Integrated Optimization of the In Vivo Heme Biosynthesis Pathway and the In Vitro Iron Concentration for 5-Aminolevulinate Production

Reactive oxygen species (ROS)-mediated oxidative stress and DNA damage have recently been recognized as contributing to the efficacy of most bactericidal antibiotics, irrespective of their primary macromolecular targets. Inhibitors of targets involved in both combating oxidative stress as well as being required for in vivo survival may exhibit powerful synergistic action. This study demonstrates that the de novo arginine biosynthetic pathway in Mycobacterium tuberculosis (Mtb) is up-regulated in the early response to the oxidative stress-elevating agent isoniazid or vitamin C. Arginine deprivation rapidly sterilizes the Mtb de novo arginine biosynthesis pathway mutants ΔargB and ΔargF without the emergence of suppressor mutants in vitro as well as in vivo. Transcriptomic and flow cytometry studies of arginine-deprived Mtb have indicated accumulation of ROS and extensive DNA damage. Metabolomics studies following arginine deprivation have revealed that these cells experienced depletion of antioxidant thiols and accumulation of the upstream metabolite substrate of ArgB or ArgF enzymes. ΔargB and ΔargF were unable to scavenge host arginine and were quickly cleared from both immunocompetent and immunocompromised mice. In summary, our investigation revealed in vivo essentiality of the de novo arginine biosynthesis pathway for Mtb and a promising drug target space for combating tuberculosis.

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Inhibition of Vibrio anguillarum byPseudomonas fluorescens AH2, a Possible Probiotic Treatment of Fish

Applied and Environmental Microbiology ◽

10.1128/aem.65.3.969-973.1999 ◽

1999 ◽

Vol 65 (3) ◽

pp. 969-973 ◽

Cited By ~ 243

Author(s):

Lone Gram ◽

Jette Melchiorsen ◽

Bettina Spanggaard ◽

Ingrid Huber ◽

Torben F. Nielsen

Keyword(s):

Iron Concentration ◽

Vibrio Anguillarum ◽

Fish Farming ◽

Fish Pathogen ◽

Probiotic Treatment ◽

Treated Fish ◽

Culture Supernatants ◽

Probiotic Effect

ABSTRACT To study the possible use of probiotics in fish farming, we evaluated the in vitro and in vivo antagonism of antibacterial strainPseudomonas fluorescens strain AH2 against the fish-pathogenic bacterium Vibrio anguillarum. As iron is important in virulence and bacterial interactions, the effect ofP. fluorescens AH2 was studied under iron-rich and iron-limited conditions. Sterile-filtered culture supernatants from iron-limited P. fluorescens AH2 inhibited the growth ofV. anguillarum, whereas sterile-filtered supernatants from iron-replete cultures of P. fluorescens AH2 did not.P. fluorescens AH2 inhibited the growth of V. anguillarum during coculture, independently of the iron concentration, when the initial count of the antagonist was 100 to 1,000 times greater that of the fish pathogen. These in vitro results were successfully repeated in vivo. A probiotic effect in vivo was tested by exposing rainbow trout (Oncorynchus mykissWalbaum) to P. fluorescens AH2 at a density of 105 CFU/ml for 5 days before a challenge with V. anguillarum at 104 to 105 CFU/ml for 1 h. Some fish were also exposed to P. fluorescens AH2 at 107 CFU/ml during the 1-h infection. The combined probiotic treatment resulted in a 46% reduction of calculated accumulated mortality; accumulated mortality was 25% after 7 days at 12°C in the probiotic-treated fish, whereas mortality was 47% in fish not treated with the probiont.

