Heme utilization in Clostridium perfringens strain 13

Heme binding in the NEAT domains of IsdA and IsdC of Staphylococcus aureus

Heme is a highly abundant prosthetic group of proteins, which are involved in oxygen transport and redox biochemistry. The strong redox reactivity of heme can cause considerable toxicity if intracellular heme levels exceed the homeostatic range. A complex network of scavenger proteins, clearance mechanisms and metabolic detoxification pathways controls heme exposure and ensures save recycling of heme-derived iron. However, in certain disease states such as during hemolysis or rhabdomyolysis extracellular heme levels can reach excessive levels that overwhelm the normal homeostatic control mechanisms. In the presented research we have explored the range of adaptive and maladaptive cellular responses to excessive heme exposure. As a general concept we could link more adaptive responses to the professional heme clearance mechanisms of macrophages such as endocytosis of hemoglobin:haptoglobin complexes. These adaptive changes include enhanced oxidative detoxification and iron handling capacity as well as an altered macrophage immune phenotype with low human leukocyte antigen-D expression. The adaptive response of macrophages is a potential target of positive and negative pharmacologic regulation. As an example we demonstrate that glucocorticoid treatment in vitro and in vivo skews macrophage polarization towards enhanced Hb-heme detoxification and iron recycling, while the anti-malaria drug chloroquine negatively interferes with the protective adaption to Hb exposure. At the other end of the heme response spectrum we characterized the cellular changes during overt heme toxicity and defined their molecular mechanisms. In these studies we defined that excess cellular heme disrupts homeostasis by oxidative reactions and porphyrin mediated impairment of the reparative proteasome pathway, respectively. These heme activities synergistically disrupt cellular protein metabolism and cause cell death.

NO potently up-regulates vascular haem oxygenase-1 (HO-1), an inducible defensive protein that degrades haem to CO, iron and the antioxidant bilirubin. Since several pathological states are characterized by increased NO production and liberation of haem from haem-containing proteins, we examined how NO influences HO-1 induction mediated by haemin. Aortic endothelial cells treated with S-nitroso-N-acetylpenicillamine (SNAP), sodium nitroprusside (SNP) or diethylenetriamine-NONOate (DETA/NO) and haemin exhibited higher levels of haem oxygenase activity compared with cells exposed to NO donors or haemin alone. This was accompanied by a marked increase in bilirubin production and, notably, by a strong magnification of cellular haem uptake. A role for haem metabolites in modulating HO-1 expression by NO was assessed by exposing cells to SNAP, SNP or DETA/NO in medium derived from cells treated with haemin, which contained increased bilirubin levels. This treatment considerably potentiated HO-1 expression and haem oxygenase activity mediated by NO and the use of a haem oxygenase inhibitor abolished this effect. Both iron liberated during haem breakdown and the formation of nitroxyl anion from NO appeared to partially contribute to the amplifying phenomenon; in addition, medium from haemin-treated cells significantly augmented the release of NO by NO donors. Thus we have identified novel mechanisms related to the induction of HO-1 by NO indicating that the signalling actions of NO vary significantly in the presence of haem and haem metabolites, ultimately increasing the defensive abilities of the endothelium to counteract oxidative and nitrosative stress.

BackgroundClostridium perfringens is a Gram-positive anaerobic bacterium causing severe diseases such as gas gangrene and pseudomembranosus colitis, that are generally due to the secretion of powerful extracellular toxins. The expression of toxin genes is mainly regulated by VirR, the response regulator of a two-component system. Up to now few targets only are known for this regulator and mainly in one strain (Strain 13). Due to the high genomic and phenotypic variability in toxin production by different strains, the development of effective strategies to counteract C. perfringens infections requires methodologies to reconstruct the VirR regulon from genome sequences.ResultsWe implemented a two step computational strategy allowing to consider available information concerning VirR binding sites in a few species to scan all genomes of the same species, assuming the VirR targets are at least partially conserved across these strains. Results obtained are in agreement with previous works where experimental validation of the promoters have been performed and showed the presence of a core and an accessory regulon of VirR in C. perfringens strains with three target genes also located on plasmids. Moreover, the type E strain JGS1987 has the largest predicted regulon with as many as 10 VirR targets not found in the other genomes.ConclusionsIn this work we exploited available experimental information concerning the targets of the VirR toxin regulator in one C. perfringens strain to obtain plausible predictions concerning target genes in genomes and plasmids of nearby strains. Our predictions are available for wet-lab researchers working on less characterized C. perfringens strains that can thus design focused experiments reducing the search space of their experiments and increasing the probability of characterizing positive targets with less efforts. Main result was that the VirR regulon is variable in different C. perfringens strains with 4 genes controlled in all but one strains and most genes controlled in one or two strains only.

