Characterization and expression of HmuR, a TonB-dependent hemoglobin receptor of Porphyromonas gingivalis.
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The gram-negative pathogen Porphyromonas gingivalis requires hemin for growth. Hemoglobin bound to haptoglobin and hemin complexed to hemopexin can be used as heme sources, indicating that P. gingivalis must have a means to remove the hemin from these host iron-binding proteins. However, the specific mechanisms utilized by P. gingivalis for the extraction of heme from heme-binding proteins and for iron transport are poorly understood. In this study we have determined that a newly identified TonB-dependent hemoglobin-hemin receptor (HmuR) is involved in hemoglobin binding and utilization in P. gingivalis A7436. HmuR shares amino acid homology with TonB-dependent outer membrane receptors of gram-negative bacteria involved in the acquisition of iron from hemin and hemoglobin, including HemR of Yersinia enterocolitica, ShuA of Shigella dysenteriae, HpuB of Neisseria gonorrhoeae and N. meningitidis, HmbR of N. meningitidis, HgbA of Haemophilus ducreyi, and HgpB of H. influenzae. Southern blot analysis confirmed the presence of the hmuR gene and revealed genetic variability in the carboxy terminus of hmuR in P. gingivalis strains 33277, 381, W50, and 53977. We also identified directly upstream of the hmuR gene a gene which we designated hmuY. Upstream of the hmuY start codon, a region with homology to the Fur binding consensus sequence was identified. Reverse transcription-PCR analysis revealed that hmuR and hmuY were cotranscribed and that transcription was negatively regulated by iron. Inactivation of hmuR resulted in a decreased ability of P. gingivalis to bind hemoglobin and to grow with hemoglobin or hemin as sole iron sources. Escherichia coli cells expressing recombinant HmuR were shown to bind hemoglobin and hemin. Furthermore, purified recombinant HmuR was demonstrated to bind hemoglobin. Taken together, these results indicate that HmuR serves as the major TonB-dependent outer membrane receptor involved in the utilization of both hemin and hemoglobin in P. gingivalis. (+info)
Effects of hemoglobin on heme oxygenase gene expression and viability of cultured smooth muscle cells.
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Ferrous Hb contributes to cerebral vasospasm after subarachnoid hemorrhage, although the mechanisms involved are uncertain. The hypothesis that cytotoxic effects of ferrous Hb on smooth muscle cells contribute to vasospasm was assessed. Cultured rat basilar artery smooth muscle cells were exposed to pure Hb, dog erythrocyte hemolysate, or Hb breakdown products; and heme oxygenase (HO-1 and HO-2) and ferritin mRNA and protein were measured. Cytotoxicity was assessed by lactate dehydrogenase release and fluorescence assays. Pure Hb or hemolysate caused dose- and time-dependent increases in HO-1 mRNA and protein. Hemin was the component of Hb that increased HO-1 mRNA. Cycloheximide inhibited the increase in HO-1 mRNA in response to hemin. Ferritin protein heavy chain but not mRNA increased upon exposure of cells to Hb. Hemin and ferric but not ferrous Hb were toxic to smooth muscle cells. Toxicity was increased by exposure to Hb plus tin protoporphyrin IX. In conclusion, exposure of smooth muscle cells to Hb induces HO-1 mRNA and protein through pathways that involve new protein synthesis. Hemin is the component of Hb that induces HO-1. Hemin and ferric but not ferrous Hb are toxic. (+info)
Hemin-binding surface protein from Bartonella quintana.
