植物血红素加氧酶生理功能研究进展(综述)

血红素加氧酶（heme oxygenase, HO）是血红素分解代谢的关键酶类,在植物体内起着重要的作用。本文从HO在植物光敏色素合成中的作用、对植物根形态建成、提高植物抗胁迫反应能力、促进植物种子萌发和调节植物气孔关闭等方面,综述植物HO生理功能的最新研究进展,并对HO未来研究方向进行展望。     

血红素加氧酶系统研究现状

血红素加氧酶(HO)是降解血红素的微粒体酶系统,目前已确定的有3种同工酶,它们降解血红素生成一氧化碳(CO)和胆绿素,并释放出铁离子.这3种产物都有重要的生物学意义.为了探讨血红素加氧酶系统的调节机制,就这种机制及3种重要产物的功能作一综述.     

Complex Structure and Biochemical Characterization of the Staphylococcus aureus Cyclic Diadenylate Monophosphate (c-di-AMP)-binding Protein PstA,the Founding Member of a New Signal Transduction Protein Family

Signaling nucleotides are integral parts of signal transduction systems allowing bacteria to cope with and rapidly respond to changes in the environment. The Staphylococcus aureus PII-like signal transduction protein PstA was recently identified as a cyclic diadenylate monophosphate (c-di-AMP)-binding protein. Here, we present the crystal structures of the apo- and c-di-AMP-bound PstA protein, which is trimeric in solution as well as in the crystals. The structures combined with detailed bioinformatics analysis revealed that the protein belongs to a new family of proteins with a similar core fold but with distinct features to classical PII proteins, which usually function in nitrogen metabolism pathways in bacteria. The complex structure revealed three identical c-di-AMP-binding sites per trimer with each binding site at a monomer-monomer interface. Although distinctly different from other cyclic-di-nucleotide-binding sites, as the half-binding sites are not symmetrical, the complex structure also highlighted common features for c-di-AMP-binding sites. A comparison between the apo and complex structures revealed a series of conformational changes that result in the ordering of two anti-parallel β-strands that protrude from each monomer and allowed us to propose a mechanism on how the PstA protein functions as a signaling transduction protein.     

Structural Studies of Potassium Transport Protein KtrA Regulator of Conductance of K+ (RCK) C Domain in Complex with Cyclic Diadenosine Monophosphate (c-di-AMP)

Although it was only recently identified as a second messenger, c-di-AMP was found to have fundamental importance in numerous bacterial functions such as ion transport. The potassium transporter protein, KtrA, was identified as a c-di-AMP receptor. However, the co-crystallization of c-di-AMP with the protein has not been studied. Here, we determined the crystal structure of the KtrA RCK_C domain in complex with c-di-AMP. The c-di-AMP nucleotide, which adopts a U-shaped conformation, is bound at the dimer interface of RCK_C close to helices α3 and α4. c-di-AMP interacts with KtrA RCK_C mainly by forming hydrogen bonds and hydrophobic interactions. c-di-AMP binding induces the contraction of the dimer, bringing the two monomers of KtrA RCK_C into close proximity. The KtrA RCK_C was able to interact with only c-di-AMP, but not with c-di-GMP, 3′,3-cGAMP, ATP, and ADP. The structure of the KtrA RCK_C domain and c-di-AMP complex would expand our understanding about the mechanism of inactivation in Ktr transporters governed by c-di-AMP.     

Heme oxygenase-1 induction may explain the antioxidant profile of pentaerythrityl trinitrate

The organic nitrate pentaerythrityl tetranitrate (PETN) is known to exert long-term antioxidant and antiatherogenic effects by as yet unidentified mechanisms. In cultured endothelial cells derived from human umbilical vein, the active PETN metabolite PETriN (0.01-1 mM) increased heme oxygenase (HO)-1 mRNA and protein levels in a concentration-dependent fashion. HO-1 induction was accompanied by a marked increase in catalytic activity of the enzyme as reflected by enhanced formation of carbon monoxide and bilirubin. Pretreatment with PETriN or bilirubin at low micromolar concentrations protected endothelial cells from hydrogen peroxide-mediated toxicity. HO-1 induction and endothelial protection by PETriN were not mimicked by isosorbide dinitrate, another long-acting nitrate. The present study demonstrates that PETriN stimulates mRNA and protein expression as well as enzymatic activity of the antioxidant defense protein HO-1 in endothelial cells. Increased HO-1 expression and ensuing formation of cytoprotective bilirubin may contribute to and explain the specific antioxidant and antiatherogenic actions of PETN.     

