幽门螺杆菌hp0318(hugZ)基因功能研究
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摘要
幽门螺杆菌(Helicobacter pylori, H.pylori),微需氧革兰氏阴性螺旋杆菌,是人类慢性胃炎、消化性溃疡的重要致病因子,与胃癌及胃MALT淋巴瘤的发生密切相关。目前,越来越多的研究证据证明,幽门螺杆菌已经适应定植于人类胃部,这种非常特殊的部位。H.pylori临床分离株普遍存在着遗传多态性,提示H.pylori存在特殊的适应性定植机制。
本室在前期研究H.pylori的适应性定植中,发现刚分离的临床分离株最初很难在蒙古沙鼠中生长。然而通过在体内传代13代后,临床株便很好地在沙鼠胃中繁殖。为了进一步阐明H.pylori这种在沙鼠体内适应性定植机制,我们通过蛋白组学研究方法比较了一株临床分离的H.pylori适应性定植蒙古沙鼠后的蛋白质变化。结果从中筛选、鉴定出4个适应性定植相关蛋白。并且,首次发现和证明功能未知基因hp0318 (hugZ)与H.pylori在沙鼠中的适应性定植存在相关性。
hugZ基因在H.pylori适应性定植中究竟发挥了什么样的作用?其编码蛋白的确切生理功能是什么?搞清楚这些问题将有助于解释H.pylori适应性定植机制和致病机制。
本研究首先通过生物信息学的方法对hp0318(hugZ)基因进行了分析,并采用DNA重组技术克隆、表达、纯化了HP0318 (HugZ)蛋白;通过免疫电镜确定HugZ蛋白在H.pylori菌体中的具体位置;在体外利用生物化学试验检测了HugZ结合和催化血红素的功能;进一步通过同源重组基因敲除技术构建了hugZ基因突变株,定量RT-PCR检测hugZ基因表达是否受到铁的调控,探讨了该基因与H.pylori利用血红素铁之间的关系。具体研究结果如下:
1.生物信息学分析hugZ基因并重组表达其编码蛋白。
生物信息学分析提示hugZ与空肠弯曲菌的血红素氧合酶cj1613c基因有较高的同源性。为了检测其在血红素铁获取中的作用,我们对HugZ进行了重组表达。首先,在大肠杆菌工程菌中表达了该基因编码蛋白,并使得LB培养基变为绿色。这种现象与其它真核和原核血红素氧合酶在大肠杆菌工程菌中表达的情况一致,支持HugZ是血红素氧合酶的推断。使用镍离子亲和层析XK1610柱纯化,可以从1L菌液中得到50mg纯化蛋白样品,纯化样品在15% SDS-PAGE胶上显示为95%的纯度。基因测序与PMF检测发现重组的HugZ与H.pylori26695 hp0318基因完全符合。
2.HugZ是幽门螺杆菌的胞浆蛋白。
我们使用了免疫电镜对HugZ在H.pylori菌体中的位置进行定位。H.pylori 26695的菌体制备成冰冻切片,使用兔抗HugZ抗体和胶体金标记的二抗检测。H.pylori菌体冰冻切片上的胶体金颗粒位置,显示HugZ蛋白分布于H.pylori胞浆中。
3.HugZ具有血红素氧合酶活性。
3.1 HugZ能够结合血红素。
为了检测HugZ是不是血红素氧合酶家族的一员,分别进行了两个血红素结合试验。HugZ能够特异地与Hemin-agarose结合,说明它有血红素结合能力。同样,在体外的血红素滴定光谱扫描研究结果也证实了HugZ这种结合血红素的能力。HugZ能够结合血红素形成典型的血红素结合蛋白光谱吸收峰,在411nm形成了Soret峰(血红素结合蛋白特征峰),540nm和580nm处的峰对应了Heme-HugZβ-卟啉和α-卟啉环。定量滴定血红素结合能力发现,20μM HugZ最多能结合等量的血红素,提示HugZ与Heme按1:1分子比例进行结合。
3.2 HugZ催化降解血红素。
体外分别使用NADPH-CPR还原系统和抗坏血酸作为供电子体时,使用350-750nm光波扫描。发现HugZ-heme形成的Soret峰随着时间延长,逐渐降低。在缺乏反应系统任何主要成份,均不能观察到这种现象。检测中发现抗坏血酸作供电子体时,催化反应的速度明显加快,能够在20min内完成催化反应。这些发现证实了HugZ的催化降解血红素的活性。
3.3 HugZ催化降解血红素生成胆绿素和CO。
胆绿素是血红素氧合酶催化降解血红素的最终产物。在光谱扫描中可以观察到随着HugZ催化反应的进行,光谱图在660nm处形成胆绿素特征吸收峰。HPLC用来检测生成何种胆绿素。与胆素标准品比较发现,HugZ催化血红素生成了胆绿素IXδ.实验中加入肌红蛋白来监测催化反应中的另外一个产物-CO。铁-氧-肌红蛋白转化成铁-CO-肌红蛋白,在光谱图上显示为411nm的Soret峰向421nm漂移,并且同样可以观察到肌蛋白β-卟啉和α-卟啉环在540nm和580nm处的吸收峰。结果表明,HugZ催化降解血红素生成胆绿素IXδ和CO。
4.hugZ基因在幽门螺杆菌对血红素铁的利用中起重要的作用,其基因表达受铁离子负调控。
4.1 hugZ突变株不能利用血红素铁来维持正常生长。
为了阐明hugZ的作用,研究首先建立了hugZ基因突变株ΔhugZ。PCR扩增hugZ基因上下游片段,在其中间插入氯霉素抗性基因(cam)克隆到质粒pBluescript SK中构建成敲除hugZ基因的自杀载体,将其转化H.pylori 26695。经同源重组,cam对hugZ基因进行置换突变,经PCR和RT-PCR鉴定和直接测序证实成功获得了hugZ基因突变株ΔhugZ。
在BBF液体培养和血平板上,hugZ基因突变株生长良好与未突变株生长无显著差异。说明在铁离子丰富的条件下,hugZ不是H.pylori正常生长必需的基因。接着,我们测试了在不同铁源条件下的生长情况。结果在血红蛋白作为唯一的铁源时,hugZ基因突变株生长不良,与未突变株比较相差显著(p<0.01)。实验提示,hugZ基因缺失株不能利用血红素铁来维持正常的生长。
4.2 hugZ基因表达受铁离子负调控。
为了检验hugZ基因表达是不是受铁离子的调控,我们使用了定量RT-PCR检测在不同的铁离子条件下hugZ转录水平。实验发现,加入FeCl3时hugZ的转录受到了抑制(参照BBF转录水平,变化倍数为0.410±0.056 (p<0.01));而在铁限制条件下hugZ的转录增高(参照BBF转录水平,变化倍数为3.90±0.010 (p<0.01))。表明hugZ基因转录表达受到铁离子负调控。
本研究结果表明,hugZ基因编码的蛋白HugZ作为血红素氧合酶参与了幽门螺杆菌利用血红素铁,并且受到铁离子的负调控。这对于解释hugZ基因在H.pylori适应性定植中发挥的作用以及揭示H.pylori致病机制具有重要的意义。
Helicobacter pylori (H. pylori), a Gram-negative microaerophilic spiral bacterium, is known as the major pathogenic agent in a wide range of gastroenteric diseases exemplified by chronic gastritis, peptic ulcer and gastric adeno-carcinoma. Increasing evidence suggests that H. pylori has adapted particularly to the niche of human stomach. Genetic diversity is widespread among the clinical isolates. This polymorphism can be attributed mainly to the consequence of adaptive changes during colonization, which in turn imply that H. pylori has a specialized adaptation mechanism.
In our earlier study, we harvested several clinical strains of H. pylori, which initially grew weakly in Mongolian gerbils but subsequently adapted after 13 serial passages in vivo. To elucidate the adaptive colonizing mechanisms of H. pylori in Mongolian gerbils further, we applied proteomic approaches to one representative H. pylori isolate. Fortunately, four adaptive colonization-associated proteins were identified, among which HugZ (heme iron utilization-related protein) was implicated in adaptive colonization by H. pylori for the first time. However, the exact physiological role of HugZ remains elusive.
What is the exact physiological role of hp0318 (hugZ) gene in H.pylori Adaptive Colonization? How does it promote H.pylori adaptive colonization ? Clarify these issues will help us to to explain the adaptability of H.pylori mechanism and pathogenesis.