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Ferritin accumulation and degradation in different organs of pea (Pisum sativum) during development

Biochemical Journal ◽

10.1042/bj2740601 ◽

1991 ◽

Vol 274 (2) ◽

pp. 601-606 ◽

Cited By ~ 98

Author(s):

S Lobreaux ◽

J F Briat

Keyword(s):

Pisum Sativum ◽

Total Protein ◽

Iron Concentration ◽

Embryo Axis ◽

Iron Supply ◽

Hydroponic Cultures ◽

Radical Damage ◽

Pea Seed

Iron concentration and ferritin distribution have been determined in different organs of pea (Pisum sativum) during development under conditions of continuous iron supply from hydroponic cultures. No ferritin was detected in total protein extracts from roots or leaves. However, a transient iron accumulation in the roots, which corresponds to an increase in iron uptake, was observed when young fruits started to develop. Ferritin was detectable in total protein extracts of flowers and pods, and it accumulated in seeds. In seeds, the same relative amount of ferritin was detected in cotyledons and in the embryo axis. In cotyledons, ferritin and iron concentration decrease progressively during the first week of germination. Ferritin in the embryo axis was processed, and disappeared, during germination, within the first 4 days of radicle and epicotyl growth. This degradation of ferritin in vivo was marked by a shortening of a 28 kDa subunit, giving 26.5 and 25 kDa polypeptides, reminiscent of the radical damage occurring in pea seed ferritin during iron exchange in vitro [Laulhere, Laboure & Briat (1989) J. Biol. Chem. 264, 3629-3635]. Developmental control of iron concentration and ferritin distribution in different organs of pea is discussed.

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The Alternative Route to Heme in the Methanogenic ArchaeonMethanosarcina barkeri

Archaea ◽

10.1155/2014/327637 ◽

2014 ◽

Vol 2014 ◽

pp. 1-13 ◽

Cited By ~ 26

Author(s):

Melanie Kühner ◽

Kristin Haufschildt ◽

Alexander Neumann ◽

Sonja Storbeck ◽

Judith Streif ◽

...

Keyword(s):

Enzyme Activity ◽

Feedback Regulation ◽

Methanosarcina Barkeri ◽

Heme Biosynthesis ◽

Alternative Route ◽

Activity Assay ◽

Enzyme Activity Assay ◽

Living Organisms

In living organisms heme is formed from the common precursor uroporphyrinogen III by either one of two substantially different pathways. In contrast to eukaryotes and most bacteria which employ the so-called “classical” heme biosynthesis pathway, the archaea use an alternative route. In this pathway, heme is formed from uroporphyrinogen III via the intermediates precorrin-2, sirohydrochlorin, siroheme, 12,18-didecarboxysiroheme, and iron-coproporphyrin III. In this study the heme biosynthesis proteins AhbAB, AhbC, and AhbD fromMethanosarcina barkeriwere functionally characterized. Using anin vivoenzyme activity assay it was shown that AhbA and AhbB (Mbar_A1459 and Mbar_A1460) together catalyze the conversion of siroheme into 12,18-didecarboxysiroheme. The two proteins form a heterodimeric complex which might be subject to feedback regulation by the pathway end-product heme. Further, AhbC (Mbar_A1793) was shown to catalyze the formation of iron-coproporphyrin IIIin vivo. Finally, recombinant AhbD (Mbar_A1458) was produced inE. coliand purified indicating that this protein most likely contains two [4Fe-4S] clusters. Using anin vitroenzyme activity assay it was demonstrated that AhbD catalyzes the conversion of iron-coproporphyrin III into heme.

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The Gonococcal Fur-Regulated tbpA and tbpB Genes Are Expressed during Natural Mucosal Gonococcal Infection

Infection and Immunity ◽

10.1128/iai.73.7.4281-4287.2005 ◽

2005 ◽

Vol 73 (7) ◽

pp. 4281-4287 ◽

Cited By ~ 19

Author(s):

Sarika Agarwal ◽

Carol A. King ◽

Ellen K. Klein ◽

David E. Soper ◽

Peter A. Rice ◽

...

