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1.
Mahamoudou Ouattara Andrea Pennati Darius J. Devlin Ya-Shu Huang Giovanni Gadda Zehava Eichenbaum 《Archives of biochemistry and biophysics》2013
The hemolytic Group A Streptococcus (GAS) is a notorious human pathogen. Shr protein of GAS participates in iron acquisition by obtaining heme from host hemoglobin and delivering it to the adjacent receptor on the surface, Shp. Heme is then conveyed to the SiaABC proteins for transport across the membrane. Using rapid kinetic studies, we investigated the role of the two heme binding NEAT modules of Shr. Stopped-flow analysis showed that holoNEAT1 quickly delivered heme to apoShp. HoloNEAT2 did not exhibit such activity; only little and slow transfer of heme from NEAT2 to apoShp was seen, suggesting that Shr NEAT domains have distinctive roles in heme transport. HoloNEAT1 also provided heme to apoNEAT2, by a fast and reversible process. To the best of our knowledge this is the first transfer observed between isolated NEAT domains of the same receptor. Sequence alignment revealed that Shr NEAT domains belong to two families of NEAT domains that are conserved in Shr orthologs from several species. Based on the heme transfer kinetics, we propose that Shr proteins modulate heme uptake according to heme availability by a mechanism where NEAT1 facilitates fast heme delivery to Shp, whereas NEAT2 serves as a temporary storage for heme on the bacterial surface. 相似文献
2.
Direct hemin transfer from IsdA to IsdC in the iron-regulated surface determinant (Isd) heme acquisition system of Staphylococcus aureus 总被引:1,自引:0,他引:1
Liu M Tanaka WN Zhu H Xie G Dooley DM Lei B 《The Journal of biological chemistry》2008,283(11):6668-6676
The iron-regulated surface determinants (Isd) of Staphylococcus aureus, including surface proteins IsdA, IsdB, IsdC, and IsdH and ATP-binding cassette transporter IsdDEF, constitute the machinery for acquiring heme as a preferred iron source. Here we report hemin transfer from hemin-containing IsdA (holo-IsdA) to hemin-free IsdC (apo-IsdC). The reaction has an equilibrium constant of 10 +/- 5 at 22 degrees C in favor of holo-IsdC formation. During the reaction, holo-IsdA binds to apo-IsdC and then transfers the cofactor to apo-IsdC with a rate constant of 54.3 +/- 1.8 s(-1) at 25 degrees C. The transfer rate is >70,000 times greater than the rate of simple hemin dissociation from holo-IsdA into solvent (k transfer = 54.3 s(-1) versus k -hemin = 0.00076 s(-1)). The standard free energy change, Delta G 0, is -27 kJ/mol for the formation of the holo-IsdA-apo-IsdC complex. IsdC has a higher affinity for hemin than IsdA. These results indicate that the IsdA-to-IsdC hemin transfer is through the activated holo-IsdA-apo-IsdC complex and is driven by the higher affinity of apo-IsdC for the cofactor. These findings demonstrate for the first time in the Isd system that heme transfer is rapid, direct, and affinity-driven from IsdA to IsdC. These results also provide the first example of heme transfer from one surface protein to another surface protein in Gram-positive bacteria and, perhaps most importantly, indicate that the mechanism of activated heme transfer, which we previously demonstrated between the streptococcal proteins Shp and HtsA, may apply in general to all bacterial heme transport systems. 相似文献
3.
In this review the superantigens (SAG) of group A Streptococcus, mainly pyrogenic toxins, and the coding genes are characterized. Specific sites on the molecules of the main histocompatibility complex, class II, and T-receptors, interacting with SAG, are described. This interaction results in hyperproduction of cytokines posing a systemic influence and leading to severe consequences. The data on the state of protective immunity in SAG-associated diseases and some approaches to their treatment are given. 相似文献
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The pathogenic bacterium Staphylococcus aureus has adopted specialized mechanisms for scavenging iron from its host. The cell-wall- and cell-membrane-associated iron-regulated surface determinant (Isd) proteins (IsdH, IsdB, IsdA, IsdC, IsdDEF, IsdG, and IsdI) allow S. aureus to scavenge iron from the heme in hemoglobin and haptoglobin-hemoglobin. Of these, IsdE chaperones heme to the ATP-binding-cassette-type transmembrane transporter (IsdF). IsdH, IsdB, IsdA, and IsdC contain at least one heme-binding near transporter (NEAT) domain. Previous studies have shown that ferric heme is transferred unidirectionally in the sequence IsdA-NEAT (Tyr-proximal amino acid)?→?IsdC-NEAT (Tyr)?→?IsdE (His). IsdA-NEAT does not transfer heme directly to IsdE. To challenge and probe this unusual unidirectional mechanism, the double mutant IsdE(M78A; H229A)-IsdE(MH)-was constructed and used in studies of heme transfer between IsdA-NEAT, IsdC-NEAT, and IsdE. This study probed the specific requirements in the heme binding site that enforce the unidirectional property of the system. Significantly, heme transfer from holo-IsdE(MH) to apo-IsdA-NEAT now occurs, breaking the established mechanism. The unique unidirectional heme-transfer properties now function under an affinity-driven mechanism. Overall, the heme proximal and distal ligands must play a crucial role controlling a gate that stops heme transfer between the native IsdE and IsdA-NEAT. We propose that these amino acids are the key control elements in the specific unidirectional protein-protein-gated release mechanism exhibited by the Isd system. 相似文献
6.