In this study, a gene homologous to hugZ, designated hp0318, identified in H. pylori ATCC 26695, exhibits high similarity to cj1613c of Campylobacter jejuni NCTC 11168. Soluble 6×His fused-HugZ protein was expressed in vitro. Hemin-agrose affinity and absorption spectroscopy analysis revealed that HugZ can bind heme at binding ratio of 1:1. Enzymatic assays showed that purified recombinant HugZ protein can degrade hemin into biliverdin and carbon monoxide in the presence of either ascorbic acid or NADPH and cytochrome P450 reductase. A hugZ deletion mutant was obtained by homologous recombination. This mutant strain showed poor growth when hemoglobin was provided as the source of iron, partly because of its failure to utilize hemoglobin efficiently. Real-time quantitative PCR also confirmed that the expression of hugZ was regulated by iron levels..Main results are listed as flows:
1. Production and evaluation of homogeneous H. pylori HugZ
Bioinformatics analysis suggested that a hugZ homologue exists in H. pylori, which is very similar to that in C. jejuni. To test its activity in iron acquisition, we prepared homogeneous H. pylori HugZ protein in vitro. Initially, soluble 6×His-tagged HugZ protein was expressed in a prokaryotic expression system; expression in Escherichia coli (E. coli) turned the LB medium green. This observation supports the hypothesis that catalytic turnover of Heme-HugZ triggers the accumulation of biliverdin, which is consistent with the expression profiles of prokaryotic/eukaryotic heme oxygenases. The recombinant HugZ protein purified by a Chelating Fastflow XK1610 column (CV=18 ml) yielded 50 mg/liter and showed about 95% purity on 15% SDS-PAGE, indicating high homogeneity. PMF-based sequencing showed that H. pylori HugZ is 251 amino acids long and shares 100% similarity to HP0318 (HugZ) protein in ATCC 26695.
2. HugZ is a cytoplasmic protein
To determine the cellular location of HugZ, Immunoelectron microscopy (IEM) was performed. Frozen sectioned samples of H. pylori 26695 strains were treated with anti-HugZ antibodies and gold-labeled secondary antibodies. Analysis of the positions of the gold particles, revealed that HugZ was predominantly located in the cytoplasm in H. H. pylori
3. HP0318 (HugZ) is a heme oxygenase..
3.1 HugZ has heme binding activity
To determine whether it is a functional member of the heme oxygenase family, two kinds of heme binding assay were performed. HugZ binding to hemin-agarose beads strongly indicated that it has heme-binding activity . Similarly, in vitro absorption spectroscopy suggested that HugZ is able to bind heme. As we expected, when HugZ was mixed with hemin, the spectrum of the complex showed a typical spectrographic curve with a prominent Soret peak at 411 nm, and a shoulder at 540 nm and a smaller peak at 580 nm, corresponding to theβ- andα-porphyrin bands of the heme-HugZ complex respectively. To quantify heme binding, HugZ solution (20μM) was titrated with increasing amounts of hemin. The increase in absorption leveled off at approximately 20μM heme, showing a 1:1 stoichiometry of heme to HugZ).
3.2 HugZ catalyzes the degradation of heme
In the first experiment, heme degradation catalyzed by HugZ was measured spectrophotometrically using human NADPH-CPR as the electron donor. NADPH was added to the reaction mixture in 10μM increments and the mixture was scanned from 350 to 800 nm after each addition. The Soret band decreased successively after addition of NADPH. Finally, the HugZ substrate-hemin was exhausted and the NADPH was not oxidized completely, so there was absorption at 340 nm due to NADPH. Heme degradation did not occur if HugZ, NADPH or CPR was omitted from the reaction mixture.
In the second experiment, the HugZ-dependent disappearance of heme was measured using 20 mM ascorbate as the reductant. Heme was degraded more rapidly with ascorbate than with human NADPH-CPR, and most of the decrease was complete by 20 min after the ascorbate was added. No degradation of heme was observed in the absence of HugZ or ascorbate. Collectively, these findings showed that HugZ catalyzes the enzymatic degradation of heme.
3.3 Biliverdin and CO produced by HugZ-catalyzed heme degradation
Biliverdin is the final product of heme degradation by heme oxygenases. When heme was degraded by HugZ, a broad absorbance peak in the 660-nm region became prominent, implying is the presence of biliverdin. To determine the kind of biliverdin formed, we subjected this product to HPLC analysis. HPLC chromatography of all four possible biliverdin isomers is shown for comparison. The HPLC profiles of the products formed during HugZ-catalyzed heme degradation with ascorbate and NADPH gave a retention time and absorption spectrum identical to that of biliverdin IXδ.
The myoglobin absorption spectrum was recorded at 2-min intervals in order to monitor the characteristic spectral changes of a myoglobin-CO complex. The transition of ferrous-dioxygen myoglobin to the ferrous-CO myoglobin complex was associated with a shift in the Soret band from 411 to 421 nm as well as the appearance of bands at 540 and 580 nm. Control reactions in the absence of the heme-HugZ complex showed no shift in the Soret band. The complete conversion indicated that carbon monoxide as well as biliverdin was generated as a product of oxidative heme cleavage in H. pylori.
4. hugZ gene play a crucial role in the acquisition of heme iron by H. pylori,and of its expression regulated by iron
4.1 The hugZ mutant fails to utilize heme iron for normal growth
In order to elucidate the role of HugZ, the mutantΔhugZ was obtained from more than 100 H. pylori transformants. The correct genotype ofΔhugZ was systemically confirmed by PCR, RT-PCR and direct DNA sequencing.
The hugZ deletion mutant (ΔhugZ) grew normally in liquid BBF and on BBF blood agar plates, indicating that HugZ is not required for bacterial growth under iron-replete conditions. Subsequently, we tested its growth in the presence of different iron sources.ΔhugZ strains showed poor growth in iron-restricted conditions while the wild type grew well. These data suggest that the hugZ mutant cannot utilize heme iron for normal growth.
4.2 Regulation of hugZ expression by iron
To test whether hugZ is regulated by iron, real-time quantitative PCR was performed. The effects of different iron levels on hugZ transcription varied. Transcription was suppressed by FeCl3 (compared to BBF, the change fold ratio was 0.410±0.056 (p<0.01, Student's t-test)) and stimulated under iron-restricted conditions (compared to BBF, the change fold ratio was 3.90±0.010 (p<0.01, Student's t-test)). These results indicated that hugZ (hp0318) is down-regulated by iron.
Taken together, these findings confirm that H. pylori HP0318 (HugZ) is a heme oxygenase. Our data imply that HugZ may play a crucial role in the acquisition of heme iron by H. pylori.