A Heme-responsive Regulator Controls Synthesis of Staphyloferrin B in Staphylococcus aureus

Staphylococcus aureus possesses a multitude of mechanisms by which it can obtain iron during growth under iron starvation conditions. It expresses an effective heme acquisition system (the iron-regulated surface determinant system), it produces two carboxylate-type siderophores staphyloferrin A and staphyloferrin B (SB), and it expresses transporters for many other siderophores that it does not synthesize. The ferric uptake regulator protein regulates expression of genes encoding all of these systems. Mechanisms of fine-tuning expression of iron-regulated genes, beyond simple iron regulation via ferric uptake regulator, have not been uncovered in this organism. Here, we identify the ninth gene of the sbn operon, sbnI, as encoding a ParB/Spo0J-like protein that is required for expression of genes in the sbn operon from sbnD onward. Expression of sbnD–I is drastically decreased in an sbnI mutant, and the mutant does not synthesize detectable SB during early phases of growth. Thus, SB-mediated iron acquisition is impaired in an sbnI mutant strain. We show that the protein forms dimers and tetramers in solution and binds to DNA within the sbnC coding region. Moreover, we show that SbnI binds heme and that heme-bound SbnI does not bind DNA. Finally, we show that providing exogenous heme to S. aureus growing in an iron-free medium results in delayed synthesis of SB. This is the first study in S. aureus that identifies a DNA-binding regulatory protein that senses heme to control gene expression for siderophore synthesis.     

Extracellular Fibrinogen-binding Protein (Efb) from Staphylococcus aureus Inhibits the Formation of Platelet-Leukocyte Complexes

Extracellular fibrinogen-binding protein (Efb) from Staphylococcus aureus inhibits platelet activation, although its mechanism of action has not been established. In this study, we discovered that the N-terminal region of Efb (Efb-N) promotes platelet binding of fibrinogen and that Efb-N binding to platelets proceeds via two independent mechanisms: fibrinogen-mediated and fibrinogen-independent. By proteomic analysis of Efb-interacting proteins within platelets and confirmation by pulldown assays followed by immunoblotting, we identified P-selectin and multimerin-1 as novel Efb interaction partners. The interaction of both P-selectin and multimerin-1 with Efb is independent of fibrinogen. We focused on Efb interaction with P-selectin. Excess of P-selectin extracellular domain significantly impaired Efb binding by activated platelets, suggesting that P-selectin is the main receptor for Efb on the surface of activated platelets. Efb-N interaction with P-selectin inhibited P-selectin binding to its physiological ligand, P-selectin glycoprotein ligand-1 (PSGL-1), both in cell lysates and in cell-free assays. Because of the importance of P-selectin-PSGL-1 binding in the interaction between platelets and leukocytes, we tested human whole blood and found that Efb abolishes the formation of platelet-monocyte and platelet-granulocyte complexes. In summary, we present evidence that in addition to its documented antithrombotic activity, Efb can play an immunoregulatory role via inhibition of P-selectin-PSGL-1-dependent formation of platelet-leukocyte complexes.     

A remote-query sensor for predictive indication of milk spoilage

Described is application of the remote-query (wireless, passive) magnetoelastic sensor platform for direct detection and monitoring of bacterium contamination of milk within hermetically sealed containers. Specific application is made to the quantification of Staphylococcus aureus ssp. anaerobius (S. aureus) concentrations in milk. S. aureus growth changes milk viscosity, in turn changing the resonance frequency of the liquid immersed sensor allowing S. aureus concentrations of 10³ to 10⁷ cells ml⁻¹ to be directly quantified.     