A carotenoid (Car), a chlorophyll (Chl(Z)), and cytochrome b(559) (Cyt b(559)) are able to donate electrons with a low quantum yield to the photooxidized chlorophyll, P680(+), when photosystem II (PSII) is illuminated at low temperatures. Three pathways for electron transfer from Cyt b(559) to P680(+) are considered: (a) the "linear pathway" in which Cyt b(559) donates via Chl(Z) to Car, (b) the "branched pathway" in which Cyt b(559) donates via Car and where Chl(Z) is also able to donate to Car, and (c) the "parallel pathway" where Cyt b(559) donates to P680 without intermediate electron carriers and electron donation from Chl(Z) and Car occurs by a competing pathway. Experiments were performed using EPR and spectrophotometry in an attempt to distinguish among these pathways, and the following observations were made. (1) Using PSII with an intact Mn cluster in which Cyt b(559) was preoxidized, Car oxidation was dominant upon illumination at < or =20 K, while electron donation from Chl dominated at >120 K. (2) When Cyt b(559) was prereduced, its light-induced oxidation occurred at < or =20 K in what appeared to be all of the centers and without the formation of a detectable Car(+) intermediate. The small and variable quantity of Car(+) photoinduced in these experiments can be attributed to the residual centers in which Cyt b(559) remained oxidized prior to illumination. (3) The relative rates for irreversible electron donation from Cyt b(559) and Car were determined indirectly at 20 K by monitoring the flash-induced loss of charge separation (i.e., the accumulation of Cyt b(559)(+)Q(A)(-) or Car(+)Q(A)(-)). Similar yields per flash were observed (13% for Cyt b(559) and 8% for Car), indicating similar donation rates. The slightly lower yield with Car as a donor is attributed at least in part to slow charge recombination occurring from the Car(+)Q(A)(-) radical pair in a fraction of centers. (4) Light-induced oxidation of Cyt b(559) and Car at 20 K was monitored directly by EPR, and the rates were found to be indistinguishable. The parallel pathway predicts that when both Cyt b(559) and Car are prereduced, the relative amounts of Cyt b(559)(+) and Car(+) produced upon illumination at 20 K should depend directly on their relative electron donation rates. The measured similarity in the donation rates thus predicts comparable yields of oxidation for both donors. However, what is observed experimentally is that Cyt b(559) oxidation occurs almost exclusively, and this argues strongly against the parallel pathway. The lack of Car(+) as a detectable intermediate is attributed to rapid electron transfer from Cyt b(559) to Car(+). The trapping of Car(+) at low temperature when Cyt b(559) is preoxidized but its absence when Cyt b(559) is prereduced is taken as an argument against the simple linear pathway. Overall, the data reported here and previously favor the branched pathway over the linear pathway, while the parallel pathway is thought to be unlikely. Structural considerations provide further arguments in favor of the branched model. 相似文献
7.