本室在前期研究H.pylori的适应性定植中,发现刚分离的临床分离株最初很难在蒙古沙鼠中生长。然而通过在体内传代13代后,临床株便很好地在沙鼠胃中繁殖。为了进一步阐明H.pylori这种在沙鼠体内适应性定植机制,我们通过蛋白组学研究方法比较了一株临床分离的H.pylori适应性定植蒙古沙鼠后的蛋白质变化。结果从中筛选、鉴定出4个适应性定植相关蛋白。并且,首次发现和证明功能未知基因hp0318 (hugZ)与H.pylori在沙鼠中的适应性定植存在相关性。
hugZ基因在H.pylori适应性定植中究竟发挥了什么样的作用?其编码蛋白的确切生理功能是什么?搞清楚这些问题将有助于解释H.pylori适应性定植机制和致病机制。
本研究首先通过生物信息学的方法对hp0318(hugZ)基因进行了分析,并采用DNA重组技术克隆、表达、纯化了HP0318 (HugZ)蛋白;通过免疫电镜确定HugZ蛋白在H.pylori菌体中的具体位置;在体外利用生物化学试验检测了HugZ结合和催化血红素的功能;进一步通过同源重组基因敲除技术构建了hugZ基因突变株,定量RT-PCR检测hugZ基因表达是否受到铁的调控,探讨了该基因与H.pylori利用血红素铁之间的关系。具体研究结果如下:
1.生物信息学分析hugZ基因并重组表达其编码蛋白。
生物信息学分析提示hugZ与空肠弯曲菌的血红素氧合酶cj1613c基因有较高的同源性。为了检测其在血红素铁获取中的作用,我们对HugZ进行了重组表达。首先,在大肠杆菌工程菌中表达了该基因编码蛋白,并使得LB培养基变为绿色。这种现象与其它真核和原核血红素氧合酶在大肠杆菌工程菌中表达的情况一致,支持HugZ是血红素氧合酶的推断。使用镍离子亲和层析XK1610柱纯化,可以从1L菌液中得到50mg纯化蛋白样品,纯化样品在15% SDS-PAGE胶上显示为95%的纯度。基因测序与PMF检测发现重组的HugZ与H.pylori26695 hp0318基因完全符合。
2.HugZ是幽门螺杆菌的胞浆蛋白。
我们使用了免疫电镜对HugZ在H.pylori菌体中的位置进行定位。H.pylori 26695的菌体制备成冰冻切片,使用兔抗HugZ抗体和胶体金标记的二抗检测。H.pylori菌体冰冻切片上的胶体金颗粒位置,显示HugZ蛋白分布于H.pylori胞浆中。
3.HugZ具有血红素氧合酶活性。
3.1 HugZ能够结合血红素。
为了检测HugZ是不是血红素氧合酶家族的一员,分别进行了两个血红素结合试验。HugZ能够特异地与Hemin-agarose结合,说明它有血红素结合能力。同样,在体外的血红素滴定光谱扫描研究结果也证实了HugZ这种结合血红素的能力。HugZ能够结合血红素形成典型的血红素结合蛋白光谱吸收峰,在411nm形成了Soret峰(血红素结合蛋白特征峰),540nm和580nm处的峰对应了Heme-HugZβ-卟啉和α-卟啉环。定量滴定血红素结合能力发现,20μM HugZ最多能结合等量的血红素,提示HugZ与Heme按1:1分子比例进行结合。
3.2 HugZ催化降解血红素。
体外分别使用NADPH-CPR还原系统和抗坏血酸作为供电子体时,使用350-750nm光波扫描。发现HugZ-heme形成的Soret峰随着时间延长,逐渐降低。在缺乏反应系统任何主要成份,均不能观察到这种现象。检测中发现抗坏血酸作供电子体时,催化反应的速度明显加快,能够在20min内完成催化反应。这些发现证实了HugZ的催化降解血红素的活性。
3.3 HugZ催化降解血红素生成胆绿素和CO。
胆绿素是血红素氧合酶催化降解血红素的最终产物。在光谱扫描中可以观察到随着HugZ催化反应的进行,光谱图在660nm处形成胆绿素特征吸收峰。HPLC用来检测生成何种胆绿素。与胆素标准品比较发现,HugZ催化血红素生成了胆绿素IXδ.实验中加入肌红蛋白来监测催化反应中的另外一个产物-CO。铁-氧-肌红蛋白转化成铁-CO-肌红蛋白,在光谱图上显示为411nm的Soret峰向421nm漂移,并且同样可以观察到肌蛋白β-卟啉和α-卟啉环在540nm和580nm处的吸收峰。结果表明,HugZ催化降解血红素生成胆绿素IXδ和CO。
4.hugZ基因在幽门螺杆菌对血红素铁的利用中起重要的作用,其基因表达受铁离子负调控。
4.1 hugZ突变株不能利用血红素铁来维持正常生长。
为了阐明hugZ的作用,研究首先建立了hugZ基因突变株ΔhugZ。PCR扩增hugZ基因上下游片段,在其中间插入氯霉素抗性基因(cam)克隆到质粒pBluescript SK中构建成敲除hugZ基因的自杀载体,将其转化H.pylori 26695。经同源重组,cam对hugZ基因进行置换突变,经PCR和RT-PCR鉴定和直接测序证实成功获得了hugZ基因突变株ΔhugZ。
在BBF液体培养和血平板上,hugZ基因突变株生长良好与未突变株生长无显著差异。说明在铁离子丰富的条件下,hugZ不是H.pylori正常生长必需的基因。接着,我们测试了在不同铁源条件下的生长情况。结果在血红蛋白作为唯一的铁源时,hugZ基因突变株生长不良,与未突变株比较相差显著(p<0.01)。实验提示,hugZ基因缺失株不能利用血红素铁来维持正常的生长。
4.2 hugZ基因表达受铁离子负调控。
为了检验hugZ基因表达是不是受铁离子的调控,我们使用了定量RT-PCR检测在不同的铁离子条件下hugZ转录水平。实验发现,加入FeCl3时hugZ的转录受到了抑制(参照BBF转录水平,变化倍数为0.410±0.056 (p<0.01));而在铁限制条件下hugZ的转录增高(参照BBF转录水平,变化倍数为3.90±0.010 (p<0.01))。表明hugZ基因转录表达受到铁离子负调控。
本研究结果表明,hugZ基因编码的蛋白HugZ作为血红素氧合酶参与了幽门螺杆菌利用血红素铁,并且受到铁离子的负调控。这对于解释hugZ基因在H.pylori适应性定植中发挥的作用以及揭示H.pylori致病机制具有重要的意义。
Helicobacter pylori (H. pylori), a Gram-negative microaerophilic spiral bacterium, is known as the major pathogenic agent in a wide range of gastroenteric diseases exemplified by chronic gastritis, peptic ulcer and gastric adeno-carcinoma. Increasing evidence suggests that H. pylori has adapted particularly to the niche of human stomach. Genetic diversity is widespread among the clinical isolates. This polymorphism can be attributed mainly to the consequence of adaptive changes during colonization, which in turn imply that H. pylori has a specialized adaptation mechanism.
In our earlier study, we harvested several clinical strains of H. pylori, which initially grew weakly in Mongolian gerbils but subsequently adapted after 13 serial passages in vivo. To elucidate the adaptive colonizing mechanisms of H. pylori in Mongolian gerbils further, we applied proteomic approaches to one representative H. pylori isolate. Fortunately, four adaptive colonization-associated proteins were identified, among which HugZ (heme iron utilization-related protein) was implicated in adaptive colonization by H. pylori for the first time. However, the exact physiological role of HugZ remains elusive.
What is the exact physiological role of hp0318 (hugZ) gene in H.pylori Adaptive Colonization? How does it promote H.pylori adaptive colonization ? Clarify these issues will help us to to explain the adaptability of H.pylori mechanism and pathogenesis.
In this study, a gene homologous to hugZ, designated hp0318, identified in H. pylori ATCC 26695, exhibits high similarity to cj1613c of Campylobacter jejuni NCTC 11168. Soluble 6×His fused-HugZ protein was expressed in vitro. Hemin-agrose affinity and absorption spectroscopy analysis revealed that HugZ can bind heme at binding ratio of 1:1. Enzymatic assays showed that purified recombinant HugZ protein can degrade hemin into biliverdin and carbon monoxide in the presence of either ascorbic acid or NADPH and cytochrome P450 reductase. A hugZ deletion mutant was obtained by homologous recombination. This mutant strain showed poor growth when hemoglobin was provided as the source of iron, partly because of its failure to utilize hemoglobin efficiently. Real-time quantitative PCR also confirmed that the expression of hugZ was regulated by iron levels..Main results are listed as flows:
1. Production and evaluation of homogeneous H. pylori HugZ
Bioinformatics analysis suggested that a hugZ homologue exists in H. pylori, which is very similar to that in C. jejuni. To test its activity in iron acquisition, we prepared homogeneous H. pylori HugZ protein in vitro. Initially, soluble 6×His-tagged HugZ protein was expressed in a prokaryotic expression system; expression in Escherichia coli (E. coli) turned the LB medium green. This observation supports the hypothesis that catalytic turnover of Heme-HugZ triggers the accumulation of biliverdin, which is consistent with the expression profiles of prokaryotic/eukaryotic heme oxygenases. The recombinant HugZ protein purified by a Chelating Fastflow XK1610 column (CV=18 ml) yielded 50 mg/liter and showed about 95% purity on 15% SDS-PAGE, indicating high homogeneity. PMF-based sequencing showed that H. pylori HugZ is 251 amino acids long and shares 100% similarity to HP0318 (HugZ) protein in ATCC 26695.