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Bartonella quintana, the agent of trench fever and a cause of endocarditis and bacillary angiomatosis in humans, has the highest reported in vitro hemin requirement for any bacterium. We determined that eight membrane-associated proteins from B. quintana bind hemin and that a approximately 25-kDa protein (HbpA) was the dominant hemin-binding protein. Like many outer membrane proteins, HbpA partitions to the detergent phase of a Triton X-114 extract of the cell and is heat modifiable, displaying an apparent molecular mass shift from approximately 25 to 30 kDa when solubilized at 100 degrees C. Immunoblots of purified outer and inner membranes and immunoelectron microscopy with whole cells show that HbpA is strictly located in the outer membrane and surface exposed, respectively. The N-terminal sequence of mature HbpA was determined and used to clone the HbpA-encoding gene (hbpA) from a lambda genomic library. The hbpA gene is 816 bp in length, encoding a predicted immature protein of approximately 29.3 kDa and a mature protein of 27.1 kDa. A Fur box homolog with 53% identity to the Escherichia coli Fur consensus is located upstream of hbpA and may be involved in regulating expression. BLAST searches indicate that the closest homologs to HbpA include the Bartonella henselae phage-associated membrane protein, Pap31 (58.4% identity), and the OMP31 porin from Brucella melitensis (31.7% identity). High-stringency Southern blots indicate that all five pathogenic Bartonella spp. possess hbpA homologs. Recombinant HbpA can bind hemin in vitro; however, it does not confer a hemin-binding phenotype upon E. coli. Intact B. quintana treated with purified anti-HbpA Fab fragments show a significant (P < 0.004) dose-dependent decrease in hemin binding relative to controls, suggesting that HbpA plays an active role in hemin acquisition and therefore pathogenesis. HbpA is the first potential virulence determinant characterized from B. quintana. (+info)
A role for linoleic acid in erythrocytes infected with Plasmodium berghei.
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Unesterified fatty acids were measured in mouse erythrocytes infected either with chloroquine-susceptible (CS) or with chloroquine-resistant (CR) lines of Plasmodium berghei. This work was undertaken to identify candidates for the lipid involved in ferriprotoporphyrin IX (FP) polymerization. Linoleic, oleic, palmitic, and stearic acids were quantified by gas chromatography/mass spectrometry. In total, they increased 4-fold with CS infections and 6-fold with CR infections. Treating infected mice with chloroquine did not affect the amounts of unesterified fatty acids in erythrocytes. Of the four fatty acids, only linoleic acid increased disproportionately to the total. It increased 16-fold for the CS line and 35-fold for the CR line. The method could detect monoglycerides but they were below the limit of detection. It could not detect diglycerides, triglycerides or phospholipids. Triglycerides and phospholipids have been tested previously, however, and found to be ineffective at promoting FP polymerization. Therefore, other than linoleic acid, the lipids most likely to be involved in FP polymerization are diglycerides. We tested dilinoleolyglycerol in the present work and found it to be an effective promoter of FP polymerization. These results suggest that linoleic acid or a diglyceride containing it has the critical role of promoting FP polymerization in malaria parasites. (+info)
Quantitative analysis of globin gene induction in single human erythroleukemic cells.
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The mechanisms involved in the normal developmental regulation of globin gene expression, and the response to pharmacological agents that elevate fetal hemoglobin, may be expected to involve either changes in each cell or a selection process affecting subsets of differentiating erythroid cells. To study these mechanisms we have developed assays to measure mRNA levels in single erythroid cells. The assay involved the use of globin-specific probes, with no detectable cross-reactivity, in real-time, fluorescence-based quantitative PCR (Q-PCR). We had previously used this Q-PCR method to measure globin mRNA levels in cultures of primary erythroid cells demonstrating that drugs like hydroxyurea, 5-azacytidine and butyric acid each yielded increases in gamma/( gamma + ss) mRNA ratios, with differential effects on ss-globin levels. We have now extended this approach to measure globin mRNA levels in single K562 cells, a human erythroleukemic cell line, with and without 30 microM hemin treatment. Hemin exposure increases total hemoglobin levels by approximately 9-fold and total alpha-, epsilon- and gamma-globin mRNA levels by 1.5-2.3-fold. Single cell analyses showed initial wide distributions of each of the three individual globin mRNA levels with most cells having detectable but very low levels of each globin transcript. Hemin induction shifted the distributions to higher levels, with a tendency to residual left skewing as some cells remained with very low expression levels despite the effect of hemin in increasing expression in most of these low expressing cells. Thus transcriptional heterogeneity remains a crucial variable, even in this extensively used model of human erythroid biology, and clearly influences strongly the response to inducing agents. These methods may enable us to define better possible molecular and/or cellular models of globin gene modulation. (+info)
Amyloid precursor proteins inhibit heme oxygenase activity and augment neurotoxicity in Alzheimer's disease.