Demonstration of the iron-regulated surface determinant (Isd) heme transfer pathway in Staphylococcus aureus 总被引:1,自引:0,他引:1
Muryoi N Tiedemann MT Pluym M Cheung J Heinrichs DE Stillman MJ 《The Journal of biological chemistry》2008,283(42):28125-28136
In this study, we report experimental results that provide the first complete challenge of a proposed model for heme acquisition by Staphylococcus aureus via the Isd pathway first put forth by Mazmanian, S. K., Skaar, E. P., Gaspar, A. H., Humayun, M., Gornicki, P., Jelenska, J., Joachmiak, A., Missiakas, D. M., and Schneewind, O. (2003) Science 299, 906-909. The heme-binding NEAT domains of Isd proteins IsdA, IsdB (domain 2), IsdC, and HarA/IsdH (domain 3), and the heme-binding IsdE protein, were overexpressed and purified in apo (heme-free) form. Absorption and magnetic circular dichroism spectral data, together with electrospray ionization mass spectrometry were used to unambiguously identify that heme transfers from NEAT-A through NEAT-C to IsdE. Heme transfer was demonstrated to occur in a unidirectional fashion in the sequence NEAT-B2 --> NEAT-A --> NEAT-C --> IsdE or, alternatively, initiating from NEAT-H3 instead of NEAT-B2: NEAT-H3 --> NEAT-A --> NEAT-C --> IsdE. Under the conditions of our experiments, only NEAT-H3 and NEAT-B2 could transfer bidirectionally, which is in the reverse direction as well, and only with each other. Whereas apo-IsdE readily accepted heme from holo-NEAT-C, it would not accept heme from holo-NEAT-A. Heme transfer to IsdE requires the presence of holo-NEAT-C, in agreement with the proposal that IsdC serves as the central conduit of the heme transfer pathway. These experimental findings corroborate the heme transfer model first proposed by the Schneewind group. Our data show that heme transport from the wall-anchored IsdH/IsdB proteins proceeds directly to IsdE at the membrane and, for this to occur, we propose that specific protein-protein interactions must take place. 相似文献
8.
《Autophagy》2013,9(3):154-155
We have identified several mammalian protein components of the autophagy pathway. By using them as powerful tools to examine the functional significance of this degradation pathway, we recently showed that autophagy efficiently kills a pathogenic bacterium, Group A Streptococcus, after it invades host cells. However, the autophagosomes induced by these bacteria have features distinct from those of the canonical pathway. 相似文献
9.
Yoshimori T 《Autophagy》2006,2(3):154-155
We have identified several mammalian protein components of the autophagy pathway. By using them as powerful tools to examine the functional significance of this degradation pathway, we recently showed that autophagy efficiently kills a pathogenic bacterium, Group A Streptococcus, after it invades host cells. However, the autophagosomes induced by these bacteria have features distinct from those of the canonical pathway. 相似文献
10.
A novel approach is described for generating reactive oxidizing centers in heme proteins, with zinc hemoglobin (Zn Hb) and zinc cytochrome c (Zn cyt c) used as examples. The reaction of 3Zn* Hb with [CoIII(NH3)5 Cl]2+, and of 3Zn* cyt c with methyl viologen are described. In the case of Zn Hb the cation radical produced decays with a rate constant of k3 = 2400s-1. Using this value the rate of the reaction (formula; see text) can be calculated to be 4500s-1. 相似文献
11.
Maximal activity of CYP3A4 is obtained using a reconstitution system consisting of NADPH-P450 reductase (CPR), dioleoylphosphatidylcholine (DOPC), an ionic detergent, and cytochrome b(5) (b(5)). The mechanism by which b(5) stimulates the catalytic activity of CYP3A4 is controversial. Recent data report that apo-cytochrome b(5) (apo-b(5)) can substitute for holo-b(5) by serving as an allosteric effector. These authors concluded that b(5) is not directly involved in electron transfer reactions to CYP3A4. We have studied the effect of apo-b(5) on the ability of purified CYP3A4 to catalyze the 6beta-hydroxylation of testosterone and horse CYP17A to catalyze the 17,20-lyase reaction. The high molecular weight form of holo-b(5) (HMW-holo-b(5)) stimulates the 6beta-hydroxylation of testosterone while the low molecular weight (truncated) form of holo-b(5) (LMW-holo-b(5)) does not. When added to the reconstituted system, HMW-apo-b(5) stimulates the activity of CYP3A4 to a level 50-60% of that obtained with HMW-holo-b(5). A similar stimulation of 17alpha-hydroxyprogesterone metabolism is seen when studying the CYP17A-catalyzed reaction. Neither LMW-holo-b(5) nor LMW-apo-b(5) stimulates the activity of CYP3A4 or CYP17A. CYP3A4 forms a complex during affinity chromatography with immobilized HMW-holo-b(5) but not with immobilized HMW-apo-b(5). Incubation of apo-b(5) with CYP3A4, using conditions required for reconstitution of enzymatic activities, results in the transfer of heme from the CYP3A4 preparation to apo-b(5), thereby forming holo-b(5). The separation of heme proteins by thiol-disulfide exchange chromatography confirms the formation of holo-b(5). A His67Ala mutant of HMW-b(5) as well as the Zn-substituted protoporphyrin derivative of HMW-b(5) do not stimulate the activity of either CYP3A4 or CYP17A. These data show that the mechanism of stimulation of CYP3A4 and CYP17A activities by apo-b(5) results from the formation of holo-b(5) by a heme transfer reaction. 相似文献