This thesis deals with the study of the properties of bispidine iron(IV)-oxo complexes. These are biomimetic model compounds for non-heme iron enzymes. The first part describes the thermal spin crossover behavior of the tetradentate bispidine ligands’ iron(II) complexes. These compounds were precisely characterized using UV-vis-NIR and Mosbauer spectroscopy as well as Evans NMR spectroscopy and SQUID measurements. The tetradentate bispidine ligands’ iron(IV)-oxo complexes were characterized using UV-vis-NIR and Mosbauer spectroscopy. They were also analyzed computationally with DFT calculations. In addition, it was possible to characterize a high spin iron(IV)-oxo species by Mosbauer spectroscopy. This species’ electron transfer properties were analyzed. The second part of this study is about the photocatalytic generation of an iron(IV) oxo species with water as oxygen source. [RuII(bpy)3] 2+ was used as a photosensitizer. After irradiating it with visible light it was oxidized with [CoIII(NH3)5(MeCN)] 3+ to [RuIII(bpy)3] 3+, which catalytically generates in situ an iron(IV)-oxo species with water as source of oxygen. This reaction is also interesting with regard to environmental issues as it seems promising to use solar energy to produce highly energetic species. The third part of this study covers the redox properties of the pentadentate bispidine ligands’ ferryl complexes when the redox inactive metal ion Sc3+ is present. The presence of Sc3+ shifts the redox potential of the ferryl complex to higher potentials. This effect can be used to manipulate the electron transfer properties of the ferryl compound. This FeIV(O)- Sc3+ complex can serve as a model compound for the Mn4CaO5 cluster in the photosystem II. The last part of this study is about the non heme iron-oxo-catalyzed methanogenesis. The ferryl complexes of the tetra- and pentadentate bispidine ligands produce methane from methionine and other thioethers. In the first step of the process, methionine is oxidized to methionine sulfoxide. A bifurcation of the reaction pathway to a sulfone or methyl radical production marks the second step of the process. The generation of methyl radicals was confirmed by spin trapping experiments. By way of detailed DFT calculations it was possible to shed light on the mechanism of methane formation in a highly oxidative environment.

Heme proteins, in particular cytochromes P450, have been extensively used in biocatalytic applications due to their high degree of regio-, chemo-, and stereoselectivity in oxene-transfer reactions. In 2013, it was shown for the first time that engineered heme proteins can also catalyze analogous carbene- and nitrene-transfer reactions. Research in this field has since grown dramatically, with emphasis on developing new heme protein variants to increase the scope of biotransformations accessible through these new transfer reactions. This thesis details the expansion of these new-to-nature carbene and nitrene-transfer reactions to include new substrate classes previously unexplored with iron-porphyrin proteins, the use of non-heme metalloproteins for these transformations, and steps toward improving the robustness of the new-to-nature biocatalytic platform. Chapter 1 introduces the steps the field of biocatalysis has taken toward engineering enzymes with new catalytic functions and the process by which these activities are discovered and enhanced. Chapter 2 details the discovery and engineering of heme proteins which catalyze the stereodivergent cyclopropanation of unactivated and electron-deficient alkenes via carbene transfer, expanding the substrate classes beyond styrenyl alkenes. Chapter 3 shows the development of engineered variants of a heme protein (Rhodothermus marinus nitric oxide dioxygenase) for the diastereodivergent synthesis of cyclopropanes functionalized with a pinacolborane moiety, enabling product diversification through standard cross-coupling reactions. In Chapter 4, a collection of non-heme metalloproteins is curated, and a non-heme iron enzyme (Pseudomonas savastanoi ethylene-forming enzyme) is shown to be both amenable to directed evolution and non-native ligand substitution to enhance its nitrene-transfer activity. Chapter 5 describes the expansion of sequence space targeted for screening in the serine-ligated cytochrome P411 from Bacillus megaterium (P411BM3) biocatalytic platform to enhance the mutational robustness of these remarkable enzymes. Overall, this work provides a framework for bringing model new-to-nature reactions to their full potential in synthetic biocatalytic reactions.

