胃十二指肠疾病高发区幽门螺杆菌毒力分析及dupA(2499)基因功能研究
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摘要
目的：
幽门螺杆菌(H. pylori)是定植于人类胃粘膜上皮细胞表面的一种细菌,与胃十二指肠疾病的发生和发展密切相关。分析胃十二指肠疾病高发区(威海)H. pylori临床分离株毒力因子的分子特征(包括cagPAI、vacA基因和dupA基因),以评价该地区H. pylori I临床分离株的毒力。研究该地区H. pylori菌株的dupA基因功能,以探讨H. pylori dupA基因的致病机制。
方法：
1.选取胃十二指肠疾病高发区(威海)临床就诊的患者,进行胃粘膜组织分离培养H. pylori菌株。采用PCR扩增结合测序的方法,分析H.pylori I临床分离株cagPAI (cagA, cagE, cagl, cagL, cagM, cagT和cagX基因)、cagA基因3’端可变区、vacA基因和dupA基因类型,与NCBI (National Center of Biotechnology Information)中相关序列进行比对。并根据基因序列获得了相应蛋白的氨基酸序列,采用MEGA4.1构建相应系统发育树。
2.研究分离于十二指肠溃疡患者的H. pylori WH-21菌株携带的dupA基因类型,并对其蛋白进行生物学信息学分析,包括该蛋白一级结构、二级结构和三级结构特征及该蛋白细胞定位和功能结构域等。
3.构建pET-32a-dupA原核表达载体,转化于E. coli Rosetta(DE3)中。经IPTG诱导后表达重组DupA蛋白,以Ni2+-NTA珠分离纯化目的蛋白。利用ATPase分解ATP生成无机磷的原理,检测重组DupA蛋白的ATPase活性。采用动物实验制备抗重组DupA蛋白多克隆抗血清。
4.利用Western blotting法检测DupA (2499)蛋白的亚细胞定位。采用免疫共沉淀实验和质谱分析方法,进行DupA (2499)蛋白的互作蛋白分析。
5.构建敲除载体pBluescript/△dupA::KMr,采用同源重组的方法构建H. pylori dupA (2499)基因缺失株。
6. H. pylori和GES-1细胞共培养后,采用Western blotting方法分析dupA (2499)基因的缺失对H. pylori CagA蛋白转运的影响,以及CFU实验和粘附率实验分析H. pylori dupA (2499)基因缺失株的粘附功能。
7.分析不同pH值液体培养基中H. pylori生长情况,研究IH. pylori dupA (2499)基因缺失株对酸性环境的抵抗力。采用Western blotting,分析培养后上清液中脲酶含量。H. pylori和GES-1细胞共培养,采用ELISA方法分析上清液中IL-8的浓度,分析H. pylori dupA (2499)基因缺失株毒力变化。
8. H. pylori和MKN-45细胞共培养后,采用MTT方法检测H. pylori dupA (2499)基因缺失株的细胞毒变化,Hoechst33258染色后显微镜下观察H. pylori dupA (2499)基因缺失株对肿瘤细胞形态变化的影响,Western blotting分析线粒体通路的凋亡相关分子标志变化,包括活化Caspase-3, PARP, Bax和Bcl-2变化。
结果：
1.经过PCR扩增后,116株H. pyloril临床分离株均携带cagA, cagL, cagM, cagT和cagX基因,而只有89.7%(104/116)和82.8%(96/116)菌株分别扩增出cagE和cagI基因。25.9%(30/116)菌株中发现cag部分缺失,其中cagPAI部分缺失株在慢性胃炎中占44.4%(24/54)显著高于胃溃疡、十二指肠溃疡和胃癌(9.7%,6/62)咖<0.001)。未发现cagPAI完全缺失的H. pylori菌株。
24株菌株cagA3'端可变区氨基酸序列可分为两大群：东亚型(91.7%)和西方型(8.3%),其中前者为EPIYA-A-B-D型,后者为EPIYA-A-B-C型。CagPAI蛋白中除了CagL蛋白外,CagI、CagM、CagT和CagX蛋白序列均为保守。系统发育树提示CagPAI蛋白也分为两个群：东亚型和西方型,大部分菌株也属于东亚型。
H. pyloril临床分离株vacA基因s区和m区的主要流行模式为s1a/m2(48.3%)和slc/m2(13.8%)。31.0%的H. pylori临床分离株携带dupA基因,其中在胃溃疡、十二指肠溃疡和胃癌携带率为40.3%,高于慢性胃炎(20.4%,p=0.02)。dupA基因序列分析显示其读码框架为2499bp,称作dupA (2499)基因。