2. HugZ is a cytoplasmic protein
To determine the cellular location of HugZ, Immunoelectron microscopy (IEM) was performed. Frozen sectioned samples of H. pylori 26695 strains were treated with anti-HugZ antibodies and gold-labeled secondary antibodies. Analysis of the positions of the gold particles, revealed that HugZ was predominantly located in the cytoplasm in H. H. pylori
3. HP0318 (HugZ) is a heme oxygenase..
3.1 HugZ has heme binding activity
To determine whether it is a functional member of the heme oxygenase family, two kinds of heme binding assay were performed. HugZ binding to hemin-agarose beads strongly indicated that it has heme-binding activity . Similarly, in vitro absorption spectroscopy suggested that HugZ is able to bind heme. As we expected, when HugZ was mixed with hemin, the spectrum of the complex showed a typical spectrographic curve with a prominent Soret peak at 411 nm, and a shoulder at 540 nm and a smaller peak at 580 nm, corresponding to theβ- andα-porphyrin bands of the heme-HugZ complex respectively. To quantify heme binding, HugZ solution (20μM) was titrated with increasing amounts of hemin. The increase in absorption leveled off at approximately 20μM heme, showing a 1:1 stoichiometry of heme to HugZ).
3.2 HugZ catalyzes the degradation of heme
In the first experiment, heme degradation catalyzed by HugZ was measured spectrophotometrically using human NADPH-CPR as the electron donor. NADPH was added to the reaction mixture in 10μM increments and the mixture was scanned from 350 to 800 nm after each addition. The Soret band decreased successively after addition of NADPH. Finally, the HugZ substrate-hemin was exhausted and the NADPH was not oxidized completely, so there was absorption at 340 nm due to NADPH. Heme degradation did not occur if HugZ, NADPH or CPR was omitted from the reaction mixture.
In the second experiment, the HugZ-dependent disappearance of heme was measured using 20 mM ascorbate as the reductant. Heme was degraded more rapidly with ascorbate than with human NADPH-CPR, and most of the decrease was complete by 20 min after the ascorbate was added. No degradation of heme was observed in the absence of HugZ or ascorbate. Collectively, these findings showed that HugZ catalyzes the enzymatic degradation of heme.
3.3 Biliverdin and CO produced by HugZ-catalyzed heme degradation
Biliverdin is the final product of heme degradation by heme oxygenases. When heme was degraded by HugZ, a broad absorbance peak in the 660-nm region became prominent, implying is the presence of biliverdin. To determine the kind of biliverdin formed, we subjected this product to HPLC analysis. HPLC chromatography of all four possible biliverdin isomers is shown for comparison. The HPLC profiles of the products formed during HugZ-catalyzed heme degradation with ascorbate and NADPH gave a retention time and absorption spectrum identical to that of biliverdin IXδ.
The myoglobin absorption spectrum was recorded at 2-min intervals in order to monitor the characteristic spectral changes of a myoglobin-CO complex. The transition of ferrous-dioxygen myoglobin to the ferrous-CO myoglobin complex was associated with a shift in the Soret band from 411 to 421 nm as well as the appearance of bands at 540 and 580 nm. Control reactions in the absence of the heme-HugZ complex showed no shift in the Soret band. The complete conversion indicated that carbon monoxide as well as biliverdin was generated as a product of oxidative heme cleavage in H. pylori.
4. hugZ gene play a crucial role in the acquisition of heme iron by H. pylori,and of its expression regulated by iron
4.1 The hugZ mutant fails to utilize heme iron for normal growth
In order to elucidate the role of HugZ, the mutantΔhugZ was obtained from more than 100 H. pylori transformants. The correct genotype ofΔhugZ was systemically confirmed by PCR, RT-PCR and direct DNA sequencing.
The hugZ deletion mutant (ΔhugZ) grew normally in liquid BBF and on BBF blood agar plates, indicating that HugZ is not required for bacterial growth under iron-replete conditions. Subsequently, we tested its growth in the presence of different iron sources.ΔhugZ strains showed poor growth in iron-restricted conditions while the wild type grew well. These data suggest that the hugZ mutant cannot utilize heme iron for normal growth.
4.2 Regulation of hugZ expression by iron
To test whether hugZ is regulated by iron, real-time quantitative PCR was performed. The effects of different iron levels on hugZ transcription varied. Transcription was suppressed by FeCl3 (compared to BBF, the change fold ratio was 0.410±0.056 (p<0.01, Student's t-test)) and stimulated under iron-restricted conditions (compared to BBF, the change fold ratio was 3.90±0.010 (p<0.01, Student's t-test)). These results indicated that hugZ (hp0318) is down-regulated by iron.
Taken together, these findings confirm that H. pylori HP0318 (HugZ) is a heme oxygenase. Our data imply that HugZ may play a crucial role in the acquisition of heme iron by H. pylori.
引文
1. Wotherspoon AC: Helicobacter pylori infection and gastric lymphoma. Br Med Bull 1998, 54(1):79-85.
2. Marshall BJ, Warren JR: Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet 1984, 1(8390):1311-1315.
3. Dunn BE, Cohen H, Blaser MJ: Helicobacter pylori. Clin Microbiol Rev 1997, 10(4):720-741.
4.胡伏莲,周殿元:幽门螺杆菌感染的基础与临床.北京:中国科学技术出版社; 2002.
5. Gottke MU, Fallone CA, Barkun AN, Vogt K, Loo V, Trautmann M, Tong JZ, Nguyen TN, Fainsilber T, Hahn HH et al: Genetic variability determinants of Helicobacter pylori: influence of clinical background and geographic origin of isolates. J Infect Dis 2000, 181(5):1674-1681.
6. Akopyants NS, Eaton KA, Berg DE: Adaptive mutation and cocolonization during Helicobacter pylori infection of gnotobiotic piglets. Infect Immun 1995, 63(1):116-121.
7. Salaun L, Ayraud S, Saunders NJ: Phase variation mediated niche adaptation during prolonged experimental murine infection with Helicobacter pylori. Microbiology 2005, 151(Pt 3):917-923.
8.郭刚,王毅超,邹全明等:幽门螺杆菌长期感染蒙古沙鼠模型的建立.中华微生物学与免疫学杂志2001, 21(6):683-684.
9. Guo G, Tong WD, Zeng H, Liu KY, Zou QM: [Comparative proteomics analysis of Helicobacter pylori after adaptive colonization in Mongolian gerbils]. Wei Sheng Wu Xue Bao 2007, 47(3):461-464.
10. Koga T, Shimada Y, Sato K, Takahashi K, Kikuchi I, Okazaki Y, Miura T, Katsuta M, Iwata M: Contribution of ferrous iron to maintenance of the gastric colonization of Helicobacter pylori in miniature pigs. Microbiol Res 2002, 157(4):323-330.
11. Waidner B, Greiner S, Odenbreit S, Kavermann H, Velayudhan J, Stahler F, Guhl J, Bisse E, van Vliet AH, Andrews SC et al: Essential role of ferritin Pfr in Helicobacter pylori iron metabolism and gastric colonization. Infect Immun 2002,70(7):3923-3929.
12. Nakao K, Imoto I, Ikemura N, Shibata T, Takaji S, Taguchi Y, Misaki M, Yamauchi K, Yamazaki N: Relation of lactoferrin levels in gastric mucosa with Helicobacter pylori infection and with the degree of gastric inflammation. Am J Gastroenterol 1997, 92(6):1005-1011.
13. Frankenberg-Dinkel N: Bacterial heme oxygenases. Antioxid Redox Signal 2004, 6(5):825-834.
14. Wilks A, Burkhard KA: Heme and virulence: how bacterial pathogens regulate, transport and utilize heme. Nat Prod Rep 2007, 24(3):511-522.
15. Payne SM: Iron acquisition in microbial pathogenesis. Trends Microbiol 1993, 1(2):66-69.
16. Andrews SC, Robinson AK, Rodriguez-Quinones F: Bacterial iron homeostasis. FEMS Microbiol Rev 2003, 27(2-3):215-237.
17. Wandersman C, Delepelaire P: Bacterial iron sources: from siderophores to hemophores. Annu Rev Microbiol 2004, 58:611-647.
18. Wyckoff EE, Schmitt M, Wilks A, Payne SM: HutZ is required for efficient heme utilization in Vibrio cholerae. J Bacteriol 2004, 186(13):4142-4151.
19. Ridley KA, Rock JD, Li Y, Ketley JM: Heme utilization in Campylobacter jejuni. J Bacteriol 2006, 188(22):7862-7875.
20. Perry RD, Shah J, Bearden SW, Thompson JM, Fetherston JD: Yersinia pestis TonB: role in iron, heme, and hemoprotein utilization. Infect Immun 2003, 71(7):4159-4162.
