RESUMO
UNLABELLED: Bacterial pathogens Neisseria meningitidis and Brucella abortus pose threats to human and animal health worldwide, causing meningococcal disease and brucellosis, respectively. Mortality from acute N. meningitidis infections remains high despite antibiotics, and brucellosis presents alimentary and health consequences. Superoxide dismutases are master regulators of reactive oxygen and general pathogenicity factors and are therefore therapeutic targets. Cu,Zn superoxide dismutases (SODs) localized to the periplasm promote survival by detoxifying superoxide radicals generated by major host antimicrobial immune responses. We discovered that passive immunization with an antibody directed at N. meningitidis SOD (NmSOD) was protective in a mouse infection model. To define the relevant atomic details and solution assembly states of this important virulence factor, we report high-resolution and X-ray scattering analyses of NmSOD and of SOD from B. abortus (BaSOD). The NmSOD structures revealed an auxiliary tetrahedral Cu-binding site bridging the dimer interface; mutational analyses suggested that this metal site contributes to protein stability, with implications for bacterial defense mechanisms. Biochemical and structural analyses informed us about electrostatic substrate guidance, dimer assembly, and an exposed C-terminal epitope in the NmSOD dimer. In contrast, the monomeric BaSOD structure provided insights for extending immunogenic peptide epitopes derived from the protein. These collective results reveal unique contributions of SOD to pathogenic virulence, refine predictive motifs for distinguishing SOD classes, and suggest general targets for antibacterial immune responses. The identified functional contributions, motifs, and targets distinguishing bacterial and eukaryotic SOD assemblies presented here provide a foundation for efforts to develop SOD-specific inhibitors of or vaccines against these harmful pathogens. IMPORTANCE: By protecting microbes against reactive oxygen insults, SODs aid survival of many bacteria within their hosts. Despite the ubiquity and conservation of these key enzymes, notable species-specific differences relevant to pathogenesis remain undefined. To probe mechanisms that govern the functioning of Neisseria meningitidis and Brucella abortus SODs, we used X-ray structures, enzymology, modeling, and murine infection experiments. We identified virulence determinants common to the two homologs, assembly differences, and a unique metal reservoir within meningococcal SOD that stabilizes the enzyme and may provide a safeguard against copper toxicity. The insights reported here provide a rationale and a basis for SOD-specific drug design and an extension of immunogen design to target two important pathogens that continue to pose global health threats.
Assuntos
Complexo Antígeno-Anticorpo/ultraestrutura , Brucella abortus/imunologia , Neisseria meningitidis/imunologia , Superóxido Dismutase/imunologia , Superóxido Dismutase/ultraestrutura , Animais , Anticorpos/administração & dosagem , Anticorpos/imunologia , Sítios de Ligação de Anticorpos , Vacina contra Brucelose/imunologia , Brucella abortus/patogenicidade , Brucelose/imunologia , Brucelose/prevenção & controle , Cristalografia por Raios X , Modelos Animais de Doenças , Imunização Passiva/métodos , Meningite Meningocócica/imunologia , Meningite Meningocócica/prevenção & controle , Vacinas Meningocócicas/imunologia , Camundongos , Neisseria meningitidis/patogenicidade , Superóxido Dismutase/genética , Fatores de Virulência/imunologiaRESUMO
BACKGROUND: Haemophilus parasuis, the causative agent of Glässer's disease, is prevalent in swine herds and clinical signs associated with this disease are meningitis, polyserositis, polyarthritis, and bacterial pneumonia. Six to eight week old pigs in segregated early weaning herds are particularly susceptible to the disease. Insufficient colostral antibody at weaning or the mixing of pigs with heterologous virulent H. parasuis strains from other farm sources in the nursery or grower-finisher stage are considered to be factors for the outbreak of Glässer's disease. Previously, a Mu-like bacteriophage portal gene was detected in a virulent swine isolate of H. parasuis by nested polymerase chain reaction. Mu-like bacteriophages are related phyologenetically to enterobacteriophage Mu and are thought to carry virulence genes or to induce host expression of virulence genes. This study characterizes the Mu-like bacteriophage, named SuMu, isolated from a virulent H. parasuis isolate. RESULTS: Characterization was done by genomic comparison to enterobacteriophage Mu and proteomic identification of various homologs by mass spectrometry. This is the first report of isolation and characterization of this bacteriophage from the Myoviridae family, a double-stranded DNA bacteriophage with a contractile tail, from a virulent field isolate of H. parasuis. The genome size of bacteriophage SuMu was 37,151 bp. DNA sequencing revealed fifty five open reading frames, including twenty five homologs to Mu-like bacteriophage proteins: Nlp, phage transposase-C-terminal, COG2842, Gam-like protein, gp16, Mor, peptidoglycan recognition protein, gp29, gp30, gpG, gp32, gp34, gp36, gp37, gpL, phage tail tube protein, DNA circulation protein, gpP, gp45, gp46, gp47, COG3778, tail fiber protein gp37-C terminal, tail fiber assembly protein, and Com. The last open reading frame was homologous to IS1414. The G + C content of bacteriophage SuMu was 41.87% while its H. parasuis host genome's G + C content was 39.93%. Twenty protein homologs to bacteriophage proteins, including 15 structural proteins, one lysogeny-related and one lysis-related protein, and three DNA replication proteins were identified by mass spectrometry. One of the tail proteins, gp36, may be a virulence-related protein. CONCLUSIONS: Bacteriophage SuMu was characterized by genomic and proteomic methods and compared to enterobacteriophage Mu.