2. H. pylori WH-21菌株携带的dupA基因全长2499bp,称作dupA (2499)基因。威海地区8株dupA碱基序列同源性为99.3%,与日本两株H. pylori菌株的dupA基因碱基序列同源性为99.25%,不同地区14株H. pylori dupA碱基序列同源性为91.65%。生物信息学分析发现DupA (2499)蛋白为稳定亲水性蛋白质,抗原性强。无信号肽且有多个跨膜区,定位于细胞内膜。a螺旋是该蛋白多肽链中主要组成结构元件,具有一个卷曲螺旋结构。三维结构预测其配体结合物质为ADP、ATP和Mg2+等。与CagE_TrbE_VirB超家族有较高的同源性,具有ATP结合位点。
3.获得了重组DupA蛋白,该蛋白ATPase活力为129.5±17.8/mgprot。成功制备了抗重组DupA蛋白多克隆抗血清,抗体效价为1：6.4x104。
4. Western blotting结果显示DupA (2499)蛋白定位于胞膜。经过免疫共沉淀实验和质谱分析,DupA (2499)蛋白的互作蛋白为脲酶和HSP60。
5.获得了H. pylori dupA (2499)基因缺失株,并通过了PCR和Western blotting的验证。
6.采用H. pylori感染GES-1细胞后,缺失株组和野生株组向胞内转运CagA蛋白无差别。CFU实验结果[(27.2±7.1)×106和(28.5±6.9)×106,p=0.772]和粘附率实验结果(81.0%和78.8%,p=0.724)均无明显差异。
7.除pH6.5-pH7.5之外,其它酸性环境下野生株组菌量显著高于缺失株组(p<0.05)。缺失株组的脲酶分泌低于野生株组,特别在酸性环境时。细菌细胞共培养后,野生株组上清液中IL-8浓度显著高于缺失株组(p<0.001)
8.与对照组比较,细菌细胞共培养12h后MKN-45细胞存活率开始降低,缺失株组MKN-45细胞存活率显著高于野生株组(p<0.05)。细菌细胞共培养12h后,Hoechst33258染色结果显示缺失株组和野生株组均可以引起MKN-45细胞凋亡,但野生株组凋亡率显著高于缺失株组(p<0.05)。6h后野生株组和缺失株组均开始出现Caspase-3和PARP的剪切活化,野生株组强于缺失株组。与缺失株组比较,野生株组Bax明显上调而Bcl-2则明显下调。
结论：
1.胃十二指肠疾病高发区(威海)H. pylori I临床分离株为强毒株,与该地区的胃十二指肠疾病高发相关。H. pylori强毒性特征与东亚型cagA基因3’端可变区、完整的cagPAI、东亚型cagFAI基因、强毒性vacA基因和dupA (2499)基因有关。
2.dupA (2499)基因保守且同源性高,适宜进行体外表达。DupA (2499)蛋白稳定且抗原性强,适合抗体的制备。
3. DupA (2499)蛋白独立于CagPAI之外,存在于H. pylori的内膜,具有ATPase活性并参与脲酶的分泌,但不参与H. pylori的CagA蛋白的转运和其粘附过程。
4. H. pylori DupA(2499)蛋白参与脲酶分泌的调节,使携带dupA (2499)基因的H. pylori菌株具有更强的耐酸特性。耐酸特性和刺激IL-8分泌的特性,与携带dupA基因H. pylori菌株引起十二指肠溃疡高发密切相关。DupA (2499)蛋白通过线粒体通路诱导肿瘤细胞凋亡,与携带dupA基因的H. pylori菌株降低胃癌发病相关。
Objectives
Helicobacter pylori (H. pylori) colonize the surface area of the gastric mucosa in the human stomach, and are associated with an increased risk for various gastroduodenal diseases. In order to estimate the virulence of H. pylori isolated from the patients in a region at high risk of gastroduodenal diseases, we investigated the prevalence of H. pylori virulence markers, including cagPAI, vac A and dupA. The function of dupA of H. pylori was analyzed to further reveal the pathogenesis of H. pylori in the study.