21. Jarosik GP, Sanders JD, Cope LD, Muller-Eberhard U, Hansen EJ: A functional tonB gene is required for both utilization of heme and virulence expression by Haemophilus influenzae type b. Infect Immun 1994, 62(6):2470-2477.
22. Worst DJ, Otto BR, de Graaff J: Iron-repressible outer membrane proteins of Helicobacter pylori involved in heme uptake. Infect Immun 1995, 63(10):4161-4165.
23. Worst DJ, Maaskant J, Vandenbroucke-Grauls CM, Kusters JG: Multiple haem-utilization loci in Helicobacter pylori. Microbiology 1999, 145 ( Pt 3):681-688.
24. F.奥斯伯著,颜子颖等译:精编分子生物学实验指南.北京:科学出版社. 1998.
25.卢圣栋主编:现代分子生物学实验技术.第1版.北京:高等教育出版社; 1993.
26. Clayton .CLaM HLT: Helicobacter pylori Protocols. In.: Humana Press; 1997.
27. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG: The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 1997, 25(24):4876-4882.
28. Tamura K, Dudley J, Nei M, Kumar S: MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 2007, 24(8):1596-1599.
29. Gouet P, Courcelle E, Stuart DI, Metoz F: ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 1999, 15(4):305-308.
30. Wilks A, Schmitt MP: Expression and characterization of a heme oxygenase (Hmu O) from Corynebacterium diphtheriae. Iron acquisition requires oxidative cleavage of the heme macrocycle. J Biol Chem 1998, 273(2):837-841.
31. Ishikawa K, Sato M, Yoshida T: Expression of rat heme oxygenase in Escherichia coli as a catalytically active, full-length form that binds to bacterial membranes. Eur J Biochem 1991, 202(1):161-165.
32.巴德年主编:当代免疫学技术与应用第1版.北京:北京医科大学中国协和医科大学联合出版社; 1998.
33.杨静,徐柏森:实用电镜技术:东南大学出版社; 2008.
34. Michie KA, Monahan LG, Beech PL, Harry EJ: Trapping of a spiral-like intermediate of the bacterial cytokinetic protein FtsZ. J Bacteriol 2006, 188(5):1680-1690.
35.甄永苏,邵荣光:现代生物技术制药丛书--抗体工程药物.北京:化学工业出版社; 2002.
36.方莹:免疫胶体金技术及其在微生物检测中的应用.中国卫生检验杂志2006, 16(11):1399-1401.
37.王志刚,王秀宏,滕悦秋,刘明,周虹:重组NADPH-细胞色素P450还原酶在大肠杆菌中的表达和纯化.哈尔滨医科大学学报2004, 38(2):99-101,106
38. Lee BC: Isolation of an outer membrane hemin-binding protein of Haemophilus influenzae type b. Infect Immun 1992, 60(3):810-816.
39. Bereswill S, Greiner S, van Vliet AH, Waidner B, Fassbinder F, Schiltz E, Kusters JG, Kist M: Regulation of ferritin-mediated cytoplasmic iron storage by the ferric uptake regulator homolog (Fur) of Helicobacter pylori. J Bacteriol 2000, 182(21):5948-5953.
40. Stahler FN, Odenbreit S, Haas R, Wilrich J, Van Vliet AH, Kusters JG, Kist M, Bereswill S: The novel Helicobacter pylori CznABC metal efflux pump is required for cadmium, zinc, and nickel resistance, urease modulation, and gastric colonization. Infect Immun 2006, 74(7):3845-3852.
41. Zhu W, Hunt DJ, Richardson AR, Stojiljkovic I: Use of heme compounds as iron sources by pathogenic neisseriae requires the product of the hemO gene. J Bacteriol 2000, 182(2):439-447.
42. Kunkle CA, Schmitt MP: Comparative analysis of hmuO function and expression in Corynebacterium species. J Bacteriol 2007, 189(9):3650-3654.
43. Puri S, O'Brian MR: The hmuQ and hmuD genes from Bradyrhizobium japonicum encode heme-degrading enzymes. J Bacteriol 2006, 188(18):6476-6482.
44. Suits MD, Pal GP, Nakatsu K, Matte A, Cygler M, Jia Z: Identification of an Escherichia coli O157:H7 heme oxygenase with tandem functional repeats. Proc Natl Acad Sci U S A 2005, 102(47):16955-16960.
45. Heuermann D, Haas R: A stable shuttle vector system for efficient genetic complementation of Helicobacter pylori strains by transformation and conjugation. Mol Gen Genet 1998, 257(5):519-528.
46. Feng Y, Li M, Zhang H, Zheng B, Han H, Wang C, Yan J, Tang J, Gao GF: Functional definition and global regulation of Zur, a zinc uptake regulator in a Streptococcus suis serotype 2 strain causing streptococcal toxic shock syndrome. J Bacteriol 2008, 190(22):7567-7578.
47. Zeng H, Guo G, Mao XH, Tong WD, Zou QM: Proteomic insights into Helicobacter pylori coccoid forms under oxidative stress. Curr Microbiol 2008, 57(4):281-286.
48.刘炯,许国铭,屠振兴,等: cag致病岛缺失的中国幽门螺杆菌突变菌株的构建及鉴定.中华微生物学和免疫学杂志2002, 22(2):206-210.
49. Throup JP, Koretke KK, Bryant AP, Ingraham KA, Chalker AF, Ge Y, Marra A, Wallis NG, Brown JR, Holmes DJ et al: A genomic analysis of two-componentsignal transduction in Streptococcus pneumoniae. Mol Microbiol 2000, 35(3):566-576.
50.金涛,吴补领,苏凌云,等:远缘链球菌ftsK基因敲除菌株的构建.牙体牙髓牙周病学杂志2005, 15(5):251-254.
51.李明:二元信号转导系统SalK/SalR调控高致病性2型猪链球菌毒力的实验研究.重庆:第三军医大学; 2008.
52. Merrell DS, Thompson LJ, Kim CC, Mitchell H, Tompkins LS, Lee A, Falkow S: Growth phase-dependent response of Helicobacter pylori to iron starvation. Infect Immun 2003, 71(11):6510-6525.
53. Gancz H, Censini S, Merrell DS: Iron and pH homeostasis intersect at the level of Fur regulation in the gastric pathogen Helicobacter pylori. Infect Immun 2006, 74(1):602-614.
1. Abraham NG, Lin JH, Schwartzman ML, Levere RD, Shibahara S: The physiological significance of heme oxygenase. Int J Biochem 1988, 20(6):543-558.
2. Frankenberg N, Mukougawa K, Kohchi T, Lagarias JC: Functional genomic analysis of the HY2 family of ferredoxin-dependent bilin reductases from oxygenic photosynthetic organisms. Plant Cell 2001, 13(4):965-978.
3. Richaud C, Zabulon G: The heme oxygenase gene (pbsA) in the red alga Rhodella violacea is discontinuous and transcriptionally activated during iron limitation. Proc Natl Acad Sci U S A 1997, 94(21):11736-11741.
4. Schmitt MP: Utilization of host iron sources by Corynebacterium diphtheriae: identification of a gene whose product is homologous to eukaryotic heme oxygenases and is required for acquisition of iron from heme and hemoglobin. J Bacteriol 1997, 179(3):838-845.
5. Zhu W, Wilks A, Stojiljkovic I: Degradation of heme in gram-negative bacteria: the product of the hemO gene of Neisseriae is a heme oxygenase. J Bacteriol 2000, 182(23):6783-6790.
6. Otto BR, Sparrius M, Verweij-van Vught AM, MacLaren DM: Iron-regulated outer membrane protein of Bacteroides fragilis involved in heme uptake. Infect Immun 1990, 58(12):3954-3958.
7. Maines MD, Trakshel GM: Purification and characterization of human biliverdin reductase. Arch Biochem Biophys 1993, 300(1):320-326.
8. Skaar EP, Gaspar AH, Schneewind O: IsdG and IsdI, heme-degrading enzymes in the cytoplasm of Staphylococcus aureus. J Biol Chem 2004, 279(1):436-443.