Assuntos
Bacteriófagos/genética , Genômica , Haemophilus parasuis/virologia , Proteômica , Animais , Bacteriófago mu/genética , Bacteriófagos/metabolismo , Bases de Dados Genéticas , Genoma Viral , Espectrometria de Massas , Fases de Leitura Aberta , Proteoma/análise , Análise de Sequência de DNA , Suínos , Proteínas Virais/metabolismo , Virulência/genéticaRESUMO
BACKGROUND: Haemophilus parasuis is the causative agent of Glässer's disease and is a pathogen of swine in high-health status herds. Reports on serotyping of field strains from outbreaks describe that approximately 30% of them are nontypeable and therefore cannot be traced. Molecular typing methods have been used as alternatives to serotyping. This study was done to compare random amplified polymorphic DNA (RAPD) profiles and whole cell protein (WCP) lysate profiles as methods for distinguishing H. parasuis reference strains and field isolates. RESULTS: The DNA and WCP lysate profiles of 15 reference strains and 31 field isolates of H. parasuis were analyzed using the Dice and neighbor joining algorithms. The results revealed unique and reproducible DNA and protein profiles among the reference strains and field isolates studied. Simpson's index of diversity showed significant discrimination between isolates when three 10 mer primers were combined for the RAPD method and also when both the RAPD and WCP lysate typing methods were combined. CONCLUSIONS: The RAPD profiles seen among the reference strains and field isolates did not appear to change over time which may reflect a lack of DNA mutations in the genes of the samples. The recent field isolates had different WCP lysate profiles than the reference strains, possibly because the number of passages of the type strains may affect their protein expression.
Assuntos
Proteínas de Bactérias/análise , DNA Bacteriano/genética , Haemophilus parasuis/química , Haemophilus parasuis/genética , Proteoma/análise , Proteômica/métodos , Técnica de Amplificação ao Acaso de DNA Polimórfico/métodos , Animais , Técnicas Bacteriológicas/métodos , Haemophilus parasuis/classificação , Haemophilus parasuis/isolamento & purificação , Reprodutibilidade dos Testes , SuínosRESUMO
A new temperature-conditional shuttle vector, pBB80C, was constructed and utilized to generate an in-frame deletion in the leukotoxin structural gene of Mannheimia haemolytica serotype 1. Culture supernatants from the mutant contained no detectable cytotoxicity to BL-3 lymphocyte targets, and contained a new protein with an approximate molecular weight of 66 kDa which was reactive to anti-leukotoxin monoclonal antibody. No protein reactive to anti-LktA monoclonal antibody was detected at the molecular weight 100-105 kDa of native LktA. Calves vaccinated mucosally by top-dressing the live mutant onto feed, or parenterally by subcutaneous injection, were resistant to virulent challenge with the parent strain. Serologic antibody response, reduction in lung lesion, and reduction in pulmonary infectious load were greater among calves mucosally vaccinated than those which were vaccinated by injection.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/imunologia , Mannheimia haemolytica/imunologia , Pasteurelose Pneumônica/imunologia , Mucosa Respiratória/imunologia , Deleção de Sequência , Animais , Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/administração & dosagem , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Bovinos , Feminino , Proteínas Hemolisinas/administração & dosagem , Imunidade nas Mucosas , Infusões Parenterais , Masculino , Mannheimia haemolytica/genética , Pasteurelose Pneumônica/microbiologia , Pasteurelose Pneumônica/prevenção & controle , VacinaçãoRESUMO
It has been demonstrated that fhaB2 (filamentous hemagglutinin) is an important virulence factor for Pasteurella multocida in development of fowl cholera disease and that vaccination with recombinant FHAB2 peptides derived from P. multocida, P-1059 (serotype A:3) protects turkeys against P-1059 challenge. Here the hypothesis that vaccination with the same rFHAB2 peptides could cross-protect turkeys against challenge with P. multocida chi73 (serotype A:1) was examined. Three rFHAB2 peptides were purified and pooled, and two doses, consisting of equal amounts of each, were administered subcutaneously to turkeys at 2-wk intervals. Simultaneously, control birds were administered sham inoculations. One week later, vaccinates and controls were challenged intranasally with P-1059 or chi73. The results showed vaccination with rFHAB2 peptides significantly protected turkeys against lethal challenge from both P. multocida serotypes (P < 0.01). The high degree of FHAB2 conservation across serotypes likely allow the observed cross-protection.