Methods
1. H. pylori strains were isolated from gastric biopsies of patients in a region at high risk of gastroduodenal diseases. cagPAI genes (cagA, cagE, cagl, cagL, cagM, cagT and cagX), the cagA3'variable region, vac A, and dupA genotypes were determined by PCR. Some amplicons of the cagA3'variable region, cagPAI genes (cagl, cagL, cagM, cagT and cagX) and dupA were sequenced to compare with deposited sequences in NCBI (National Center of Biotechnology Information). The deduced amino acid sequence of proteins was obtained from the nucleotide sequence. The full-length amino acid sequences were aligned, and the phylo genetic trees were constructed by the program MEGA4.1.
2. dupA genotype of H. pylori WH-21strain was analyzed, which was isolated from the patient with duodenal ulcer. The biological functions of the deduced DupA were analyzed using the structural bioinformatics analysis methods, including the primary structure, secondary structure, tertiary structure, the prediction of protein subcellular localization and the functional domains.
3. After recombinant expression vector pET32a (+)-dupA was constructed and transformed into E. coli Rosetta (DE3), recombinant DupA fusion protein was induced by IPTG and affinity purified using Ni-nitrilotriacetic acid resin. ATPase activity of recombinant DupA fusion protein was tested using the assay based on the inorganic phosphate released by ATP hydrolysis. The polyclonal antibody to recombinant DupA fusion protein was produced by animal experiment.
4. The subcellular localization of DupA (2499) was analyzed by Western blotting. The interaction proteins of DupA (2499) were investigated using Immunoprecipitation assay and MS.
5. After the pBluescript/△dupA::KMr was constructed and introduced into H. pylori WH21strain by electroporation, an isogenic mutant of H. pylori dupA was generated by allelic exchange mutagenesis.
6. The CagA translocation activity of the dupA-deleted mutant was analyzed after GES-1cells infected with H. pylori by Western blotting. The adhesion activity of the dupA-deleted mutant was quantified by colony-forming units (CFU) assay and adhesion rate assay.
7. Susceptibility of dupA-deleted mutant to low pH was measured by the bacterial growth in the different pH liquid media. The supernatants of the different pH liquid media were collected for analyzing urease levels using Western blotting. IL-8levels in the supernatants were determined after GES-1cells co-cultured with H. pylori strains to estimate the virulence of the dupA-deleted mutant.
8. After MKN-45cells co-cultured with H. pylori strains, the death of MKN-45cells induced by H. pylori was studied in cytotoxicity assay by MTT assay, and Hoechst33258staining was performed for morphology analysis. Protein-involved mitochondria mediated apoptosis were analyzed by Western blotting, including Caspase-3, PARP, Bax and Bcl-2.