- Hiatus hernia (HH) is the herniation of stomach into the chest cavity through diaphragm. HH is idiopathic and hence its exact cause is not known; possible causes are age related changes in diaphragm and inborn large hiatus. [1] Cameron lesions are yet another indicative for hiatus hernia. The purpose of this case report is highlighting various sources of the above condition which lead to worsened patient condition leading to hospitalization. Current case is a presentation of heme fibroid, duodenal Cameron lesion and type 1 hiatus hernia leading to worsened iron deficiency anaemia. For the efficient management of patient a proton pump inhibitor and vitamin B complex was advised. Key words: Hiatus Hernia, Iron deficiency Anemia, Proton Pump Inhibitor, Heme Fibroid

Storage iron kinetics. 3. Study of desferrioxamine action by selective radioiron labels of RE and parenchymal cells.

Sideroblastic anemia is characterized by anemia with ring sideroblasts produced by the bone marrow. Sideroblasts are formed by disutilization and deposit of iron in the mitochondoria. There are two forms of sideroblastic anemia: congenital and acquired. Congenital sideroblastic anemia is caused by mutations in genes involved in heme biosynthesis, iron-sulfur (Fe-S) cluster biogenesis, or mitochondrial metabolism. Although there is a variation in the mutated genes among races, the most common congenital sideroblastic anemia is X-linked sideroblastic anemia caused by mutations in the erythroid-specific δ-aminolevulinate synthase gene, which is the first enzyme of heme biosynthesis in erythroid cells. The most commonly acquired sideroblastic anemia is myelodysplastic syndrome with ring sideroblasts (MDS-RS). It has been shown that the splicing factor 3b subunit 1 (SF3B1) gene, which is a core component of the RNA splicing complex, is highly mutated in MDS-RS, although the underlying mechanism of the onset of the disease by the mutation of the SF3B1 gene remains unclear. Molecular analysis will contribute to the development of effective treatment for congenital and acquired sideroblastic anemia, which are intractable diseases.

The cytochrome P450 sterol 14α-demethylase enzyme (CYP51) is the target of azole antifungals. Azoles block ergosterol synthesis, and thereby inhibit fungal growth, by binding in the active-site cavity of the enzyme and ligating the iron atom of the heme cofactor through a nitrogen atom of the azole.In this work, homology models of the CYP51 enzymes from Candida albicans were constructed based on the X-ray crystal structure of CYP51 from Saccharomyces cerevisiae .

Bacterioferritin-cytochrome from Azotobacter vinelandii is an unusual protein containing haem groups as well as iron core like other ferritin. This paper reports the purification of bacterioferritin by affinity chromatography and the formation of brick-red crystals from a solution containing MgCl2. The crystals are optical isotropic with maximum dimensions of 0.4 X 0.4 X 0.1 mm3. The preliminary X-ray crystallographic studies have been performed. 1.5 degrees unscreened precession photographs show that the crystals of bacterioferritin belong to the cubic system, space group I432, with cell dimension 230 A. There are probably 8 molecules in one cubic unit cell and the molecule might have 32 symmetry. A molecular diameter of 115 A is derived from the packing of the molecules and a molecular weight of 826,000 is estimated for bacterioferritin.

