RESUMEN
A series of PdII complexes with bis-(2-pyridylmethyl)glycine as a ligand of formula [PdX(bis-(2-pyridylmethyl)glycine)] where Xâ¯=â¯Cl, Br, I were prepared and the effect of the halogen nature in the antitumor activity of eight tumorigenic and one non-tumorigenic cell line was evaluated. The chloride derivative was further functionalized with a transferrin receptor binding peptide, generating the first PdII based metallopeptide. Its antitumor activity was also evaluated. However, among all the complexes, the chloride and iodine parent compounds showed the lowest GI50 values in the panel evaluated, and lowest GI50 than cisplatin in several cell lines. In contrast, the bromine derivative showed higher values of GI50 than chloride and iodine (around 30 - 50⯵M). The same trend was observed for the bovine serum albumin binding constant with higher values for iodine, chlorine, and bromine in this order. In aqueous solution, the chloride is exchanged by water while the bromine and iodine are not. DNA was evaluated as a target and showed no significative interaction for all the compounds. The results suggest sulfur-rich proteins and not DNA as a target. This report represents the first PdII metallopeptide reported, its evaluation in solution and antitumor activity. This work opens the possibilities for further functionalization of PdII complexes and the importance of the halogen coordination in the design of novel metallodrugs.
Asunto(s)
Complejos de Coordinación/química , Complejos de Coordinación/farmacología , Halógenos/química , Paladio/química , Péptidos/química , Péptidos/farmacología , Proteínas de Unión a Transferrina/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Complejos de Coordinación/síntesis química , Células HT29 , Humanos , Células MCF-7 , Espectroscopía de Resonancia Magnética , Péptidos/síntesis química , Relación Estructura-Actividad , Difracción de Rayos XRESUMEN
Gram-negative porcine pathogens from the Pasteurellaceae family possess a surface receptor complex capable of acquiring iron from porcine transferrin (pTf). This receptor consists of transferrin-binding protein A (TbpA), a transmembrane iron transporter, and TbpB, a surface-exposed lipoprotein. Questions remain as to how the receptor complex engages pTf in such a way that iron is positioned for release, and whether divergent strains present distinct recognition sites on Tf. In this study, the TbpB-pTf interface was mapped using a combination of mass shift analysis and molecular docking simulations, localizing binding uniquely to the pTf C lobe for multiple divergent strains of Actinobacillus plueropneumoniae and suis. The interface was further characterized and validated with site-directed mutagenesis. Although targeting a common lobe, variants differ in preference for the two sublobes comprising the iron coordination site. Sublobes C1 and C2 participate in high affinity binding, but sublobe C1 contributes in a minor fashion to the overall affinity. Further, the TbpB-pTf complex does not release iron independent of other mediators, based on competitive iron binding studies. Together, our findings support a model whereby TbpB efficiently captures and presents iron-loaded pTf to other elements of the uptake pathway, even under low iron conditions.
Asunto(s)
Proteínas de Unión a Transferrina/química , Transferrina/química , Actinobacillus/metabolismo , Actinobacillus suis/metabolismo , Animales , Análisis Mutacional de ADN , Haemophilus influenzae/metabolismo , Hierro/química , Lipoproteínas/química , Espectrometría de Masas/métodos , Conformación Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Estructura Terciaria de Proteína , Resonancia por Plasmón de Superficie , PorcinosRESUMEN
Members of the Actinobacillus minor/"porcitonsillarum" complex are common inhabitants of the swine respiratory tract. Although avirulent or of low virulence for pigs, these organisms, like pathogens, do grow in vivo and must, therefore, be able to acquire iron within the host. Here, we investigated the abilities of six members of the A. minor/"porcitonsillarum" complex to acquire iron from transferrin and various haemoglobins. Using growth assays, all six strains were shown to acquire iron from porcine, bovine and human haemoglobins but not from porcine transferrin. Analyses of whole genome sequences revealed that A. minor strains NM305(T) and 202, unlike the swine-pathogenic actinobacilli, A. pleuropneumoniae and A. suis, lack not only the transferrin-binding protein genes, tbpA and tbpB, but also the haemoglobin-binding protein gene, hgbA. Strains NM305(T) and 202, however, were found to possess other putative haemin/haemoglobin-binding protein genes that were predicted to encode mature proteins of â¼ 72 and â¼ 75 kDa, respectively. An affinity procedure based on haemin-agarose allowed the isolation of â¼ 65 and â¼ 67 kDa iron-repressible outer membrane polypeptides from membranes derived from strains NM305(T) and 202, respectively, and mass spectrometry revealed that these polypeptides were the products of the putative haemin/haemoglobin-binding protein genes. PCR approaches allowed the amplification and sequencing of homologues of both haemin/haemoglobin-binding protein genes from each of the other four strains, strains 33PN and 7ATS of the A. minor/"porcitonsillarum" complex and "A. porcitonsillarum" strains 9953L55 and 0347, suggesting that such proteins are involved in the utilization of haemoglobin-bound iron, presumably as surface receptors, by all six strains investigated.