Keyword(s):

Gene Expression ◽

Expression Profiles ◽

Iron Concentration ◽

Pcr Analysis ◽

Positive Trend ◽

Gonococcal Infection ◽

Rt Pcr ◽

Male Subjects

ABSTRACT Iron is limiting in the human host, and bacterial pathogens respond to this environment by regulating gene expression through the ferric uptake regulator protein (Fur). In vitro studies have demonstrated that Neisseria gonorrhoeae controls the expression of several critical genes through an iron- and Fur-mediated mechanism. While most in vitro experiments are designed to determine the response of N. gonorrhoeae to an exogenous iron concentration of zero, these organisms are unlikely to be exposed to such severe limitations of iron in vivo. To determine if N. gonorrhoeae expresses iron- and Fur-regulated genes in vivo during uncomplicated gonococcal infection, we examined gene expression profiles of specimens obtained from male subjects with urethral infections. RNA was isolated from urethral swab specimens and used as a template to amplify, by reverse transcriptase PCR (RT-PCR), gonococcal genes known to be regulated by iron and Fur (tbpA, tbpB, and fur). The constitutively expressed gonococcal rmp gene was used as a positive control. RT-PCR analysis indicated that gonorrhea-positive specimens where rmp expression was seen were also 93% (51/55) fbpA positive, 87% (48/55) tbpA positive, and 86% (14 of 16 tested) tbpB positive. In addition, we detected a fur transcript in 79% (37 of 47 tested) of positive specimens. We also measured increases in levels of immunoglobulin G antibody against TbpA (91%) and TbpB (73%) antigens in sera from infected male subjects compared to those in uninfected controls. A positive trend between tbpA gene expression and TbpA antibody levels in sera indicated a relationship between levels of gene expression and immune response in male subjects infected with gonorrhea for the first time. These results indicate that gonococcal iron- and Fur-regulated tbpA and tbpB genes are expressed in gonococcal infection and that male subjects with mucosal gonococcal infections exhibit antibodies to these proteins.

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Thiol Redox Sensitivity of Two Key Enzymes of Heme Biosynthesis and Pentose Phosphate Pathways: Uroporphyrinogen Decarboxylase and Transketolase

Oxidative Medicine and Cellular Longevity ◽

10.1155/2013/932472 ◽

2013 ◽

Vol 2013 ◽

pp. 1-13 ◽

Cited By ~ 8

Author(s):

Brian McDonagh ◽

José Rafael Pedrajas ◽

C. Alicia Padilla ◽

José Antonio Bárcena

Keyword(s):

Redox Regulation ◽

Tumour Progression ◽

Pentose Phosphate ◽

Heme Biosynthesis ◽

Label Free ◽

Uroporphyrinogen Decarboxylase ◽

Cell Extracts ◽

Thiol Redox

Uroporphyrinogen decarboxylase (Hem12p) and transketolase (Tkl1p) are key mediators of two critical processes within the cell, heme biosynthesis, and the nonoxidative part of the pentose phosphate pathway (PPP). The redox properties of both Hem12p and Tkl1p fromSaccharomyces cerevisiaewere investigated using proteomic techniques (SRM and label-free quantification) and biochemical assays in cell extracts andin vitrowith recombinant proteins. Thein vivoanalysis revealed an increase in oxidized Cys-peptides in the absence of Grx2p, and also after treatment with H2O2in the case of Tkl1p, without corresponding changes in total protein, demonstrating a true redox response. Out of three detectable Cys residues in Hem12p, only the conserved residue Cys52 could be modified by glutathione and efficiently deglutathionylated by Grx2p, suggesting a possible redox control mechanism for heme biosynthesis. On the other hand, Tkl1p activity was sensitive to thiol redox modification and although Cys622 could be glutathionylated to a limited extent, it was not a natural substrate of Grx2p. The human orthologues of both enzymes have been involved in certain cancers and possess Cys residues equivalent to those identified as redox sensitive in yeast. The possible implication for redox regulation in the context of tumour progression is put forward.

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Duodenal cytochrome b: a novel ferrireductase in airway epithelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00342.2005 ◽

2006 ◽

Vol 291 (2) ◽

pp. L272-L280 ◽

Cited By ~ 17

Author(s):

Jennifer L. Turi ◽

Xinchao Wang ◽

Andrew T. McKie ◽

Eva Nozik-Grayck ◽

Lisa B. Mamo ◽

...

