 cells re-infected with viral constructs containing the empty vector. Moreover, transient transfection of CTR4 or CTR5 into HEK293 cells resulted in significantly higher creatine uptake. CONCLUSIONS: CTR4 and CTR5 are possible regulators of the creatine transporter since their overexpression results in upregulated CTR1 protein and creatine uptake. GENERAL SIGNIFICANCE: Provides added insight into the mechanism(s) of creatine transport regulation.
Assuntos
Processamento Alternativo , Regulação da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Células 3T3 , Animais , Sequência de Bases , Creatina/metabolismo , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana Transportadoras/fisiologia , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Transcrição GênicaRESUMO
Creatine transporter (SLC6A8) deficiency is the most common cause of cerebral creatine syndromes, and is characterized by depletion of creatine in the brain. Manifestations of this X-linked disorder include intellectual disability, speech/language impairment, behavior abnormalities, and seizures. At the moment, no effective treatment is available. In order to investigate the molecular pathophysiology of this disorder, we performed RNA sequencing on fibroblasts derived from patients. The transcriptomes of fibroblast cells from eight unrelated individuals with SLC6A8 deficiency and three wild-type controls were sequenced. SLC6A8 mutations with different effects on the protein product resulted in different gene expression profiles. Differential gene expression analysis followed by gene ontology term enrichment analysis revealed that especially the expression of genes encoding components of the extracellular matrix and cytoskeleton are altered in SLC6A8 deficiency, such as collagens, keratins, integrins, and cadherins. This suggests an important novel role for creatine in the structural development and maintenance of cells. It is likely that the (extracellular) structure of brain cells is also impaired in SLC6A8-deficient patients, and future studies are necessary to confirm this and to reveal the true functions of creatine in the brain.
Assuntos
Encefalopatias Metabólicas Congênitas/genética , Encefalopatias Metabólicas Congênitas/metabolismo , Creatina/deficiência , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Proteínas de Membrana Transportadoras/genética , Deficiência Intelectual Ligada ao Cromossomo X/genética , Deficiência Intelectual Ligada ao Cromossomo X/metabolismo , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/deficiência , Linhagem Celular , Creatina/genética , Creatina/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Masculino , Mutação , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/genética , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/metabolismo , Análise de Sequência de RNA , Sinapses/genética , Sinapses/metabolismoRESUMO
A reduced response of cystathionine beta-synthase (CBS) to its allosteric activator S-adenosylmethionine (SAM) has been reported to be a cause of CBS dysfunction in homocystinuria patients. In this work we performed a retrospective analysis of fibroblast data from 62 homocystinuria patients and found that 13 of them presented a disturbed SAM activation. Their genotypic background was identified and the corresponding CBS mutant proteins were produced in E. coli. Nine distinct mutations were detected in 22 independent alleles: the novel mutations p.K269del, p.P427L, p.S500L and p.L540Q; and the previously described mutations p.P49L, p.C165Rfs*2, p.I278T, p.R336H and p.D444N. Expression levels and residual enzyme activities, determined in the soluble fraction of E. coli lysates, strongly correlated with the localization of the affected amino acid residue. C-terminal mutations lead to activities in the range of the wild-type CBS and to oligomeric forms migrating faster than tetramers, suggesting an abnormal conformation that might be responsible for the lack of SAM activation. Mutations in the catalytic core were associated with low protein expression levels, decreased enzyme activities and a higher content of high molecular mass forms. Furthermore, the absence of SAM activation found in the patients' fibroblasts was confirmed for all but one of the characterized recombinant proteins (p.P49L). Our study experimentally supports a deficient regulation of CBS by SAM as a frequently found mechanism in CBS deficiency, which should be considered not only as a valuable diagnostic tool but also as a potential target for the development of new therapeutic approaches in classical homocystinuria.