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Amyloid precursor protein (APP) generates the beta-amyloid peptide, postulated to participate in the neurotoxicity of Alzheimer's disease. We report that APP and APLP bind to heme oxygenase (HO), an enzyme whose product, bilirubin, is antioxidant and neuroprotective. The binding of APP inhibits HO activity, and APP with mutations linked to the familial Alzheimer's disease (FAD) provides substantially greater inhibition of HO activity than wild-type APP. Cortical cultures from transgenic mice expressing Swedish mutant APP have greatly reduced bilirubin levels, establishing that mutant APP inhibits HO activity in vivo. Oxidative neurotoxicity is markedly greater in cerebral cortical cultures from APP Swedish mutant transgenic mice than wild-type cultures. These findings indicate that augmented neurotoxicity caused by APP-HO interactions may contribute to neuronal cell death in Alzheimer's disease. (+info)
Construction and consequences of directed mutations affecting the hemin receptor in pathogenic Corynebacterium species.
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Genes encoding an ATP-binding cassette transporter system involved in hemin iron utilization from Corynebacterium ulcerans were cloned and characterized. The genes are homologous to a hemin transport system previously identified in Corynebacterium diphtheriae. Disruption of the hmuT gene, which encodes the putative hemin receptor, resulted in greatly reduced ability of C. ulcerans to use hemin or hemoglobin as an iron source. Inactivation of hmuT in C. diphtheriae by site-specific recombination had no effect on hemin utilization, which suggests that C. diphtheriae has an additional system for transporting hemin. (+info)
Bioactivity of [6R]-5-formyltetrahydrofolate, an unusual isomer, in humans and Enterococcus hirae, and cytochrome c oxidation of 10-formytetrahydrofolate to 10-formyldihydrofolate.
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The bio-inactive C-6 isomer, [6R]-5-formyl-tetrahydrofolate (5-HCO-H(4)F), is not found in Nature. An oral dose of 13.5 micromol of [6R]-5-HCO-H(4)F in humans results in the appearance of the naturally occurring [6S]-5-methyl-tetrahydrofolate and relatively large amounts of other bioactive folates in plasma. The removal of the asymmetry at C-6 could account for these results. Two oxidized cytochrome c [cyt c (Fe3+)] molecules oxidize one 10-formyl-tetrahydrofolate (10-HCO-H(4)F) with second-order kinetics and a rate constant of 1.3 x 10(4) M(-1) x s(-1). The folate product of this oxidation reaction is 10-formyl-dihydrofolate (10-HCO-H(2)F), which has no C-6 asymmetric centre and is therefore bioactive. The folate-requiring bacterium, Enterococcus hirae, does not normally biosynthesize cytochromes but does so when given an exogenous source of haem (e.g. haemin). E. hirae grown in haemin-supplemented media for 3 days utilizes both [6R]- and [6S]-5-HCO-H(4)F in contrast to that grown in control medium, which utilizes only the [6S] isomer. Since known chemical reactions form 10-HCO-H(4)F from 5-HCO-H(4)F, the unusually large rate constant for the oxidation of 10-HCO-H(4)F by cyt c (Fe3+) may account for the unexpected bioactivity of [6R]-5-HCO-H(4)F in humans and in E. hirae grown in haemin-containing media. We used an unnatural C-6 folate isomer as a tool to reveal the possible in vivo oxidation of 10-HCO-H(4)F to 10-HCO-H(2)F; however, nothing precludes this oxidation from occurring in vivo with the natural C-6 isomer. (+info)