Asunto(s)
Actinobacillus/metabolismo , Hemoglobinas/química , Hierro/metabolismo , Transferrina/química , Actinobacillus/genética , Actinobacillus/crecimiento & desarrollo , Animales , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Bovinos , ADN Bacteriano/genética , Humanos , Sideróforos/química , Porcinos , Proteínas de Unión a Transferrina/genéticaRESUMEN
Transferrin binding protein (TfBP) is a cytoplasmic glycoprotein that was originally isolated from the chick oviduct. As we previously demonstrated the constitutive expression of TfBP in the avian nervous system, in this study we examined whether TfBP is expressed in the reptilian nervous system. In accordance with previous findings in the chicken, oligodendrocytes were most prominently labeled by antiserum to TfBP. Great variability was observed between different regions of the central nervous system (CNS) in terms of TfBP-labeled oligodendrocyte numbers. In the retina, TfBP was localized specifically in the cells that are morphologically oligodendrocytes and present in the optic nerve and the ganglion cell layer. TfBP staining was also seen in the Schwann cells of peripheral nerves. Furthermore, choroid plexus cells and capillary endothelial cells similarly exhibited strong reactions. These results may reflect the fact that the homology of nervous system genes is conserved between close phylogenetic lines, and proove the potential of TfBP as a marker for oligodendrocytes in avian as well as reptile.
Asunto(s)
Sistema Nervioso/metabolismo , Proteínas de Unión a Transferrina/metabolismo , Tortugas/metabolismo , Animales , Anticuerpos/inmunología , Western Blotting , Encéfalo/citología , Encéfalo/metabolismo , Pollos , Inmunohistoquímica , Disco Óptico/citología , Disco Óptico/metabolismo , Retina/citología , Retina/metabolismo , Nervio Ciático/citología , Nervio Ciático/metabolismo , Médula Espinal/citología , Médula Espinal/metabolismoRESUMEN
Transferrin-binding protein (TfBP) has been shown to be a novel protein, structurally related to the chicken heat shock protein 108. The physiological function of this protein, however, has not yet been established. Antiserum to TfBP selectively stains transferrin- and iron-rich oligodendrocytes and choroidal epithelium in the adult and embryonic chick brain, suggesting a role for this protein in transferrin and iron storage in these cells. In this study, we further demonstrate TfBP-immunoreactivity (IR) in the blood vessels of the embryonic chick central nervous system. A strong TfBP-IR was present in blood vessels from E6, declined from E10 and was absent by E18. Thus, the expression of the TfBP in the blood vessels precedes its expression in the oligodendrocytes. At the subcellular level, TfBP-IR was confined to the cytoplasm of capillary pericytes while the Tf-receptor IR was associated with the capillary endothelium of the brain. The up-regulated expression of TfBP, together with the Tf-receptor of the brain capillaries, suggests that pericytes may be associated with the high iron uptake required for the metabolic demands of the developing brain.
