The Function Research Of Helicobacter Pylori Hp0318 (hugZ) Gene

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 HugZBioinformatics 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 proteinTo 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. pylori3. HP0318 (HugZ) is a heme oxygenase..3.1 HugZ has heme binding activityTo 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 hemeIn 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 degradationBiliverdin 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 iron4.1 The hugZ mutant fails to utilize heme iron for normal growthIn 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 ironTo 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.