9. Migita CT, Zhang X, Yoshida T: Expression and characterization of cyanobacterium heme oxygenase, a key enzyme in the phycobilin synthesis. Properties of the heme complex of recombinant active enzyme. Eur J Biochem 2003, 270(4):687-698.
10. Loeb MR: Ferrochelatase activity and protoporphyrin IX utilization in Haemophilus influenzae. J Bacteriol 1995, 177(12):3613-3615.
11. Wilks A, Burkhard KA: Heme and virulence: how bacterial pathogens regulate, transport and utilize heme. Nat Prod Rep 2007, 24(3):511-522.
12. Clarke TE, Tari LW, Vogel HJ: Structural biology of bacterial iron uptake systems. Curr Top Med Chem 2001, 1(1):7-30.
13. Andrews SC, Robinson AK, Rodriguez-Quinones F: Bacterial iron homeostasis. FEMS Microbiol Rev 2003, 27(2-3):215-237.
14. Vasil ML, Ochsner UA: The response of Pseudomonas aeruginosa to iron: genetics, biochemistry and virulence. Mol Microbiol 1999, 34(3):399-413.
15. Hirotsu S, Chu GC, Unno M, Lee DS, Yoshida T, Park SY, Shiro Y, Ikeda-Saito M: The crystal structures of the ferric and ferrous forms of the heme complex of HmuO, a heme oxygenase of Corynebacterium diphtheriae. J Biol Chem 2004, 279(12):11937-11947.
16. Tai SS, Wang TR, Lee CJ: Characterization of hemin binding activity of Streptococcus pneumoniae. Infect Immun 1997, 65(3):1083-1087.
17. Drazek ES, Hammack CA, Schmitt MP: Corynebacterium diphtheriae genes required for acquisition of iron from haemin and haemoglobin are homologous to ABC haemin transporters. Mol Microbiol 2000, 36(1):68-84.
18. Mazmanian SK, Skaar EP, Gaspar AH, Humayun M, Gornicki P, Jelenska J, Joachmiak A, Missiakas DM, Schneewind O: Passage of heme-iron across the envelope of Staphylococcus aureus. Science 2003, 299(5608):906-909.
19. Engel RR, Matsen JM, Chapman SS, Schwartz S: Carbon monoxide production from heme compounds by bacteria. J Bacteriol 1972, 112(3):1310-1315.
20. Schmitt MP: Transcription of the Corynebacterium diphtheriae hmuO gene is regulated by iron and heme. Infect Immun 1997, 65(11):4634-4641.
21. Zhu W, Hunt DJ, Richardson AR, Stojiljkovic I: Use of heme compounds as iron sources by pathogenic neisseriae requires the product of the hemO gene. J Bacteriol 2000, 182(2):439-447.
22. Ratliff M, Zhu W, Deshmukh R, Wilks A, Stojiljkovic I: Homologues of neisserial heme oxygenase in gram-negative bacteria: degradation of heme by the product of the pigA gene of Pseudomonas aeruginosa. J Bacteriol 2001, 183(21):6394-6403.
23. Frankenberg-Dinkel N: Bacterial heme oxygenases. Antioxid Redox Signal 2004, 6(5):825-834.
24. Wilks A: Heme oxygenase: evolution, structure, and mechanism. Antioxid RedoxSignal 2002, 4(4):603-614.
25. Unno M, Matsui T, Ikeda-Saito M: Structure and catalytic mechanism of heme oxygenase. Nat Prod Rep 2007, 24(3):553-570.
26. Sakamoto H, Sugishima M, Higashimoto Y, Fukuyama K, Noguchi M: [Structure and mechanism of heme oxygenase]. Seikagaku 2005, 77(7):634-638.
27. Cannon JB: Pharmaceutics and drug delivery aspects of heme and porphyrin therapy. J Pharm Sci 1993, 82(5):435-446.
28. Muramoto T, Tsurui N, Terry MJ, Yokota A, Kohchi T: Expression and biochemical properties of a ferredoxin-dependent heme oxygenase required for phytochrome chromophore synthesis. Plant Physiol 2002, 130(4):1958-1966.
29. Takahashi S, Wang J, Rousseau DL, Ishikawa K, Yoshida T, Host JR, Ikeda-Saito M: Heme-heme oxygenase complex. Structure of the catalytic site and its implication for oxygen activation. J Biol Chem 1994, 269(2):1010-1014.
30. Guo Y, Guo G, Mao X, Zhang W, Xiao J, Tong W, Liu T, Xiao B, Liu X, Feng Y et al: Functional identification of HugZ, a heme oxygenase from Helicobacter pylori. BMC Microbiol 2008, 8:226.
31. Ridley KA, Rock JD, Li Y, Ketley JM: Heme utilization in Campylobacter jejuni. J Bacteriol 2006, 188(22):7862-7875.
32. Lill R, Kispal G: Maturation of cellular Fe-S proteins: an essential function of mitochondria. Trends Biochem Sci 2000, 25(8):352-356.
33. Caignan GA, Deshmukh R, Wilks A, Zeng Y, Huang HW, Moenne-Loccoz P, Bunce RA, Eastman MA, Rivera M: Oxidation of heme to beta- and delta-biliverdin by Pseudomonas aeruginosa heme oxygenase as a consequence of an unusual seating of the heme. J Am Chem Soc 2002, 124(50):14879-14892.
34. Chu GC, Park SY, Shiro Y, Yoshida T, Ikeda-Saito M: Crystallization and preliminary X-ray diffraction analysis of a recombinant bacterial heme oxygenase (Hmu O) from Corynebacterium diphtheriae. J Struct Biol 1999, 126(2):171-174.
35. Schuller DJ, Wilks A, Ortiz de Montellano PR, Poulos TL: Crystal structure of human heme oxygenase-1. Nat Struct Biol 1999, 6(9):860-867.
36. Sugishima M, Omata Y, Kakuta Y, Sakamoto H, Noguchi M, Fukuyama K: Crystal structure of rat heme oxygenase-1 in complex with heme. FEBS Lett 2000,471(1):61-66.
37. Kakuta Y, Horio T, Takahashi Y, Fukuyama K: Crystal structure of Escherichia coli Fdx, an adrenodoxin-type ferredoxin involved in the assembly of iron-sulfur clusters. Biochemistry 2001, 40(37):11007-11012.
38. Schuller DJ, Zhu W, Stojiljkovic I, Wilks A, Poulos TL: Crystal structure of heme oxygenase from the gram-negative pathogen Neisseria meningitidis and a comparison with mammalian heme oxygenase-1. Biochemistry 2001, 40(38): 11552-11558.
2. Marshall BJ, Warren JR: Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet 1984, 1(8390):1311-1315.
3. Dunn BE, Cohen H, Blaser MJ: Helicobacter pylori. Clin Microbiol Rev 1997, 10(4):720-741.
4.胡伏莲,周殿元:幽门螺杆菌感染的基础与临床.北京:中国科学技术出版社; 2002.
5. Gottke MU, Fallone CA, Barkun AN, Vogt K, Loo V, Trautmann M, Tong JZ, Nguyen TN, Fainsilber T, Hahn HH et al: Genetic variability determinants of Helicobacter pylori: influence of clinical background and geographic origin of isolates. J Infect Dis 2000, 181(5):1674-1681.
6. Akopyants NS, Eaton KA, Berg DE: Adaptive mutation and cocolonization during Helicobacter pylori infection of gnotobiotic piglets. Infect Immun 1995, 63(1):116-121.
7. Salaun L, Ayraud S, Saunders NJ: Phase variation mediated niche adaptation during prolonged experimental murine infection with Helicobacter pylori. Microbiology 2005, 151(Pt 3):917-923.
8.郭刚,王毅超,邹全明等:幽门螺杆菌长期感染蒙古沙鼠模型的建立.中华微生物学与免疫学杂志2001, 21(6):683-684.
9. Guo G, Tong WD, Zeng H, Liu KY, Zou QM: [Comparative proteomics analysis of Helicobacter pylori after adaptive colonization in Mongolian gerbils]. Wei Sheng Wu Xue Bao 2007, 47(3):461-464.
10. Koga T, Shimada Y, Sato K, Takahashi K, Kikuchi I, Okazaki Y, Miura T, Katsuta M, Iwata M: Contribution of ferrous iron to maintenance of the gastric colonization of Helicobacter pylori in miniature pigs. Microbiol Res 2002, 157(4):323-330.