Assuntos
Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Infecções por Pasteurella/veterinária , Pasteurella multocida/imunologia , Doenças das Aves Domésticas/prevenção & controle , Perus , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Feminino , Masculino , Infecções por Pasteurella/microbiologia , Infecções por Pasteurella/prevenção & controle , Pasteurella multocida/metabolismo , Doenças das Aves Domésticas/microbiologia , Proteínas Recombinantes/imunologiaRESUMO
Many pathogenic bacteria employ systems to incorporate sialic acid into their membranes as a means of protection against host defense mechanisms. In Pasteurella multocida, an opportunistic pathogen which causes diseases of economic importance in a wide range of animal species, sialic acid uptake plays a role in a mouse model of systemic pasteurellosis. To further investigate the importance of sialic acid uptake in pathogenesis, sialic acid uptake mutants of an avian strain of P. multocida P-1059 (A:3) were constructed, characterized, and an in-frame sialic acid uptake deletion mutant was assessed for virulence in turkeys. Inactivation of sialic acid uptake resulted in a high degree of attenuation when turkeys were challenged either intranasally or intravenously. Resistance of the sialic acid uptake mutant to killing by turkey serum complement was similar to that of the parent, suggesting other mechanisms are responsible for attenuation of virulence in turkeys.
Assuntos
Ácido N-Acetilneuramínico/imunologia , Infecções por Pasteurella/veterinária , Pasteurella multocida/patogenicidade , Doenças das Aves Domésticas/microbiologia , Animais , Infecções por Pasteurella/imunologia , Infecções por Pasteurella/microbiologia , Pasteurella multocida/genética , Pasteurella multocida/imunologia , Doenças das Aves Domésticas/imunologia , Perus , VirulênciaRESUMO
Three gene fragments, derived from Pasteurella multocida strain P-1059 (serotype A:3), encoding approximately the 5' one-third of fhaB2 were overexpressed individually in Escherichia coli. The recombinant peptides were purified, pooled, and administered to turkey poults to evaluate immunity. The results showed that turkeys immunized twice with the recombinant peptides were significantly protected against intranasal challenge with P. multocida strain P-1059. Vaccination elicited antibody responses, based on Western blotting, that were reactive with a wild-type P-1059 cellular product approximately 170 kDa in size and multiple high molecular weight products in culture supernatant. These antibodies did not react with cell or supernatant blots of a P-1059 fhaB2 isogenic mutant. Pasteurella multocida fhaB2 genes of a bovine strain (A:3) and an avian strain (F:3) are highly conserved as is the portion of P-1059 fhaB2 examined here (>99% identities). These findings suggest that broad cross-protection against this heterogeneous pathogen may be achievable through immunization with specific recombinant FHAB2 peptides.