Asunto(s)
Encéfalo/metabolismo , Capilares/metabolismo , Proteínas de Unión a Transferrina/metabolismo , Animales , Encéfalo/embriología , Embrión de Pollo , Inmunohistoquímica , Microscopía Fluorescente , Microscopía InmunoelectrónicaRESUMEN
Oligodendrocytes develop from precursor cells in the neuroepithelium of the ventral ventricular zone. Oligodendrocytes in the different stages of development are characterized by expression of a number of different marker molecules such as myelin genes, growth factors, and specific antigens. We have previously identified that transferrin binding protein (TfBP), a member of heat shock protein 90 families, is a novel avian ER-associated membrane protein that is specifically localized in oligodendrocytes in adult chicken CNS. In this study we describe the developmental expression of TfBP in the embryonic chick spinal cord. A few, distinct, TfBP+ cells appeared at the lateral margin of the subventricular neuroepithelium of the spinal cord at E7. Thereafter, some TfBP+ cells, exhibited a migrative form of unipolar or bipolar shape occurred around E8 in the mantle layer, midway between the neuroepithelium and the marginal layer of the primitive spinal cord. Thereafter, the TfBP+ cells rapidly increased in number as well as their staining intensity, and overall distribution of TfBP+ cells at E15 was comparable to that of a mature spinal cord. Our observations suggest that TfBP is expressed in the subpopulation of oligodendrocyte lineage in the development and a putative role of TfBP in relation to transferrin and iron trafficking is considered.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Oligodendroglía/metabolismo , Proteínas de Unión a Transferrina/biosíntesis , Animales , Western Blotting , Linaje de la Célula , Embrión de Pollo , Inmunohistoquímica , Microscopía Fluorescente , Oligodendroglía/citología , Oligodendroglía/inmunología , Médula Espinal/embriologíaRESUMEN
Entamoeba histolytica is an enteric protozoan that exclusively infects human beings. This parasite requires iron for its metabolic functions. Lactoferrin is a mammalian glycoprotein that chelates extracellular iron on mucosal surfaces, including the surface of the large intestine, where E. histolytica initiates infection. This work examined the interaction in vitro of E. histolytica trophozoites with human hololactoferrin (iron-saturated lactoferrin). A minimum concentration of 50 microM Fe from hololactoferrin supported growth of the amoeba. Amoebic binding sites for hololactoferrin were different from those for human apolactoferrin, holotransferrin and haemoglobin. One amoebic hololactoferrrin-binding polypeptide of 90 kDa was found, which was not observed after treatment of trophozoites with trypsin. Hololactoferrin-binding-protein levels increased in amoebas starved of iron, or grown in hololactoferrin. Internalization of hololactoferrin was inhibited by filipin. Endocytosed hololactoferrin colocalized with an anti-chick embryo caveolin mAb in amoebic vesicles, and lactoferrin was further detected in acidic vesicles; amoebic caveolin of 22 kDa was detected by Western blotting using this antibody. Cysteine proteases from amoebic extracts were able to cleave hololactoferrin. Together, these data indicate that E. histolytica trophozoites bind to hololactoferrin through specific membrane lactoferrin-binding proteins. This ferric protein might be internalized via caveolae-like microdomains, then used as an iron source, and degraded.
Asunto(s)
Anticuerpos Antiprotozoarios/inmunología , Endocitosis/fisiología , Entamoeba histolytica/metabolismo , Hierro/metabolismo , Lactoferrina/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Entamoeba histolytica/crecimiento & desarrollo , Humanos , Proteínas de Unión a Hierro , Proteínas de Unión a TransferrinaRESUMEN
Cyclic AMP response element (CRE) is a specific DNA sequence, which mediates transcriptional activation in the response to the cyclic AMP-activated and protein kinase A dependent signaling pathway. In the present study, phosphorylated CRE binding protein (CREB) immunoreactivity was mainly localized in the white matter of chick central nervous system (CNS). We have further confirmed the specificity of phospho-CREB immunoreaction in myelin using demyelinated optic nerve induced by lysophophatidylcholine (LPC), which is known to produce demyelination with little axonal damage. Double immunofluorescent analyses with myelin basic protein (MBP) and transferrin binding protein (TfBP), oligodendrocyte marker showed that phospho-CREB recognized a myelin-related protein in chick. Immunoblot analyses showed that phospho-CREB recognized a protein with molecular weights of approximately 70 kDa. Our data suggest that the antigen recognized by phospho-CREB is a myelin-associated protein in the chick CNS.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Vaina de Mielina/metabolismo , Animales , Encéfalo/citología , Encéfalo/metabolismo , Pollos , Enfermedades Desmielinizantes , Humanos , Inmunohistoquímica , Lisofosfatidilcolinas/metabolismo , Proteína Básica de Mielina/metabolismo , Nervio Óptico/citología , Nervio Óptico/metabolismo , Fosforilación , Ratas , Proteínas de Unión a Transferrina/metabolismoRESUMEN
The developmental expression of heat shock protein 108 (HSP108) mRNA was mapped in chicken brain using in situ hybridization and reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR showed that HSP108 mRNA increased from embryonic day 5 (E5) to 13 (E13), significantly decreased from E17 to E21 and then increased again at the adult stage. In situ hybridization showed that while intense HSP108 positive (HSP108+) signals were localized in the cerebellum from E7 to E14, the intensities of these signals were significantly decreased at E17. However, at the adult stage, HSP108 expression increased in a cell type dependent manner. A decrease in HSP108 mRNA expression during differentiation was also observed in an in vitro study of brain cells treated with nerve growth factor (NGF).