Keyword(s):

Epithelial Cells ◽

Cytochrome B ◽

Iron Transport ◽

Iron Concentration ◽

Airway Epithelial Cells ◽

Airway Epithelial ◽

Cellular Iron ◽

Duodenal Cytochrome B

Catalytically active iron in the lung causes oxidative stress and promotes microbial growth that can be limited by intracellular sequestration of iron within ferritin. Because cellular iron uptake requires membrane ferrireductase activity that in the gut can be provided by duodenal cytochrome b (Dcytb), we sought Dcytb in the lung to test the hypothesis that it contributes to epithelial iron regulation by reducing Fe3+ for cellular iron transport. Dcytb expression was found in respiratory epithelium in vitro and in vivo and was responsive to iron concentration. Iron transport was measured in human bronchial epithelial (HBE) cells using inductively coupled plasma atomic emission spectroscopy and was demonstrated to be partially inhibited in the presence of Dcytb-blocking antibody, suggesting that Dcytb reduces Fe3+ for cellular iron transport. A definite source of reducing equivalents for Dcytb was sought but not identified. We found no evidence that ascorbate was involved but did demonstrate that O2−· production decreased when Dcytb function was blocked. The presence of Dcytb in airway epithelial cells and its regulation by iron therefore may contribute to pulmonary cytoprotection.

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Differential Fluorescence Induction Analysis of Streptococcus pneumoniae Identifies Genes Involved in Pathogenesis

Infection and Immunity ◽

10.1128/iai.70.3.1422-1433.2002 ◽

2002 ◽

Vol 70 (3) ◽

pp. 1422-1433 ◽

Cited By ~ 77

Author(s):

Andrea Marra ◽

Jyoti Asundi ◽

Magdalena Bartilson ◽

Stacey Lawson ◽

Flora Fang ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Fluorescent Protein ◽

Iron Concentration ◽

Fluorescence Induction ◽

Infection Model ◽

Infection Models ◽

Promoter Trap ◽

Green Fluorescent

ABSTRACT Differential fluorescence induction (DFI) technology was used to identify promoters of Streptococcus pneumoniae induced under various in vitro and in vivo conditions. A promoter-trap library using green fluorescent protein as the reporter was constructed in S. pneumoniae, and the entire library was screened for clones exhibiting increased gfp expression under the chosen conditions. The in vitro conditions used were chosen to mimic aspects of the in vivo environment encountered by the pathogen once it enters a host: changes in temperature, osmolarity, oxygen, and iron concentration, as well as blood. In addition, the library was used to infect animals in three different models, and clones induced in these environments were identified. Several promoters were identified in multiple screens, and genes whose promoters were induced twofold or greater under the inducing condition were mutated to assess their roles in virulence. A total of 25 genes were mutated, and the effects of the mutations were assessed in at least two different infection models. Over 50% of these mutants were attenuated in at least one infection model. We show that DFI is a useful tool for identifying bacterial virulence factors as well as a means of elucidating the microenvironment encountered by pathogens upon infection.

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Biliary Iron Excretion in Rats Following Treatment With Analogs of Pyridoxal Isonicotinoyl Hydrazone

Blood ◽

10.1182/blood.v91.11.4368 ◽

1998 ◽

Vol 91 (11) ◽

pp. 4368-4372 ◽

Cited By ~ 20

Author(s):

Karel Bláha ◽

Miroslav Cikrt ◽

Jana Nerudová ◽

Helena Fornusková ◽

Premysl Ponka

Keyword(s):