Glucose Activates TORC2-Gad8 Protein via Positive Regulation of the cAMP/cAMP-dependent Protein Kinase A (PKA) Pathway and Negative Regulation of the Pmk1 Protein-Mitogen-activated Protein Kinase Pathway

The target of rapamycin (TOR) kinase belongs to the highly conserved eukaryotic family of phosphatidylinositol 3-kinase-related kinases. TOR proteins are found at the core of two evolutionary conserved complexes, known as TORC1 and TORC2. In fission yeast, TORC2 is dispensable for proliferation under optimal growth conditions but is required for starvation and stress responses. TORC2 has been implicated in a wide variety of functions; however, the signals that regulate TORC2 activity have so far remained obscure. TORC2 has one known direct substrate, the AGC kinase Gad8, which is related to AKT in human cells. Gad8 is phosphorylated by TORC2 at Ser-546 (equivalent to AKT Ser-473), leading to its activation. Here, we show that glucose is necessary and sufficient to induce Gad8 Ser-546 phosphorylation in vivo and Gad8 kinase activity in vitro. The glucose signal that activates TORC2-Gad8 is mediated via the cAMP/PKA pathway, a major glucose-sensing pathway. By contrast, Pmk1, similar to human extracellular signal-regulated kinases and a major stress-induced mitogen activated protein kinase (MAPK) in fission yeast, inhibits TORC2-dependent Gad8 phosphorylation and activation. Inhibition of TORC2-Gad8 also occurs in response to ionic or osmotic stress, in a manner dependent on the cAMP/PKA and Pmk1-MAPK signaling pathways. Our findings highlight the significance of glucose availability in regulation of TORC2-Gad8 and indicate a novel link between the cAMP/PKA, Pmk1/MAPK, and TORC2-Gad8 signaling.     

Induction of Heme Oxygenase-1 by Na+-H+ Exchanger 1 Protein Plays a Crucial Role in Imatinib-resistant Chronic Myeloid Leukemia Cells

Resistance toward imatinib (IM) and other BCR/ABL tyrosine kinase inhibitors remains troublesome in the treatment of advanced stage chronic myeloid leukemia (CML). The aim of this study was to estimate the reversal effects of down-regulation of Na⁺/H⁺ exchanger 1 (NHE1) on the chemoresistance of BCR-ABL-positive leukemia patients'' cells and cell lines. After treatment with the specific NHE1 inhibitor cariporide to decrease intracellular pH (pH_i), the heme oxygenase-1 (HO-1) levels of the K562R cell line and cells from IM-insensitive CML patients decreased. HO-1, as a Bcr/Abl-dependent survival molecule in CML cells, is important for the resistance to tyrosine kinase inhibitors in patients with newly diagnosed CML or IM-resistant CML. Silencing PKC-β and Nrf-2 or treatment with inhibitors of p38 pathways obviously blocked NHE1-induced HO-1 expression. Furthermore, treatment with HO-1 or p38 inhibitor plus IM increased the apoptosis of the K562R cell line and IM-insensitive CML patients'' cells. Inhibiting HO-1 enhanced the activation of caspase-3 and poly(ADP-ribose) polymerase-1. Hence, the results support the anti-apoptotic role of HO-1 induced by NHE1 in the K562R cell line and IM-insensitive CML patients and provide a mechanism by which inducing HO-1 expression via the PKC-β/p38-MAPK pathway may promote tumor resistance to oxidative stress.     

Comparison of the Virulence of Methicillin-Resistant and Methicillin-Sensitive Staphylococcus aureus

The virulence of methicillin-resistant Staphylococcus aureus (MRSA) was compared with that of methicillin-sensitive S. aureus (MSSA), using 13 MRSA and 7 MSSA strains isolated from clinical specimens. The infectivity and lethality of the two groups were examined as to the inoculum required to infect 50% of guinea pigs (ID₅₀) and to kill 50% of mice (LD₅₀), respectively. The mean ID₅₀ [log₁₀ colony forming units (CFU)] for MRSA strains was 7.1 ± 0.60 standard deviation, which was 1.5 higher than that for MSSA strains (P < 0.001). The mean LD₅₀ (log₁₀ CFU) for MRSA strains was 9.0 ± 0.42, being 1.1 higher than that for MSSA strains (P = 0.001). Pretreatment of mice with cyclophosphamide decreased the mean LD₅₀ for MRSA strains more than that for MSSA strains, resulting in the difference in the mean LD₅₀ being insignificant (P = 0.502). These results indicate that MRSA is less virulent than MSSA in normal hosts, but that they are equally virulent in immunocompromised hosts. The growth of MRSA strains was much slower than that of MSSA strains in the lag phase, although their growth rates were almost the same in the exponential growth phase, suggesting that the difference in virulence between them may be at least partly due to such a difference in growth.     