Assuntos
Vacinas Bacterianas/imunologia , Hemaglutininas/imunologia , Infecções por Pasteurella/veterinária , Pasteurella multocida/imunologia , Proteínas Recombinantes/imunologia , Perus , Animais , Anticorpos Antibacterianos/sangue , Vacinas Bacterianas/administração & dosagem , Infecções por Pasteurella/prevenção & controle , VacinaçãoRESUMO
Fowl cholera continues to be of concern to the poultry industry, especially for turkey growers. This disease costs the turkey industry millions of dollars annually. In order to develop improved live attenuated vaccines or subunit vaccines, the outer-membrane proteins of Pasteurella multocida were examined with the use of proteomics. Of the 11 proteins total present in an outer-membrane subfraction of P. multocida, four additional proteins were identified, completing the composition of the detergent-soluble cross-protective protein fraction. These additional four proteins include protective bacterial surface antigen, OMA87 (Accession no. 15603857); heme-hemopexin receptor, HemR (Accession no. 15602441); lactate permease, LctP (Accession no. 15603717); and heptosyl transferase F, RfaF (Accession no. 15603709). Both the Oma87 and the HemR proteins would be of interest for subunit and modified live vaccine studies, respectively, because of their purported roles as virulence factors for P. multocida.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Ácidos Cólicos/química , Detergentes/química , Pasteurella multocida/metabolismo , Mapeamento de Peptídeos , Solubilidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Ornithobacterium rhinotracheale (ORT) is an emerging respiratory pathogen of poultry in North America that is causing millions of dollars in economic losses to the poultry industry. Ornithobacterium rhinotracheale is associated with airsacculitis, pleuritis, pneumonia, and consolidation of lungs. Little is known about the molecular mechanisms of infection. In this study, the mechanism of iron acquisition by O. rhinotracheale was explored. O. rhinotracheale strains grown under iron deprivation in media containing 200 microM 2,2'-dipyridyl did not secrete siderophores as measured by the chrome azurol S (CAS) agar and CAS solution assays. Filter disks impregnated with various protein-bound iron compounds and inorganic iron salts of Fe(III) and Fe(II) placed on iron-restricted agar inoculated with a lawn of O. rhinotracheale supported growth from sheep and porcine hemoglobins, ovotransferrin, Fe(III), and Fe(II), but they did not support growth from bovine transferrin, bovine apo-transferrin, bovine lactoferrin, and hemin. However, both bovine hemoglobin and transferrin supported growth of O. rhinotracheale serotype C. Four immunoreactive proteins involved in iron acquisition were identified in an O. rhinotracheale membrane extract by using mass spectrometry. Furthermore, O. rhinotracheale field strains showed differential sensitivity to 2,2'-dipyridyl. Of the 72 field strains tested, 22 strains were resistant to the iron chelator at concentrations of 50 microM and 100 microM, suggesting this attribute may be related to disease-producing potential of these strains. This is the first report on the identification of the iron acquisition mechanism of O. rhinotracheale.
Assuntos
Ferro/metabolismo , Ornithobacterium/crescimento & desenvolvimento , Ornithobacterium/metabolismo , Sideróforos/metabolismo , 2,2'-Dipiridil/toxicidade , Eletroforese em Gel Bidimensional , Hemoglobinas , Ornithobacterium/efeitos dos fármacos , Especificidade da Espécie , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , TransferrinaRESUMO
The selective accumulation of lutein in the macula of the human retina is likely to be mediated by specific transport and/or binding proteins. Our objective was to determine whether transthyretin (TTR) is a plasma transport protein for lutein. We used a biosynthetic 13C-lutein tracer and GC-combustion interfaced-isotope ratio MS to gain the requisite sensitivity to detect the minute amounts of lutein expected as a physiological ligand for TTR. Subjects (n = 4) each ingested 1 mg of 13C-lutein daily for 3 d and donated blood 24 h after the final dose. For three subjects, the plasma TTR-retinol-binding protein (RBP) complex was partially purified by anion-exchange (diethylaminoethyl, DEAE) chromatography and then dissociated by hydrophobic-interaction chromatography to yield the TTR component. For subject 4, the initial DEAE purification step was omitted and total plasma TTR (RBP-bound and free) was isolated by hydrophobic-interaction chromatography. In each case, the crude TTR fractions were then purified to homogeneity by RBP-Sepharose affinity chromatography. Pure TTR was extracted with chloroform, and unlabeled lutein was added to the extract as a carrier. The mean 13C/12C ratio (expressed in delta notation, delta13C) of the lutein fraction isolated from the plasma TTR extracts of the four subjects was -30.53 +/- 3.29 per thousand. The delta13C value of the unlabeled lutein carrier was -30.97 +/- 0.27per thousand. Thus, no 13C enrichment was detected in association with TTR. We conclude that lutein is not associated with TTR in human plasma after being ingested in physiological amounts.