Asunto(s)
Encéfalo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Choque Térmico/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión a Transferrina/metabolismo , Factores de Edad , Análisis de Varianza , Animales , Northern Blotting/métodos , Encéfalo/citología , Encéfalo/embriología , Células Cultivadas , Embrión de Pollo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Proteínas de Choque Térmico/genética , Hibridación in Situ/métodos , Factor de Crecimiento Nervioso/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Proteínas de Unión a Transferrina/genéticaRESUMEN
Human serum transferrin is an essential bilobal protein that transports iron in the circulation for delivery to iron-requiring cells. Obtaining the C-terminal lobe of human transferrin in verified native conformation has been problematic, possibly because its 11 disulfide bonds lead to misfolding when the lobe is expressed without its accompanying N-lobe. A recently reported method for preparing the C-lobe free of extraneous residues, with normal iron-binding properties and capable of delivering iron to cells, makes use of a Factor Xa cleavage site inserted into the interlobal connecting strand of the full-length protein. An inefficient step in this method requires the use of ConA chromatography to separate the cleaved lobes from each other, since only the C-lobe is glycosylated. Inserting a 6-His sequence near the start of the N-lobe enhances recovery of the recombinant transferrin from other proteins in the culture medium of the BHK21 cells expressing the mutant transferrin. The new procedure is more economical in time and effort than its predecessor, and offers the additional advantage of isolating C-lobe expressed with or without its glycan chains.
Asunto(s)
Histidina/metabolismo , Transferrina/química , Transferrina/aislamiento & purificación , Western Blotting , Espectroscopía de Resonancia por Spin del Electrón , Electroforesis en Gel de Poliacrilamida , Humanos , Cinética , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Transferrina/metabolismo , Proteínas de Unión a Transferrina/metabolismoRESUMEN
The pathogenic Neisseria have a siderophore-independent iron-uptake system reliant on a direct interaction between the bacterial cell and transferrin. In the meningococcus this uptake system is dependent on two surface-exposed transferrin-binding proteins. This short account will review our current knowledge of the transferrin-mediated iron-acquisition system of pathogenic Neisseria.
Asunto(s)
Hierro/metabolismo , Neisseria meningitidis/metabolismo , Receptores de Transferrina/metabolismo , Transferrina/metabolismo , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Neisseria meningitidis/patogenicidad , Receptores de Transferrina/química , Proteínas de Unión a Transferrina/química , Proteínas de Unión a Transferrina/metabolismo , VirulenciaRESUMEN
Analysis of haplotypes is an important tool in population genetics, familial heredity and gene mapping. Determination of haplotypes of multiple single nucleotide polymorphisms (SNPs) or other simple mutations is time consuming and expensive when analyzing large populations, and often requires the help of computational and statistical procedures. Based on double PCR amplification of specific alleles, described previously, we have developed a simple, rapid and low-cost method for direct haplotyping of multiple SNPs and simple mutations found within relatively short specific regions or genes (micro-haplotypes). Using this method, it is possible to directly determine the physical linkage of multiple heterozygous alleles, by conducting a series of double allele-specific PCR amplification sets with simple analysis by gel electrophoresis. Application of the method requires prior information as to the sequence of the segment to be haplotyped, including the polymorphic sites. We applied the method to haplotyping of nine sites in the chicken HSP108 gene. One of the haplotypes in the population apparently arose by recombination between two existing haplotypes, and we were able to locate the point of recombination within a segment of 19 bp. We anticipate rapidly growing needs for SNP haplotyping in human (medical and pharmacogenetics), animal and plant genetics; in this context, the multiple double PCR amplifications of specific alleles (MD-PASA) method offers a useful haplotyping tool.