Assuntos
Cistationina beta-Sintase/genética , Homocistinúria/enzimologia , Homocistinúria/genética , Mutação , S-Adenosilmetionina/genética , Alelos , Células Cultivadas , Cistationina beta-Sintase/metabolismo , Escherichia coli/genética , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Genótipo , Homocistinúria/metabolismo , Homocistinúria/patologia , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estudos Retrospectivos , S-Adenosilmetionina/metabolismoRESUMO
BACKGROUND: Mosaic IDH1 mutations are described as the cause of metaphyseal chondromatosis with increased urinary excretion of D-2-hydroxyglutarate (MC-HGA), and mutations in IDH2 as the cause of D-2-hydroxyglutaric aciduria (D-2HGA) type II. Mosaicism for IDH2 mutations has not previously been reported as a cause of D-2HGA. Here we describe three cases: one MC-HGA case with IDH1 mosaic mutations, and two D-2HGA type II cases. In one D-2HGA case we identified mosaicism for an IDH2 mutation as the genetic cause of this disorder; the other D-2HGA case was caused by a heterozygous IDH2 mutation, while the unaffected mother was a mosaic carrier. METHODS: We performed amplicon deep sequencing using the 454 GS Junior platform, next to Sanger sequencing, to identify and confirm mosaicism of IDH1 or IDH2 mutations in MC-HGA or D-2HGA, respectively. RESULTS AND CONCLUSIONS: We identified different mutant allele percentages in DNA samples derived from different tissues (blood vs fibroblasts). Furthermore, we found that mutant allele percentages of IDH1 decreased after more passages had occurred in fibroblast cell cultures. We describe a method for the detection and validation of mosaic mutations in IDH1 and IDH2, making quantification with laborious cloning techniques obsolete.
Assuntos
Encefalopatias Metabólicas Congênitas/genética , Isocitrato Desidrogenase/genética , Mosaicismo , Encefalopatias Metabólicas Congênitas/diagnóstico , Células Cultivadas , Criança , Feminino , Frequência do Gene , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Mutação , PaisRESUMO
A DPD deficiency should be considered in case of severe toxicity even in the absence of common risk variants in DPYD.
Assuntos
Antimetabólitos Antineoplásicos , Capecitabina , Deficiência da Di-Hidropirimidina Desidrogenase , Di-Hidrouracila Desidrogenase (NADP) , Humanos , Deficiência da Di-Hidropirimidina Desidrogenase/genética , Capecitabina/efeitos adversos , Di-Hidrouracila Desidrogenase (NADP)/genética , Antimetabólitos Antineoplásicos/efeitos adversos , Antimetabólitos Antineoplásicos/uso terapêutico , Masculino , Feminino , Evolução Fatal , Pessoa de Meia-IdadeRESUMO
L-2-Hydroxyglutaric aciduria (L2HGA) is a rare, neurometabolic disorder with an autosomal recessive mode of inheritance. Affected individuals only have neurological manifestations, including psychomotor retardation, cerebellar ataxia, and more variably macrocephaly, or epilepsy. The diagnosis of L2HGA can be made based on magnetic resonance imaging (MRI), biochemical analysis, and mutational analysis of L2HGDH. About 200 patients with elevated concentrations of 2-hydroxyglutarate (2HG) in the urine were referred for chiral determination of 2HG and L2HGDH mutational analysis. All patients with increased L2HG (n=106; 83 families) were included. Clinical information on 61 patients was obtained via questionnaires. In 82 families the mutations were detected by direct sequence analysis and/or multiplex ligation dependent probe amplification (MLPA), including one case where MLPA was essential to detect the second allele. In another case RT-PCR followed by deep intronic sequencing was needed to detect the mutation. Thirty-five novel mutations as well as 35 reported mutations and 14 nondisease-related variants are reviewed and included in a novel Leiden Open source Variation Database (LOVD) for L2HGDH variants (http://www.LOVD.nl/L2HGDH). Every user can access the database and submit variants/patients. Furthermore, we report on the phenotype, including neurological manifestations and urinary levels of L2HG, and we evaluate the phenotype-genotype relationship.