Assuntos
Luteína/administração & dosagem , Pré-Albumina/metabolismo , Administração Oral , Adulto , Western Blotting , Isótopos de Carbono , Proteínas de Transporte/sangue , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Cromatografia de Afinidade , Feminino , Humanos , Ligantes , Luteína/metabolismo , Espectrometria de Massas , Pré-Albumina/isolamento & purificação , Proteínas de Ligação ao Retinol/análise , Proteínas de Ligação ao Retinol/metabolismo , Proteínas Plasmáticas de Ligação ao Retinol , Espectrofotometria , Espectrofotometria UltravioletaRESUMO
OBJECTIVE: To use antibodies produced by calves in response to infection with Mannheimia haemolytica in immunoaffinity chromatography for the identification and subsequent isolation of the dominant immunogenic antigens from bacteria grown in iron-deficient media. SAMPLE POPULATION: Serum from 10 calves actively infected with M haemolytica. PROCEDURE: An outer membrane protein fraction was obtained from sonicated salt-extracted M haemolytica cells by extraction with N-lauroyl sarcosinate. The immunoglobulin fraction of serum from calves actively infected with M haemolytica was used to prepare an immunoaffinity column. The immunoaffinity column was used to isolate the dominant immunogenic proteins from the outer membrane protein fraction. The resultant immunogenic protein fraction was subjected to ELISA and immunoblot methods as well as carbohydrate quantification. Sequencing of the N-terminal was performed on the most prominent protein. RESULTS: 5 immunogenic proteins with molecular weights of 42, 30, 24, 20, and 15 kd were isolated. The immunogenic protein fraction was found to contain 51% carbohydrate. The immunoaffinity column capacity was 1 microg of immunogenic protein/mL of gel. The N-terminal sequence of the 42-kd protein was Tyr-Gln-Thr-Tyr-Gln-Ser-X-Leu-Gln, where X could not be identified. CONCLUSIONS AND CLINICAL RELEVANCE: lmmunogenic proteins were isolated by use of immunoaffinity chromatography. A substantial amount of carbohydrates was co-purified in the process. Additional experiments are needed to determine whether the carbohydrates would hinder or enhance development of vaccine preparations. This method could potentially allow a more rapid production of antigens for use in vaccines.
Assuntos
Antígenos de Bactérias/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Doenças dos Bovinos/imunologia , Mannheimia haemolytica/imunologia , Infecções por Pasteurellaceae/veterinária , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/normas , Western Blotting/veterinária , Carboidratos/análise , Bovinos , Doenças dos Bovinos/microbiologia , Cromatografia de Afinidade/veterinária , Eletroforese em Gel de Poliacrilamida/veterinária , Ensaio de Imunoadsorção Enzimática/veterinária , Infecções por Pasteurellaceae/imunologia , Infecções por Pasteurellaceae/microbiologia , Análise de Sequência de ProteínaRESUMO
The Gram-negative, pleomorphic, rod-shaped bacterium Ornithobacterium rhinotracheale is a cause of pneumonia and airsacculitis in poultry. It is a member of the family Flavobacteriaceae of the phylum "Bacteroidetes". O. rhinotracheale strain H06-030791 was isolated from the lung of a turkey in North Carolina in 2006. Its genome consists of a circular chromosome of 2,319,034 bp in length with a total of 2243 protein-coding genes and nine RNA genes. Genome sequences are available for two additional strains of O. rhinotracheale, isolated in 1988 and 1995, the latter described in a companion genome report in this issue of SIGS. The genome sequence of O. rhinotracheale strain H06-030791, a more contemporary isolate, will be of value in establishing core and pan-genomes for O. rhinotracheale and elucidating its evolutionary history.
RESUMO
Ornithobacterium rhinotracheale strain ORT-UMN 88 is a Gram-negative, pleomorphic, rod-shaped bacterium and an etiologic agent of pneumonia and airsacculitis in poultry. It is a member of the family Flavobacteriaceae of the phylum Bacteroidetes. O. rhinotracheale strain ORT-UMN 88 was isolated from the pneumonic lung of a turkey in 1995. It was the isolate first used to experimentally reproduce disease in turkeys and has since been the focus of investigations characterizing potential virulence factors of the bacterium. The genome of O. rhinotracheale strain ORT-UMN 88 consists of a circular chromosome of 2,397,867 bp with a total of 2300 protein-coding genes, nine RNA genes, and one noncoding RNA gene. A companion paper in this issue of SIGS reports the non-contiguous finished genome sequence of an additional strain of O. rhinotracheale, isolated in 2006.