11. Waidner B, Greiner S, Odenbreit S, Kavermann H, Velayudhan J, Stahler F, Guhl J, Bisse E, van Vliet AH, Andrews SC et al: Essential role of ferritin Pfr in Helicobacter pylori iron metabolism and gastric colonization. Infect Immun 2002,70(7):3923-3929.
12. Nakao K, Imoto I, Ikemura N, Shibata T, Takaji S, Taguchi Y, Misaki M, Yamauchi K, Yamazaki N: Relation of lactoferrin levels in gastric mucosa with Helicobacter pylori infection and with the degree of gastric inflammation. Am J Gastroenterol 1997, 92(6):1005-1011.
13. Frankenberg-Dinkel N: Bacterial heme oxygenases. Antioxid Redox Signal 2004, 6(5):825-834.
14. Wilks A, Burkhard KA: Heme and virulence: how bacterial pathogens regulate, transport and utilize heme. Nat Prod Rep 2007, 24(3):511-522.
15. Payne SM: Iron acquisition in microbial pathogenesis. Trends Microbiol 1993, 1(2):66-69.
16. Andrews SC, Robinson AK, Rodriguez-Quinones F: Bacterial iron homeostasis. FEMS Microbiol Rev 2003, 27(2-3):215-237.
17. Wandersman C, Delepelaire P: Bacterial iron sources: from siderophores to hemophores. Annu Rev Microbiol 2004, 58:611-647.
18. Wyckoff EE, Schmitt M, Wilks A, Payne SM: HutZ is required for efficient heme utilization in Vibrio cholerae. J Bacteriol 2004, 186(13):4142-4151.
19. Ridley KA, Rock JD, Li Y, Ketley JM: Heme utilization in Campylobacter jejuni. J Bacteriol 2006, 188(22):7862-7875.
20. Perry RD, Shah J, Bearden SW, Thompson JM, Fetherston JD: Yersinia pestis TonB: role in iron, heme, and hemoprotein utilization. Infect Immun 2003, 71(7):4159-4162.
21. Jarosik GP, Sanders JD, Cope LD, Muller-Eberhard U, Hansen EJ: A functional tonB gene is required for both utilization of heme and virulence expression by Haemophilus influenzae type b. Infect Immun 1994, 62(6):2470-2477.
22. Worst DJ, Otto BR, de Graaff J: Iron-repressible outer membrane proteins of Helicobacter pylori involved in heme uptake. Infect Immun 1995, 63(10):4161-4165.
23. Worst DJ, Maaskant J, Vandenbroucke-Grauls CM, Kusters JG: Multiple haem-utilization loci in Helicobacter pylori. Microbiology 1999, 145 ( Pt 3):681-688.
24. F.奥斯伯著,颜子颖等译:精编分子生物学实验指南.北京:科学出版社. 1998.
25.卢圣栋主编:现代分子生物学实验技术.第1版.北京:高等教育出版社; 1993.
26. Clayton .CLaM HLT: Helicobacter pylori Protocols. In.: Humana Press; 1997.
27. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG: The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 1997, 25(24):4876-4882.
28. Tamura K, Dudley J, Nei M, Kumar S: MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 2007, 24(8):1596-1599.
29. Gouet P, Courcelle E, Stuart DI, Metoz F: ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 1999, 15(4):305-308.
30. Wilks A, Schmitt MP: Expression and characterization of a heme oxygenase (Hmu O) from Corynebacterium diphtheriae. Iron acquisition requires oxidative cleavage of the heme macrocycle. J Biol Chem 1998, 273(2):837-841.
31. Ishikawa K, Sato M, Yoshida T: Expression of rat heme oxygenase in Escherichia coli as a catalytically active, full-length form that binds to bacterial membranes. Eur J Biochem 1991, 202(1):161-165.
32.巴德年主编:当代免疫学技术与应用第1版.北京:北京医科大学中国协和医科大学联合出版社; 1998.
33.杨静,徐柏森:实用电镜技术:东南大学出版社; 2008.
34. Michie KA, Monahan LG, Beech PL, Harry EJ: Trapping of a spiral-like intermediate of the bacterial cytokinetic protein FtsZ. J Bacteriol 2006, 188(5):1680-1690.
35.甄永苏,邵荣光:现代生物技术制药丛书--抗体工程药物.北京:化学工业出版社; 2002.
36.方莹:免疫胶体金技术及其在微生物检测中的应用.中国卫生检验杂志2006, 16(11):1399-1401.
37.王志刚,王秀宏,滕悦秋,刘明,周虹:重组NADPH-细胞色素P450还原酶在大肠杆菌中的表达和纯化.哈尔滨医科大学学报2004, 38(2):99-101,106
38. Lee BC: Isolation of an outer membrane hemin-binding protein of Haemophilus influenzae type b. Infect Immun 1992, 60(3):810-816.
39. Bereswill S, Greiner S, van Vliet AH, Waidner B, Fassbinder F, Schiltz E, Kusters JG, Kist M: Regulation of ferritin-mediated cytoplasmic iron storage by the ferric uptake regulator homolog (Fur) of Helicobacter pylori. J Bacteriol 2000, 182(21):5948-5953.
40. Stahler FN, Odenbreit S, Haas R, Wilrich J, Van Vliet AH, Kusters JG, Kist M, Bereswill S: The novel Helicobacter pylori CznABC metal efflux pump is required for cadmium, zinc, and nickel resistance, urease modulation, and gastric colonization. Infect Immun 2006, 74(7):3845-3852.
41. Zhu W, Hunt DJ, Richardson AR, Stojiljkovic I: Use of heme compounds as iron sources by pathogenic neisseriae requires the product of the hemO gene. J Bacteriol 2000, 182(2):439-447.
42. Kunkle CA, Schmitt MP: Comparative analysis of hmuO function and expression in Corynebacterium species. J Bacteriol 2007, 189(9):3650-3654.
43. Puri S, O'Brian MR: The hmuQ and hmuD genes from Bradyrhizobium japonicum encode heme-degrading enzymes. J Bacteriol 2006, 188(18):6476-6482.
44. Suits MD, Pal GP, Nakatsu K, Matte A, Cygler M, Jia Z: Identification of an Escherichia coli O157:H7 heme oxygenase with tandem functional repeats. Proc Natl Acad Sci U S A 2005, 102(47):16955-16960.
45. Heuermann D, Haas R: A stable shuttle vector system for efficient genetic complementation of Helicobacter pylori strains by transformation and conjugation. Mol Gen Genet 1998, 257(5):519-528.
46. Feng Y, Li M, Zhang H, Zheng B, Han H, Wang C, Yan J, Tang J, Gao GF: Functional definition and global regulation of Zur, a zinc uptake regulator in a Streptococcus suis serotype 2 strain causing streptococcal toxic shock syndrome. J Bacteriol 2008, 190(22):7567-7578.
47. Zeng H, Guo G, Mao XH, Tong WD, Zou QM: Proteomic insights into Helicobacter pylori coccoid forms under oxidative stress. Curr Microbiol 2008, 57(4):281-286.
48.刘炯,许国铭,屠振兴,等: cag致病岛缺失的中国幽门螺杆菌突变菌株的构建及鉴定.中华微生物学和免疫学杂志2002, 22(2):206-210.
49. Throup JP, Koretke KK, Bryant AP, Ingraham KA, Chalker AF, Ge Y, Marra A, Wallis NG, Brown JR, Holmes DJ et al: A genomic analysis of two-componentsignal transduction in Streptococcus pneumoniae. Mol Microbiol 2000, 35(3):566-576.
50.金涛,吴补领,苏凌云,等:远缘链球菌ftsK基因敲除菌株的构建.牙体牙髓牙周病学杂志2005, 15(5):251-254.
51.李明:二元信号转导系统SalK/SalR调控高致病性2型猪链球菌毒力的实验研究.重庆:第三军医大学; 2008.
52. Merrell DS, Thompson LJ, Kim CC, Mitchell H, Tompkins LS, Lee A, Falkow S: Growth phase-dependent response of Helicobacter pylori to iron starvation. Infect Immun 2003, 71(11):6510-6525.
53. Gancz H, Censini S, Merrell DS: Iron and pH homeostasis intersect at the level of Fur regulation in the gastric pathogen Helicobacter pylori. Infect Immun 2006, 74(1):602-614.