Results
1. After PCR amplification of the cagPAI genes, the results showed that100%(116/116) of the isolates were positive for cagA, cagL, cagM, cagT and cagX, whereas89.7%(104/116) and82.8%(96/116)strains had cagE and cagl, respectively. Isolates with partial cagPAI were found in25.9%(30/116) strains, which present at a higher frequency in chronic gastritis (44.4%,24/54) than that of gastric ulcer, duodenal ulcer and gastric cancer (9.7%,6/62)(p<0.001). None of the isolates completely lacked the cagPAI.
cagA3'variable regions of24strains fell into two types:East Asian-type (91.7%) classified as EPIYA-A-B-D and Western-type (28.3%) classified as EPIYA-A-B-C. The proteins encoded by cagPAI genes (CagI、CagM、CagT、CagX) were conserved with high homology except CagL.The phylogenetic trees of the full-length CagPAI proteins (CagI, CagL, CagM, CagT and CagX) demonstrated that all CagPAI proteins in the isolates could be placed into two major groups (East Asian-group and Western-group), and most of them were included in the East Asian-group.
It showed that sla/m2(48.3%) and s1c/m2(13.8%) strains were the most prevalent H. pylori strains in this region. Thirty-one percent of strains (31.0%) possessed dupA, and the prevalence of dupA was significantly higher in strains from patients with gastric ulcer, duodenal ulcer and gastric cancer (40.3%) than that from chronic gastritis (20.4%, p=0.02). Sequencing of dupA revealed an ORF of2,449-bp, which was called as dupA (2499).
2. Sequencing of dupA in H. pylori WH-21revealed an ORF of2,449-bp, which was dupA (2499). There was homology among dupA sequences of the nine strains from Weihai, approximately99.3%. The homology among strains from Weihai and two strains from Japan was99.25%, while it was91.65%among fourteen strians isolated from different regions. Using the bioinformatics software, DupA (2499) was classified as stable one with high hydrophilicity and antigenicity. There was transmembrane helices but no signal peptide in DupA (2499), which located in membrane. The major structural element of polypeptide chain was α-helix, and one coiled coil was predicted in DupA (2499). It contained combined sites of ADP、ATP and Mg2+in the tertiary structure. The protein was predicted to have high homology with the CagE_TrbE_VirB superfamily with ATP binding site.
3. The expressed recombination DupA fusion protein was obtained, and the protein exhibited a clear ATPase activity (129.5±17.8U/mgproft. The polyclonal antibody to recombinant DupA fusion protein was produced in the study, and the antibody titer was1:6.4×104.
4. DupA (2499) was located in bacterial membrane using Western blotting, which interacted urease and HSP60by IP and MS.
5. The isogenic dupA mutant of H. pylori was obtained and identified by PCR using one pair of primers (P1and P4) and Western blotting using anti-DupA antibody.
6. After used H. pylori to infect GES-1cells, the quantities of CagA protein translocated into the cells were similar between the wild type strain and the dupA-deleted mutant. No Significantly decreased numbers of the dupA-deleted mutant bound to GES-1cells were compared with the wild type strain both in CFU assay [(27.2±7.1)×106and (28.5±6.9)×106, p=0.772] and adhesion rate assay (81.0%and78.8%,p=0.724).
7. The result revealed that the wild type strain had a stronger growth than the dupA-deleted mutant in low pH (p<0.05). Reduced urease secretion levels were found in dup A-deleted mutant especially in low pH comparing to the wild type strain.
After the two strains infected GES-1cells, IL-8levels were significantly higher in supernatants co-cultured with the wild type strain than in those with its dupA-deleted mutant (p<0.001)
8. The amounts of vital cells were decreased which were infected with wild type strain, compared to those for the mutant-infected cells after12h (p<0.05). After12h, apoptosis was observed in MKN-45cells treated both with the wild type strain and the deleted-dupA mutant. The number of apoptotic cells resulting from treatment with the wild type strain was significantly higher than in cells treated with the deleted-dupA mutant (p<0.05). The increase of cleaved Caspase-3, cleaved PARP and Bax in MKN-45cells exposed to the wild type strain was significantly higher than that exposed to the deleted-dupA mutant, while the decrease of Bcl-2in the wild type strain was more obvious from6h.