Assuntos
Oxirredutases do Álcool/genética , Encefalopatias Metabólicas Congênitas/enzimologia , Encefalopatias Metabólicas Congênitas/genética , Estudos de Associação Genética , Mutação/genética , Animais , Encefalopatias Metabólicas Congênitas/patologia , Modelos Animais de Doenças , HumanosRESUMO
Creatine transporter deficiency is an X-linked mental retardation disorder caused by mutations in the creatine transporter gene (SLC6A8). So far, 20 mutations in the SLC6A8 gene have been described. We have developed a diagnostic assay to test creatine uptake in fibroblasts. Additionally, we expanded the assay to characterize novel SLC6A8 missense variants. A total of 13 variants were introduced in the SLC6A8 cDNA by site-directed mutagenesis. All variants were transiently transfected in SLC6A8-deficient fibroblasts and tested for restoration of creatine uptake in deficient primary fibroblasts. Thus, we proved that nine variants (p.Gly87Arg, p.Phe107del, p.Tyr317X, p.Asn336del, p.Cys337Trp, p.Ile347del, p.Pro390Leu, p.Arg391Trp, and p.Pro554Leu) are pathogenic mutations and four variants (p.Lys4Arg, p.Gly26Arg, p.Met560Val, and p.Val629Ile) are nonpathogenic. The present study provides an improved diagnostic tool to classify sequence variants of unknown significance.
Assuntos
Deficiência Intelectual Ligada ao Cromossomo X/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/genética , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/genética , Algoritmos , Células Cultivadas , Creatina/farmacocinética , Técnicas de Diagnóstico por Radioisótopos , Cromatografia Gasosa-Espectrometria de Massas , Proteínas de Fluorescência Verde/genética , Humanos , Deficiência Intelectual Ligada ao Cromossomo X/genética , Proteínas do Tecido Nervoso/deficiência , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/deficiência , Proteínas Recombinantes de Fusão/genética , Valores de Referência , TransfecçãoRESUMO
A haemoglobin-degrading enzyme from pathogenic Escherichia coli has been cloned, expressed and purified to homogeneity. The pure protein proteolyses haemoglobin and binds haem. In vivo, its role is to remove haem from haemoglobin and pass it to the bacteria, allowing them to overcome the limiting concentration of iron available in the body. The protein has been crystallized using polyethylene glycol to give crystals in a hexagonal space group with unit-cell parameters a = b = 114.6, c = 434.3 A. X-ray data have been collected to 2.5 A resolution. This is the first member of the SPATE (serine protease autotransporters of Enterobacteriaceae) family of autotransporter proteins to be crystallized.
Assuntos
Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Escherichia coli/enzimologia , Proteínas de Helminto , Cristalografia por Raios X , Heme/metabolismo , Peso Molecular , Ligação ProteicaRESUMO
Intra-abdominal infections (IAI) continue to be a serious clinical problem. Bacterial synergism is an important factor that influences the shift from contamination to IAI, leading to the development of lesions and abscess formation. Escherichia coli and Bacteroides fragilis are particularly abundant in IAI. The underlying molecular mechanisms of this pathogenic synergy are still unclear. The role of the hemoglobin protease (Hbp) autotransporter protein from E. coli in the synergy of IAI was investigated. Hbp is identical to Tsh, a temperature-sensitive hemagglutinin associated with avian pathogenic E. coli. Clinical isolates from miscellaneous extraintestinal infections were phenotypically and genotypically screened for Hbp. The presence of Hbp was significantly associated with E. coli isolated from IAI and other extraintestinal infections. In a murine infection model, Hbp was shown to contribute to the pathogenic synergy of abscess development. Mice immunized with Hbp were protected against mixed infections and did not develop abscess lesions. Furthermore, an E. coli wild-type strain that did not induce abscess formation in the synergy model was transformed with a plasmid encoding the hbp gene, and mixed infections with this strain lead to increased growth of B. fragilis and induction of abscess lesions. Growth-promoting studies showed that purified Hbp is able to deliver heme to B. fragilis strain BE1. In conclusion, results suggest the synergy of abscess formation by E. coli and B. fragilis can be partly explained by the capacity of B. fragilis to intercept Hbp and iron from heme to overcome the iron restrictions imposed by the host.