Structure-Function Analysis of Heterodimer Formation,Oligomerization, and Receptor Binding of the Staphylococcus aureus Bi-component Toxin LukGH

The bi-component leukocidins of Staphylococcus aureus are important virulence factors that lyse human phagocytic cells and contribute to immune evasion. The γ-hemolysins (HlgAB and HlgCB) and Panton-Valentine leukocidin (PVL or LukSF) were shown to assemble from soluble subunits into membrane-bound oligomers on the surface of target cells, creating barrel-like pore structures that lead to cell lysis. LukGH is the most distantly related member of this toxin family, sharing only 30–40% amino acid sequence identity with the others. We observed that, unlike other leukocidin subunits, recombinant LukH and LukG had low solubility and were unable to bind to target cells, unless both components were present. Using biolayer interferometry and intrinsic tryptophan fluorescence we detected binding of LukH to LukG in solution with an affinity in the low nanomolar range and dynamic light scattering measurements confirmed formation of a heterodimer. We elucidated the structure of LukGH by x-ray crystallography at 2.8-Å resolution. This revealed an octameric structure that strongly resembles that reported for HlgAB, but with important structural differences. Structure guided mutagenesis studies demonstrated that three salt bridges, not found in other bi-component leukocidins, are essential for dimer formation in solution and receptor binding. We detected weak binding of LukH, but not LukG, to the cellular receptor CD11b by biolayer interferometry, suggesting that in common with other members of this toxin family, the S-component has the primary contact role with the receptor. These new insights provide the basis for novel strategies to counteract this powerful toxin and Staphylococcus aureus pathogenesis.     

The Capsular Polysaccharide of Staphylococcus aureus Is Attached to Peptidoglycan by the LytR-CpsA-Psr (LCP) Family of Enzymes

Envelope biogenesis in bacteria involves synthesis of intermediates that are tethered to the lipid carrier undecaprenol-phosphate. LytR-CpsA-Psr (LCP) enzymes have been proposed to catalyze the transfer of undecaprenol-linked intermediates onto the C6-hydroxyl of MurNAc in peptidoglycan, thereby promoting attachment of wall teichoic acid (WTA) in bacilli and staphylococci and capsular polysaccharides (CPS) in streptococci. S. aureus encodes three lcp enzymes, and a variant lacking all three genes (Δlcp) releases WTA from the bacterial envelope and displays a growth defect. Here, we report that the type 5 capsular polysaccharide (CP5) of Staphylococcus aureus Newman is covalently attached to the glycan strands of peptidoglycan. Cell wall attachment of CP5 is abrogated in the Δlcp variant, a defect that is best complemented via expression of lcpC in trans. CP5 synthesis and peptidoglycan attachment are not impaired in the tagO mutant, suggesting that CP5 synthesis does not involve the GlcNAc-ManNAc linkage unit of WTA and may instead utilize another Wzy-type ligase to assemble undecaprenyl-phosphate intermediates. Thus, LCP enzymes of S. aureus are promiscuous enzymes that attach secondary cell wall polymers with discrete linkage units to peptidoglycan.     

耐甲氧西林金黄色葡萄球菌的两种新SCCmec型别及其抗药性研究

为探明本地区耐甲氧西林金黄色葡萄球菌(Staphylococcusaureus)的耐药性、流行病学分布状况及携带的葡萄球菌染色体mec盒(SCCmec)型别,用K-B琼脂扩散法、E-test和多位点PCR,对临床分离的金黄色葡萄球菌菌株进行了SCCmec分型及耐药性测定。结果发现了两种新的SCCmec型别,新1型含Ⅱ型的mecA上游特异性位点B和位于mecA内的M位点以及Ⅲ型的下游位点F,缺乏Ⅱ型上游位点C和下游位点D、G;新2型含Ⅰ、Ⅱ型的上游特异性位点A、B和两个Ⅲ型的下游位点F、H,同样缺乏位点C、D、G,可能分别为原有Ⅱ型和Ⅰ、Ⅱ型与Ⅲ型的基因重组株;且携带有新SCCmec型别的MRSA菌株,其流行病学分布特点及抗药性也与国外已报导的菌株不同,多分自门诊病人,且耐药性高,抗药谱广,值得引起高度重视和关注。     