Asunto(s)
Alelos , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Nucleótido Simple , Animales , Secuencia de Bases , Proteínas Portadoras/genética , Pollos , Haplotipos , Proteínas de Unión a Hierro , Datos de Secuencia Molecular , Mutación , Proteínas de Unión a TransferrinaRESUMEN
One hundred and twenty individual sera obtained from healthy persons of different age groups were studied for the presence of antibodies to meningococcal iron-regulated proteins (IRP). The study revealed that occurrence of such antibodies in sera under study was IRP nature- and age-dependent. Antibodies to two IRP were found to occur most frequently: 85 kD (TbpB) and 72 kD (FrpB). Antibodies to the former IRP were detected in more than 50% and antibodies to the latter IRP, in more than 90% of sera. This was probably due to the presence of epitopes common with those in protein antigens of some other microorganisms, such as Moraxella catarrhalis and Haemophilus influenzae. The occurrence of antibodies to periplasmatic IRP with 34 kD (FbpA) in blood sera varied within the range of 5 to 30%. At the same time the occurrence of antibodies to this protein in the sera under study was age-depended: children until five years exhibited the minimal occurrence (about 5%), while in adults it reached 30%.
Asunto(s)
Envejecimiento/inmunología , Anticuerpos Antibacterianos/sangre , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Bacterianas/inmunología , Proteínas Portadoras/inmunología , Neisseria meningitidis/inmunología , Adulto , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/sangre , Niño , Preescolar , Haemophilus influenzae/inmunología , Humanos , Proteínas de Unión a Hierro , Peso Molecular , Moraxella catarrhalis/inmunología , Proteínas de Unión a TransferrinaRESUMEN
Three genetically defined Actinobacillus pleuropneumoniae serotype 7 mutants with deletions in the small (tbpB), the large (tbpA), and both transferrin binding protein genes were constructed and examined in an aerosol infection model. Neither mutant caused clinical disease or could be reisolated, and no immune response could be detected 21 days after infection. This result clearly implies that each transferrin binding protein on its own is a virulence factor of A. pleuropneumoniae serotype 7.
Asunto(s)
Infecciones por Actinobacillus/microbiología , Infecciones por Actinobacillus/veterinaria , Actinobacillus pleuropneumoniae/patogenicidad , Proteínas Portadoras/genética , Enfermedades de los Porcinos/microbiología , Actinobacillus pleuropneumoniae/clasificación , Actinobacillus pleuropneumoniae/genética , Aerosoles , Animales , Proteínas Portadoras/metabolismo , Eliminación de Gen , Proteínas de Unión a Hierro , Serotipificación , Porcinos , Proteínas de Unión a Transferrina , VirulenciaRESUMEN
The dpr gene is an antioxidant gene which was isolated from the Streptococcus mutans chromosome by its ability to complement an alkyl hydroperoxide reductase-deficient mutant of Escherichia coli, and it was proven to play an indispensable role in oxygen tolerance in S. mutans. Here, we purified the 20-kDa dpr gene product, Dpr, from a crude extract of S. mutans as an iron-binding protein and found that Dpr formed a spherical oligomer about 9 nm in diameter. Molecular weight determinations of Dpr in solution by analytical ultracentrifugation and light-scattering analyses gave values of 223,000 to 292,000, consistent with a subunit composition of 11.5 to 15 subunits per molecule. The purified Dpr contained iron and zinc atoms and had an ability to incorporate up to 480 iron and 11.2 zinc atoms per molecule. Unlike E. coli Dps and two other members of the Dps family, Dpr was unable to bind DNA. One hundred nanomolar Dpr prevented by more than 90% the formation of hydroxyl radical generated by 10 microM iron(II) salt in vitro. The data shown in this study indicate that Dpr may act as a ferritin-like iron-binding protein in S. mutans and may allow this catalase- and heme-peroxidase-deficient bacterium to grow under air by limiting the iron-catalyzed Fenton reaction.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Radical Hidroxilo/metabolismo , Hierro/metabolismo , Streptococcus mutans/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/farmacología , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión a Hierro , Peso Molecular , Oxígeno/metabolismo , Proteínas de Unión a Transferrina , ZincRESUMEN