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Ankita J. Sachla Mahamoudou Ouattara Elvira Romero Johnson Agniswamy Irene T. Weber Giovanni Gadda 《Biometals》2016,29(4):593-609
In Group A streptococcus (GAS), the metallorepressor MtsR regulates iron homeostasis. Here we describe a new MtsR-repressed gene, which we named hupZ (heme utilization protein). A recombinant HupZ protein was purified bound to heme from Escherichia coli grown in the presence of 5-aminolevulinic acid and iron. HupZ specifically binds heme with stoichiometry of 1:1. The addition of NADPH to heme-bound HupZ (in the presence of cytochrome P450 reductase, NADPH-regeneration system and catalase) triggered progressive decrease of the HupZ Soret band and the appearance of an absorption peak at 660 nm that was resistance to hydrolytic conditions. No spectral changes were observed when ferredoxin and ferredoxin reductase were used as redox partners. Differential spectroscopy with myoglobin or with the ferrous chelator, ferrozine, confirmed that carbon monoxide and free iron are produced during the reaction. ApoHupZ was crystallized as a homodimer with a split β-barrel conformation in each monomer comprising six β strands and three α helices. This structure resembles the split β-barrel domain shared by the members of a recently described family of heme degrading enzymes. However, HupZ is smaller and lacks key residues found in the proteins of the latter group. Phylogenetic analysis places HupZ on a clade separated from those for previously described heme oxygenases. In summary, we have identified a new GAS enzyme-containing split β-barrel and capable of heme biotransformation in vitro; to the best of our knowledge, this is the first enzyme among Streptococcus species with such activity. 相似文献
14.
Fischetti VA 《Trends in microbiology》2007,15(7):297-300
Genomic analysis of 12 Streptococcus pyogenes genomes representing six different serotypes reveals that they are poly-lysogenized, with as many as seven separate phage genomes (some of which are defective). Sequence alignments of these genomes (excluding incorporated prophage) have revealed that they are approximately 90% conserved, indicating that their diversity and disease capacity might be phage related. However, because S. pyogenes are only found in humans, how are new phages acquired? In vitro and in vivo experiments show that efficient phage transfer from donor to recipient streptococci occurs in the presence of mammalian cells. This suggests that, through evolution, phage have devised a system whereby progeny phage are induced and transferred to host streptococci at a site where host organisms are more prevalent. 相似文献
15.
Nozawa T Furukawa N Aikawa C Watanabe T Haobam B Kurokawa K Maruyama F Nakagawa I 《PloS one》2011,6(5):e19543
Streptococcus pyogenes, one of the major human pathogens, is a unique species since it has acquired diverse strain-specific virulence properties mainly through the acquisition of streptococcal prophages. In addition, S. pyogenes possesses clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems that can restrict horizontal gene transfer (HGT) including phage insertion. Therefore, it was of interest to examine the relationship between CRISPR and acquisition of prophages in S. pyogenes. Although two distinct CRISPR loci were found in S. pyogenes, some strains lacked CRISPR and these strains possess significantly more prophages than CRISPR harboring strains. We also found that the number of spacers of S. pyogenes CRISPR was less than for other streptococci. The demonstrated spacer contents, however, suggested that the CRISPR appear to limit phage insertions. In addition, we found a significant inverse correlation between the number of spacers and prophages in S. pyogenes. It was therefore suggested that S. pyogenes CRISPR have permitted phage insertion by lacking its own spacers. Interestingly, in two closely related S. pyogenes strains (SSI-1 and MGAS315), CRISPR activity appeared to be impaired following the insertion of phage genomes into the repeat sequences. Detailed analysis of this prophage insertion site suggested that MGAS315 is the ancestral strain of SSI-1. As a result of analysis of 35 additional streptococcal genomes, it was suggested that the influences of the CRISPR on the phage insertion vary among species even within the same genus. Our results suggested that limitations in CRISPR content could explain the characteristic acquisition of prophages and might contribute to strain-specific pathogenesis in S. pyogenes. 相似文献
16.