Identification of a domain critical for Staphylococcus aureus LukED receptor targeting and lysis of erythrocytes

Leukocidin ED (LukED) is a pore-forming toxin produced by Staphylococcus aureus, which lyses host cells and promotes virulence of the bacteria. LukED enables S. aureus to acquire iron by lysing erythrocytes, which depends on targeting the host receptor Duffy antigen receptor for chemokines (DARC). The toxin also targets DARC on the endothelium, contributing to the lethality observed during bloodstream infection in mice. LukED is comprised of two monomers: LukE and LukD. LukE binds to DARC and facilitates hemolysis, but the closely related Panton–Valentine leukocidin S (LukS-PV) does not bind to DARC and is not hemolytic. The interaction of LukE with DARC and the role this plays in hemolysis are incompletely characterized. To determine the domain(s) of LukE that are critical for DARC binding, we studied the hemolytic function of LukE–LukS-PV chimeras, in which areas of sequence divergence (divergence regions, or DRs) were swapped between the toxins. We found that two regions of LukE''s rim domain contribute to hemolysis, namely residues 57–75 (DR1) and residues 182–196 (DR4). Interestingly, LukE DR1 is sufficient to render LukS-PV capable of DARC binding and hemolysis. Further, LukE, by binding DARC through DR1, promotes the recruitment of LukD to erythrocytes, likely by facilitating LukED oligomer formation. Finally, we show that LukE targets murine Darc through DR1 in vivo to cause host lethality. These findings expand our biochemical understanding of the LukE–DARC interaction and the role that this toxin-receptor pair plays in S. aureus pathophysiology.     

MRSA的7种新SCCmec型别及其抗药特性

为探明海口地区耐甲氧西林金黄色葡萄球菌(MRSA)的耐药性和携带的葡萄球菌染色体mec盒(SCCmec)型别,对收集的1174株金黄色葡萄球菌用PBP2a检测法确证为MRSA有686株,用多重PCR对58株进行SCCmec分型测定,并用K-B琼脂扩散法和E-test法测定其对临床常用7类抗生素的代表性药物耐药性。结果在17株中又发现了7种新的SCCmec型别,其结构特点为:New3含A、F、H、M4个位点,New4型含F、H、M3个位点,New5含D、B、M3个位点,New6型含A、B、M3个位点,New7型含H、E、C、M4个位点,New8型含A、M两个位点,New9型含A、C、M3个位点;它们均与报道型别的结构特点存在明显差异;且携带新型的MRSA菌株,其分布特点及抗药性也与已报道的菌株存在差异:多分自门诊病人,且耐药性高,抗药谱较广,值得引起高度重视和关注。     

MRSA的7种新SCCmec型别及其抗药特性

为探明海口地区耐甲氧西林金黄色葡萄球菌(MRSA)的耐药性和携带的葡萄球菌染色体mec盒(SCCmec)型别,对收集的1174株金黄色葡萄球菌用PBP2a检测法确证为MRSA有686株,用多重PCR对58株进行SCCmec分型测定,并用K-B琼脂扩散法和E-test法测定其对临床常用7类抗生素的代表性药物耐药性。结果在17株中又发现了7种新的SCCmec型别,其结构特点为:New3含A、F、H、M4个位点,New4型含F、H、M3个位点,New5含D、B、M3个位点,New6型含A、B、M3个位点,New7型含H、E、C、M4个位点,New8型含A、M两个位点,New9型含A、C、M3个位点;它们均与报道型别的结构特点存在明显差异;且携带新型的MRSA菌株,其分布特点及抗药性也与已报道的菌株存在差异:多分自门诊病人,且耐药性高,抗药谱较广,值得引起高度重视和关注。