Iron Overload ◽

Iron Concentration ◽

Clinical Manifestations ◽

Thalassemia Major ◽

Iron Chelator ◽

Atomic Absorption Spectrophotometry ◽

Major Life ◽

Pyridoxal Isonicotinoyl Hydrazone

Abstract Iron overload is a major life-threatening complication of thalassemia major and other iron-loading anemias treated by regular blood transfusions. Although the clinical manifestations of iron overload may be prevented by desferrioxamine, the only iron-chelating drug in routine clinical use, this treatment requires subcutaneous infusion of desferrioxamine for 12 hours each day. New orally effective iron chelators are urgently needed, and pyridoxal isonicotinoyl hydrazone (PIH), which was first recognized as an effective iron chelator in vitro and subsequently in vivo, shows promise for the treatment of iron overload. More recently, over 40 analogs of PIH were synthesized, and some of them proved to be very potent in mobilizing 59Fe in vitro from 59Fe-labeled cells. In this study, we show that PIH analogs such as pyridoxal benzoyl hydrazone, pyridoxal p-methoxybenzoyl hydrazone (PMBH), pyridoxal m-fluorobenzoyl hydrazone (PFBH), and pyridoxal-2-thiophenecarboxyl hydrazone, compounds previously shown to mobilize iron from cells in vitro, are also effective in vivo. All of these chelators significantly enhanced biliary excretion of iron (measured by atomic absorption spectrophotometry) following their intraperitoneal (IP) and/or oral administration to rats. The most effective was PFBH, which increased iron concentration in the bile about 150-fold, as compared with basal biliary iron concentration, within 1 hour following a single IP dose of 0.2 mmol/kg body weight. In contrast, desferrioxamine increased the biliary iron concentration only 20-fold to 30-fold under the same conditions. Moreover, while control rats excreted ≈ 0.8 μg Fe in 2 hours, treatment with PFBH, PMBH, and desferrioxamine resulted in cumulative excretions of 87, 59, and 22 μg Fe, respectively, in the same period of time. Interestingly, PMBH was also quite effective following gastric administration, resulting in a 6-hour cumulative value of 34 μg Fe. These compounds are nontoxic and are inexpensive and easy to make. Their further evaluation as candidate drugs for the treatment of iron overload is warranted.

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Copper-dependent activation of hypoxia-inducible factor (HIF)-1: implications for ceruloplasmin regulation

Blood ◽

10.1182/blood-2004-10-3980 ◽

2005 ◽

Vol 105 (12) ◽

pp. 4613-4619 ◽

Cited By ~ 184

Author(s):

Falk Martin ◽

Tobias Linden ◽

Dörthe M. Katschinski ◽

Felix Oehme ◽

Ingo Flamme ◽

...

Keyword(s):

Gene Expression ◽

Glucose Transporter ◽

Iron Concentration ◽

Hypoxia Inducible Factor ◽

Sensory System ◽

Mrna Levels ◽

Copper Binding ◽

Glucose Transporter 1

Abstract Cellular oxygen partial pressure is sensed by a family of prolyl-4-hydroxylase domain (PHD) enzymes that modify hypoxia-inducible factor (HIF)α subunits. Upon hydroxylation under normoxic conditions, HIFα is bound by the von Hippel-Lindau tumor suppressor protein and targeted for proteasomal destruction. Since PHD activity is dependent on oxygen and ferrous iron, HIF-1 mediates not only oxygen- but also iron-regulated transcriptional gene expression. Here we show that copper (CuCl2) stabilizes nuclear HIF-1α under normoxic conditions, resulting in hypoxia-response element (HRE)-dependent reporter gene expression. In in vitro hydroxylation assays CuCl2 inhibited prolyl-4-hydroxylation independently of the iron concentration. Ceruloplasmin, the main copper transport protein in the plasma and a known HIF-1 target in vitro, was also induced in vivo in the liver of hypoxic mice. Both hypoxia and CuCl2 increased ceruloplasmin (as well as vascular endothelial growth factor [VEGF] and glucose transporter 1 [Glut-1]) mRNA levels in hepatoma cells, which was due to transcriptional induction of the ceruloplasmin gene (CP) promoter. In conclusion, our data suggest that PHD/HIF/HRE-dependent gene regulation can serve as a sensory system not only for oxygen and iron but also for copper metabolism, regulating the oxygen-, iron- and copper-binding transport proteins hemoglobin, transferrin, and ceruloplasmin, respectively. (Blood. 2005;105:4613-4619)

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