The disruption of the blood-brain barrier (BBB) occurs after ischemic and hemorrhagic stroke and contributes to secondary brain damage. Matrix metalloproteinase-9 (MMP9) has been identified to be the main mediator of post-stroke BBB disruption. It is unknown whether deposition of heme/iron in the brain following stroke would affect MMP9 expression. In this study, I have demonstrated that heme/iron up-regulated MMP9 expression in rat astrocytes and that this upregulation was most likely due to reactive oxygen species (ROS) generated by heme/iron deposition on cells. ROS can activate AP-1 and NFκB signaling pathways which were responsible for increased MMP9 expression. Inhibiting AP-1 and NFκB decreased MMP9 expression. Heme/iron deposition also activated Nrf-2 and increased the expression of neuroprotective heme oxygenase-1. My study suggests that heme and iron deposition generates ROS and increases MMP9 expression through AP-1 and NFκB signaling pathways and that targeting these pathways or clearance of heme and iron may modulate MMP9 expression for reduced damage.

The Reverse Vaccinology (RV) approach allows using genomic information for the delineation of new protein-based vaccines starting from an in silico analysis. The first powerful example of the application of the RV approach is given by the development of a protein-based vaccine against serogroup B Meningococcus. A similar approach was also used to identify new Staphylococcus aureus vaccine candidates, including the ferric hydroxamate-binding lipoprotein FhuD2. S. aureus is a widespread human pathogen, which employs various different strategies for iron uptake, including: (i) siderophore-mediated iron acquisition using the endogenous siderophores staphyloferrin A and B, (ii) siderophore-mediated iron acquisition using xeno-siderophores (the pathway exploited by FhuD2) and (iii) heme-mediated iron acquisition. In this work the high resolution crystal structure of FhuD2 in the iron (III)-siderophore-bound form was determined. FhuD2 belongs to the Periplasmic Binding Protein family (PBP ) class III, and is principally formed by two globular domains, at the N- and C-termini of the protein, that make up a cleft where ferrichrome-iron (III) is bound. The N- and C-terminal domains, connected by a single long α-helix, present Rossmann-like folds, showing a β-stranded core and an α-helical periphery, which do not undergo extensive structural rearrangement when they interact with the ligand, typical of class III PBP members. The structure shows that ferrichrome-bound iron does not come directly into contact with the protein; rather, the metal ion is fully coordinated by six oxygen donors of the hydroxamate groups of three ornithine residues, which, with the three glycine residues, make up the peptide backbone of ferrichrome. Furthermore, it was found that iron-free ferrichrome is able to subtract iron from transferrin. This study shows for the first time the structure of FhuD2, which was found to bind to siderophores ,and that the protein plays an important role in S. aureus colonization and infection phases.

The ability of a pathogenic bacterium to scavenge iron from its host is important for its growth and survival during an infection. Our studies on C. perfringens gas gangrene strain JIR325, a derivative of strain 13, showed that it is capable of utilizing both human hemoglobin and ferric chloride, but not human holo-transferrin, as an iron source for in vitro growth. Analysis of the C. perfringens strain 13 genome sequence identified a putative heme acquisition system encoded by an iron-regulated surface gene region that we have named the Cht (Clostridium perfringens heme transport) locus. This locus comprises eight genes that are co-transcribed and includes genes that encode NEAT domain-containing proteins (ChtD and ChtE) and a putative sortase (Srt). The ChtD, ChtE and Srt proteins were shown to be expressed in JIR325 cells grown under iron-limited conditions and were localized to the cell envelope. Moreover, the NEAT proteins, ChtD and ChtE, were found to bind heme. Both chtDE and srt mutants were constructed, but these mutants were not defective in hemoglobin or ferric chloride utilization. They were, however, attenuated for virulence when tested in a mouse myonecrosis model, although the virulence phenotype could not be restored via complementation and, as is common with such systems, secondary mutations were identified in these strains. In summary, this study provides evidence for the functional redundancies that occur in the heme transport pathways of this life threatening pathogen.