1. Abraham NG, Lin JH, Schwartzman ML, Levere RD, Shibahara S: The physiological significance of heme oxygenase. Int J Biochem 1988, 20(6):543-558.
2. Frankenberg N, Mukougawa K, Kohchi T, Lagarias JC: Functional genomic analysis of the HY2 family of ferredoxin-dependent bilin reductases from oxygenic photosynthetic organisms. Plant Cell 2001, 13(4):965-978.
3. Richaud C, Zabulon G: The heme oxygenase gene (pbsA) in the red alga Rhodella violacea is discontinuous and transcriptionally activated during iron limitation. Proc Natl Acad Sci U S A 1997, 94(21):11736-11741.
4. Schmitt MP: Utilization of host iron sources by Corynebacterium diphtheriae: identification of a gene whose product is homologous to eukaryotic heme oxygenases and is required for acquisition of iron from heme and hemoglobin. J Bacteriol 1997, 179(3):838-845.
5. Zhu W, Wilks A, Stojiljkovic I: Degradation of heme in gram-negative bacteria: the product of the hemO gene of Neisseriae is a heme oxygenase. J Bacteriol 2000, 182(23):6783-6790.
6. Otto BR, Sparrius M, Verweij-van Vught AM, MacLaren DM: Iron-regulated outer membrane protein of Bacteroides fragilis involved in heme uptake. Infect Immun 1990, 58(12):3954-3958.
7. Maines MD, Trakshel GM: Purification and characterization of human biliverdin reductase. Arch Biochem Biophys 1993, 300(1):320-326.
8. Skaar EP, Gaspar AH, Schneewind O: IsdG and IsdI, heme-degrading enzymes in the cytoplasm of Staphylococcus aureus. J Biol Chem 2004, 279(1):436-443.
9. Migita CT, Zhang X, Yoshida T: Expression and characterization of cyanobacterium heme oxygenase, a key enzyme in the phycobilin synthesis. Properties of the heme complex of recombinant active enzyme. Eur J Biochem 2003, 270(4):687-698.
10. Loeb MR: Ferrochelatase activity and protoporphyrin IX utilization in Haemophilus influenzae. J Bacteriol 1995, 177(12):3613-3615.
11. Wilks A, Burkhard KA: Heme and virulence: how bacterial pathogens regulate, transport and utilize heme. Nat Prod Rep 2007, 24(3):511-522.
12. Clarke TE, Tari LW, Vogel HJ: Structural biology of bacterial iron uptake systems. Curr Top Med Chem 2001, 1(1):7-30.
13. Andrews SC, Robinson AK, Rodriguez-Quinones F: Bacterial iron homeostasis. FEMS Microbiol Rev 2003, 27(2-3):215-237.
14. Vasil ML, Ochsner UA: The response of Pseudomonas aeruginosa to iron: genetics, biochemistry and virulence. Mol Microbiol 1999, 34(3):399-413.
15. Hirotsu S, Chu GC, Unno M, Lee DS, Yoshida T, Park SY, Shiro Y, Ikeda-Saito M: The crystal structures of the ferric and ferrous forms of the heme complex of HmuO, a heme oxygenase of Corynebacterium diphtheriae. J Biol Chem 2004, 279(12):11937-11947.
16. Tai SS, Wang TR, Lee CJ: Characterization of hemin binding activity of Streptococcus pneumoniae. Infect Immun 1997, 65(3):1083-1087.
17. Drazek ES, Hammack CA, Schmitt MP: Corynebacterium diphtheriae genes required for acquisition of iron from haemin and haemoglobin are homologous to ABC haemin transporters. Mol Microbiol 2000, 36(1):68-84.
18. Mazmanian SK, Skaar EP, Gaspar AH, Humayun M, Gornicki P, Jelenska J, Joachmiak A, Missiakas DM, Schneewind O: Passage of heme-iron across the envelope of Staphylococcus aureus. Science 2003, 299(5608):906-909.
19. Engel RR, Matsen JM, Chapman SS, Schwartz S: Carbon monoxide production from heme compounds by bacteria. J Bacteriol 1972, 112(3):1310-1315.
20. Schmitt MP: Transcription of the Corynebacterium diphtheriae hmuO gene is regulated by iron and heme. Infect Immun 1997, 65(11):4634-4641.
21. Zhu W, Hunt DJ, Richardson AR, Stojiljkovic I: Use of heme compounds as iron sources by pathogenic neisseriae requires the product of the hemO gene. J Bacteriol 2000, 182(2):439-447.
22. Ratliff M, Zhu W, Deshmukh R, Wilks A, Stojiljkovic I: Homologues of neisserial heme oxygenase in gram-negative bacteria: degradation of heme by the product of the pigA gene of Pseudomonas aeruginosa. J Bacteriol 2001, 183(21):6394-6403.
23. Frankenberg-Dinkel N: Bacterial heme oxygenases. Antioxid Redox Signal 2004, 6(5):825-834.
24. Wilks A: Heme oxygenase: evolution, structure, and mechanism. Antioxid RedoxSignal 2002, 4(4):603-614.
25. Unno M, Matsui T, Ikeda-Saito M: Structure and catalytic mechanism of heme oxygenase. Nat Prod Rep 2007, 24(3):553-570.
26. Sakamoto H, Sugishima M, Higashimoto Y, Fukuyama K, Noguchi M: [Structure and mechanism of heme oxygenase]. Seikagaku 2005, 77(7):634-638.
27. Cannon JB: Pharmaceutics and drug delivery aspects of heme and porphyrin therapy. J Pharm Sci 1993, 82(5):435-446.
28. Muramoto T, Tsurui N, Terry MJ, Yokota A, Kohchi T: Expression and biochemical properties of a ferredoxin-dependent heme oxygenase required for phytochrome chromophore synthesis. Plant Physiol 2002, 130(4):1958-1966.
29. Takahashi S, Wang J, Rousseau DL, Ishikawa K, Yoshida T, Host JR, Ikeda-Saito M: Heme-heme oxygenase complex. Structure of the catalytic site and its implication for oxygen activation. J Biol Chem 1994, 269(2):1010-1014.
30. Guo Y, Guo G, Mao X, Zhang W, Xiao J, Tong W, Liu T, Xiao B, Liu X, Feng Y et al: Functional identification of HugZ, a heme oxygenase from Helicobacter pylori. BMC Microbiol 2008, 8:226.
31. Ridley KA, Rock JD, Li Y, Ketley JM: Heme utilization in Campylobacter jejuni. J Bacteriol 2006, 188(22):7862-7875.
32. Lill R, Kispal G: Maturation of cellular Fe-S proteins: an essential function of mitochondria. Trends Biochem Sci 2000, 25(8):352-356.
33. Caignan GA, Deshmukh R, Wilks A, Zeng Y, Huang HW, Moenne-Loccoz P, Bunce RA, Eastman MA, Rivera M: Oxidation of heme to beta- and delta-biliverdin by Pseudomonas aeruginosa heme oxygenase as a consequence of an unusual seating of the heme. J Am Chem Soc 2002, 124(50):14879-14892.
34. Chu GC, Park SY, Shiro Y, Yoshida T, Ikeda-Saito M: Crystallization and preliminary X-ray diffraction analysis of a recombinant bacterial heme oxygenase (Hmu O) from Corynebacterium diphtheriae. J Struct Biol 1999, 126(2):171-174.
35. Schuller DJ, Wilks A, Ortiz de Montellano PR, Poulos TL: Crystal structure of human heme oxygenase-1. Nat Struct Biol 1999, 6(9):860-867.
36. Sugishima M, Omata Y, Kakuta Y, Sakamoto H, Noguchi M, Fukuyama K: Crystal structure of rat heme oxygenase-1 in complex with heme. FEBS Lett 2000,471(1):61-66.
37. Kakuta Y, Horio T, Takahashi Y, Fukuyama K: Crystal structure of Escherichia coli Fdx, an adrenodoxin-type ferredoxin involved in the assembly of iron-sulfur clusters. Biochemistry 2001, 40(37):11007-11012.
38. Schuller DJ, Zhu W, Stojiljkovic I, Wilks A, Poulos TL: Crystal structure of heme oxygenase from the gram-negative pathogen Neisseria meningitidis and a comparison with mammalian heme oxygenase-1. Biochemistry 2001, 40(38): 11552-11558.