RESUMO
A nested polymerase chain reaction (nPCR) assay was developed to determine the presence of a gene encoding a bacteriophage Mu-like portal protein, gp29, in 15 reference strains and 31 field isolates of Haemophilus parasuis. Specific primers, based on the gene's sequence, were utilized. A majority of the virulent reference strains and field isolates tested harbored the gene. The results suggest that the nPCR technique described in the current report could serve as a tool for epidemiological studies of H. parasuis.
Assuntos
Bacteriófagos/genética , Genes Virais/genética , Haemophilus parasuis/virologia , Reação em Cadeia da Polimerase/veterinária , Animais , Infecções por Haemophilus/microbiologia , Infecções por Haemophilus/veterinária , Infecções por Haemophilus/virologia , Haemophilus parasuis/isolamento & purificação , SuínosRESUMO
Haemophilus parasuis is a Gram-negative respiratory pathogen of young pigs that colonizes the upper respiratory tract and produces a number of symptoms collectiviely described as Glässer's disease. Recently, an H. parasuis P5-like outer membrane adhesin protein homologous to H. influenzae P5 was identified. The P5 adhesin was partially purified by anion exchange and size-exclusion chromatography. Final purification for functional studies was performed by elution of the protein from a polyacrylamide gel. Identification of the protein as a P5 adhesin homolog of H. influenzae was confirmed by N-terminal sequencing. The P5 protein had a molecular mass of 32,000 and a pI of 5.5. Unlike the H. influenzae P5 adhesin, the H. parasuis P5 protein did not bind carcinoembryonic antigen.
Assuntos
Adesinas Bacterianas/química , Haemophilus parasuis/metabolismo , Adesinas Bacterianas/isolamento & purificação , Antígeno Carcinoembrionário/química , Immunoblotting , Mapeamento de Interação de Proteínas , Análise de Sequência de ProteínaRESUMO
Fowl cholera is caused by Pasteurella multocida serovars A:1, A:3, and A:4. The 39-kDa cross-protective factor protein and four other membrane proteins of the membrane proteome of P. multocida were identified. We determined that the 39-kDa cross-protective protein was Pasteurella lipoprotein B, or PlpB.
Assuntos
Proteínas da Membrana Bacteriana Externa/análise , Pasteurella multocida/química , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/química , Dados de Sequência Molecular , Peso Molecular , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The objective of this study was to isolate and characterize beef muscle proteins that enhance nonheme iron bioavailability. Beef sirloin was cooked, lyophilized and reconstituted with water before in vitro digestion. After centrifugation, the digest supernatant was sequentially ultrafiltered using 10- and 1-kDa molecular weight cut-off membranes. Nonheme iron bioavailability was assessed by Caco-2 cell monolayer (59)Fe uptake using an extrinsic labeling method. All ultrafiltration fractions significantly (P < 0.001) increased iron solubility at pH 6.0, compared with the blank. However, iron uptake was significantly (P < 0.001) greater than the blank only in the presence of the 1-kDa retentate (1KR). Therefore, the 1KR was chosen for further analysis. Immobilized metal affinity chromatography (IMAC) of the 1KR yielded four fractions, i.e., three distinct fractions (F1, F3, F4) and one fraction (F2) comprised of a few closely associated peaks. All four IMAC fractions resulted in significantly (P < 0.001) greater (two- to fivefold) iron solubility at pH 6.0, compared with the blank. Iron uptake with F2 and F4 was significantly greater than the blank (P < 0.001 and P < 0.05, respectively). Gel electrophoresis and matrix-assisted laser desorption/ionization analysis illustrated that F1-F4 contained many peptides ranging from 1- to 7-kDa. Amino acid composition analysis revealed that histidine concentration increased progressively from F1 to F4, corresponding to a general, but not parallel increase in iron solubility and uptake. Our results suggest that the enhancement of nonheme iron absorption by beef may be due to peptides produced during gastrointestinal digestion and that histidine content may be important.