Heme biosynthesis represents one of the most essential metabolic pathways in living organisms, providing the precursors for cytochrome prosthetic groups, photosynthetic pigments, and vitamin B(12). Using genomic data, we have compared the heme pathway in the diatom Thalassiosira pseudonana and the red alga Cyanidioschyzon merolae to those of green algae and higher plants, as well as to those of heterotrophic eukaryotes (fungi, apicomplexans, and animals). Phylogenetic analyses showed the mosaic character of this pathway in photosynthetic eukaryotes. Although most of the algal and plant enzymes showed the expected plastid (cyanobacterial) origin, at least one of them (porphobilinogen deaminase) appears to have a mitochondrial (alpha-proteobacterial) origin. Another enzyme, glutamyl-tRNA synthase, obviously originated in the eukaryotic nucleus. Because all the plastid-targeted sequences consistently form a well-supported cluster, this suggests that genes were either transferred from the primary endosymbiont (cyanobacteria) to the primary host nucleus shortly after the primary endosymbiotic event or replaced with genes from other sources at an equally early time, i.e., before the formation of three primary plastid lineages. The one striking exception to this pattern is ferrochelatase, the enzyme catalyzing the first committed step to heme and bilin pigments. In this case, two red algal sequences do not cluster either with the other plastid sequences or with cyanobacterial sequences and appear to have a proteobacterial origin like that of the apicomplexan parasites Plasmodium and Toxoplasma. Although the heterokonts also acquired their plastid via secondary endosymbiosis from a red alga, the diatom has a typical plastid-cyanobacterial ferrochelatase. We have not found any remnants of the plastidlike heme pathway in the nonphotosynthetic heterokonts Phytophthora ramorum and Phytophthora sojae. 相似文献
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A型链球菌是一类革兰氏阳性病原菌,其产生的多种毒素因子是导致人体多重感染的重要原因。这些菌毒素因子的表达直接或间接地受多个毒素调控系统调控,各个调控系统之间存在相互关联并对病原菌与宿主的相互作用产生影响。干扰毒素的产生或调控通路对于发展特异的抗A型链球菌感染药物具有重要的意义。 相似文献
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Carbamazepine, a drug which is widely used in neurological diseases, has a porphyrogenic effect in chick embryo liver cells in culture. It increased the concentration of cellular porphyrins by 80-fold and delta-aminolevulinate synthase activity by 4-fold. The increase in the accumulation of porphyrins preceded that of ALAS activity. Measurements of the activities of aminolevulinate dehydrase, porphobilinogen deaminase, and uroporphyrinogen decarboxylase showed that C inhibits UROD up to nearly 50% and PBGD activity up to 20%, but does not affect the activity of ALAD. The pattern of accumulation of porphyrins, mainly uro- and heptacarboxylporphyrin, is compatible with an inhibition of UROD. We may, therefore, conclude that the porphyrogenic effect of C in monolayers of chick embryo liver cells is the result of its inhibitory effect on the activity of UROD. 相似文献
20.
Anna Henningham Masaya Yamaguchi Ramy K. Aziz Kirsten Kuipers Cosmo Z. Buffalo Samira Dahesh Biswa Choudhury Jeremy Van Vleet Yuka Yamaguchi Lisa M. Seymour Nouri L. Ben Zakour Lingjun He Helen V. Smith Keith Grimwood Scott A. Beatson Partho Ghosh Mark J. Walker Victor Nizet Jason N. Cole 《The Journal of biological chemistry》2014,289(46):32303-32315
A recent analysis of group A Streptococcus (GAS) invasive infections in Australia has shown a predominance of M4 GAS, a serotype recently reported to lack the antiphagocytic hyaluronic acid (HA) capsule. Here, we use molecular genetics and bioinformatics techniques to characterize 17 clinical M4 isolates associated with invasive disease in children during this recent epidemiology. All M4 isolates lacked HA capsule, and whole genome sequence analysis of two isolates revealed the complete absence of the hasABC capsule biosynthesis operon. Conversely, M4 isolates possess a functional HA-degrading hyaluronate lyase (HylA) enzyme that is rendered nonfunctional in other GAS through a point mutation. Transformation with a plasmid expressing hasABC restored partial encapsulation in wild-type (WT) M4 GAS, and full encapsulation in an isogenic M4 mutant lacking HylA. However, partial encapsulation reduced binding to human complement regulatory protein C4BP, did not enhance survival in whole human blood, and did not increase virulence of WT M4 GAS in a mouse model of systemic infection. Bioinformatics analysis found no hasABC homologs in closely related species, suggesting that this operon was a recent acquisition. These data showcase a mutually exclusive interaction of HA capsule and active HylA among strains of this leading human pathogen. 相似文献