Induced sputum (IS) and bronchoalveolar lavage (BAL) sample different lung compartments, with IS obtaining secretions from the surfaces of the bronchial airways and BAL sampling secretions from the alveolar airspaces. Deposition of iron-containing particulate matter occurs preferentially in the bronchial airways compared to the distal airspaces. Iron-binding and storage proteins have been measured in BAL in healthy humans, where they act to sequester and detoxify available iron; however, their comparative levels in the airways are not well defined. Seventeen (n = 17) healthy, nonsmoking volunteers underwent sputum induction and fiber-optic bronchoscopy with lavage in order to measure and compare the levels of iron and iron-binding and storage proteins in BAL, bronchial lavage (BL), and IS. Relative to BAL and BL specimens, concentrations of total iron, ferritin, lactoferrin, and total iron binding capacity (TIBC) were significantly increased and transferrin decreased in IS. Immunohistochemical staining showed increased ferritin, lactoferrin, and TIBC in IS. Constitutive levels of iron and iron-binding and storage proteins (except for transferrin) are in greatest concentration in the bronchial airways relative to the alveolar airspaces in healthy humans. We speculate that these proteins reflect activated antioxidant defense mechanisms resulting from the preferential deposition of iron-containing particulate matter in the airways. The finding of decreased transferrin with increased ferritin suggests a local cellular response via iron-response elements (IRE) associated with the mRNAs for these two iron-binding proteins.
Asunto(s)
Contaminantes Atmosféricos/efectos adversos , Antioxidantes , Proteínas Portadoras/análisis , Adolescente , Adulto , Líquido del Lavado Bronquioalveolar/química , Broncoscopía , Humanos , Inmunohistoquímica , Proteínas de Unión a Hierro , Tamaño de la Partícula , Esputo/química , Proteínas de Unión a TransferrinaRESUMEN
The major yolk protein (MYP) in sea urchins has historically been classified as a vitellogenin based on its abundance in the yolk platelets. Curiously, it is found in both sexes of sea urchins where it is presumed to play a physiological role in gametogenesis, embryogenesis, or both. Here we present the primary structure of MYP as predicted from cDNAs of two sea urchins species, Strongylocentrotus purpuratus and Lytechinus variegatus. The sequence from these two species share identity to one another, but bear no resemblance to other known vitellogenins. Instead the sequence shares identity to members of the transferrin superfamily of proteins. In vitro iron binding assays, including both (59)Fe overlay assays of MYP enriched coelomic fluid and immunoprecipitation of native iron-bound MYP from coelomic fluid, support this classification. We suggest that one of MYP's transferrin-like properties is to shuttle iron to developing germ cells.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas del Huevo/metabolismo , Hierro/metabolismo , Transferrina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/química , Proteínas Portadoras/genética , Clonación Molecular , ADN Complementario , Proteínas del Huevo/química , Proteínas del Huevo/genética , Proteínas de Unión a Hierro , Datos de Secuencia Molecular , Pruebas de Precipitina , Erizos de Mar , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Proteínas de Unión a TransferrinaRESUMEN
Neisseria gonorrhoeae is capable of utilizing a variety of iron sources in vitro, including human transferrin, human lactoferrin, hemoglobin, hemoglobin-haptoglobin complexes, heme, and heterologous siderophores. Transferrin has been implicated as a critical iron store for N. gonorrhoeae in the human male urethra. The demonstration that gonococci can infect the lower genital tracts of estradiol-treated BALB/c mice in the absence of human transferrin, however, suggests that other usable iron sources are present in the murine genital tract. Here we demonstrate that gonococcal transferrin and hemoglobin receptor mutants are not attenuated in mice, thereby ruling out transferrin and hemoglobin as essential for murine infection. An increased frequency of phase variants with the hemoglobin receptor "on" (Hg(+)) occurred in ca. 50% of infected mice; this increase was temporally associated with an influx of neutrophils and detectable levels of hemoglobin in the vagina, suggesting that the presence of hemoglobin in inflammatory exudates selects for Hg(+) phase variants during infection. We also demonstrate that commensal lactobacilli support the growth of N. gonorrhoeae in vitro unless an iron chelator is added to the medium. We hypothesize that commensal lactobacilli may enhance growth of gonococci in vivo by promoting the solubilization of iron on mucosal surfaces through the production of metabolic intermediates. Finally, transferrin-binding lipoprotein (TbpB) was detected on gonococci in vaginal smears, suggesting that although gonococci replicate within the genital tracts of mice, they may be sufficiently iron-stressed to express iron-repressible proteins. In summary, these studies support the potential role of nontransferrin, nonhemoglobin iron sources during gonococcal infection of the female genital tract.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/fisiología , Proteínas Bacterianas , Lactobacillus/fisiología , Neisseria gonorrhoeae/crecimiento & desarrollo , Receptores de Superficie Celular/fisiología , Transferrina/fisiología , Vagina/microbiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/análisis , Femenino , Proteínas de Unión a Hierro , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas de Unión a TransferrinaRESUMEN
tbpA, fur, and fldA homologs from two strains (9L and 3384Y) of the sheep pathogen Histophilus ovis were sequenced. The predicted TbpA proteins of these strains are homologs of the Pasteurella multocida TbpA protein and collectively represent the second example of a new subfamily of TonB-dependent receptors. tbpA transcripts were readily detected by reverse transcription (RT)-PCR with RNA isolated from strain 9L grown under iron-restricted conditions in the presence or absence of bovine transferrin (Tf). However, with strain 3384Y and depending on the primer pair, tbpA transcripts were detected by RT-PCR predominantly when the RNA was from cells grown under iron-restricted conditions in the presence of bovine Tf. In both strains, the fldA homolog was found to be immediately upstream of fur and, based on RT-PCR, these genes are transcribed as a single unit; the availability of iron and the presence or absence of bovine Tf in the growth medium had no apparent effect on the relative amounts of the fldA-fur transcripts.
Asunto(s)
Proteínas Bacterianas/aislamiento & purificación , Proteínas Portadoras/aislamiento & purificación , Flavoproteínas , Bacterias Gramnegativas/química , Proteínas Represoras/aislamiento & purificación , Transcripción Genética/efectos de los fármacos , Proteínas Bacterianas/genética , Secuencia de Bases , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Medios de Cultivo , Expresión Génica/efectos de los fármacos , Bacterias Gramnegativas/genética , Bacterias Gramnegativas/crecimiento & desarrollo , Hierro/farmacología , Proteínas de Unión a Hierro , Datos de Secuencia Molecular , Pasteurella multocida/química , ARN Bacteriano/análisis , ARN Bacteriano/genética , ARN Mensajero/genética , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transferrina , Proteínas de Unión a TransferrinaRESUMEN
Pathogenic bacteria of the genus Neisseria have a siderophore-independent iron-uptake system reliant on a direct interaction between the bacterial cell and human transferrin (hTf), a serum protein. In the meningococcus, this uptake system is dependent on two surface-exposed, transferrin-binding proteins (Tbps), TbpA and TbpB. TbpA is highly conserved among meningococcal strains, and is thought to be a porin-like integral protein that functions as a gated channel for the passage of iron into the periplasm. TbpB is more variable in size, lipidated and fully surface-exposed. Given its location on the cell surface, its role in pathogenicity and interstrain sequence conservation, TbpA is currently being regarded for inclusion in a meningococcal vaccine effective against all serogroups. This requires gaining knowledge of the ligand-receptor interactions. In the present study we have optimized a procedure for obtaining purified, functionally active recombinant TbpA at a level and stability necessary for the initiation of such studies.