Conclusions
1. H. pylori isolated from patients are highly virulent strains, which associates with the high risk of gastroduodenal diseases in the region at high risk of gastroduodenal diseases. The virulent characteristics of H. pylori involve the more active East Asian-type cagA, intact cagPAI, East Asian-type cagPAI, highly virulent vacA genotypes, and dupA (2499).
2. dupA (2499) is conserved and the homology is high, which is suitable for cloning and expression in vitro. DupA (2499) is classified as stability and has strong antigenicity, which is suitable for inducing antibody.
3. DupA (2499) locates in membrane independently of CagPAI to involved the urease secretion as ATPase, which is not involved the adhesion and CagA delivery to host cells
4. DupA interacted and involved the urease secretion in H. pylori cells, and the characteristic makes dupA-positive strains resistant to high acid conditions. dupA-positive strain is resistant to high acid conditions and induces IL-8secretion, and the characteristics associated with duodenal ulcer development. DupA inhibits tumor cell growth, and leads to mitochondria mediated apoptosis to reduce the risk for gastric cancer.
幽门螺杆菌(H. pylori)是定植于人类胃粘膜上皮细胞表面的一种细菌,与胃十二指肠疾病的发生和发展密切相关。分析胃十二指肠疾病高发区(威海)H. pylori临床分离株毒力因子的分子特征(包括cagPAI、vacA基因和dupA基因),以评价该地区H. pylori I临床分离株的毒力。研究该地区H. pylori菌株的dupA基因功能,以探讨H. pylori dupA基因的致病机制。
方法：
1.选取胃十二指肠疾病高发区(威海)临床就诊的患者,进行胃粘膜组织分离培养H. pylori菌株。采用PCR扩增结合测序的方法,分析H.pylori I临床分离株cagPAI (cagA, cagE, cagl, cagL, cagM, cagT和cagX基因)、cagA基因3’端可变区、vacA基因和dupA基因类型,与NCBI (National Center of Biotechnology Information)中相关序列进行比对。并根据基因序列获得了相应蛋白的氨基酸序列,采用MEGA4.1构建相应系统发育树。
2.研究分离于十二指肠溃疡患者的H. pylori WH-21菌株携带的dupA基因类型,并对其蛋白进行生物学信息学分析,包括该蛋白一级结构、二级结构和三级结构特征及该蛋白细胞定位和功能结构域等。
3.构建pET-32a-dupA原核表达载体,转化于E. coli Rosetta(DE3)中。经IPTG诱导后表达重组DupA蛋白,以Ni2+-NTA珠分离纯化目的蛋白。利用ATPase分解ATP生成无机磷的原理,检测重组DupA蛋白的ATPase活性。采用动物实验制备抗重组DupA蛋白多克隆抗血清。
4.利用Western blotting法检测DupA (2499)蛋白的亚细胞定位。采用免疫共沉淀实验和质谱分析方法,进行DupA (2499)蛋白的互作蛋白分析。
5.构建敲除载体pBluescript/△dupA::KMr,采用同源重组的方法构建H. pylori dupA (2499)基因缺失株。
6. H. pylori和GES-1细胞共培养后,采用Western blotting方法分析dupA (2499)基因的缺失对H. pylori CagA蛋白转运的影响,以及CFU实验和粘附率实验分析H. pylori dupA (2499)基因缺失株的粘附功能。
7.分析不同pH值液体培养基中H. pylori生长情况,研究IH. pylori dupA (2499)基因缺失株对酸性环境的抵抗力。采用Western blotting,分析培养后上清液中脲酶含量。H. pylori和GES-1细胞共培养,采用ELISA方法分析上清液中IL-8的浓度,分析H. pylori dupA (2499)基因缺失株毒力变化。
8. H. pylori和MKN-45细胞共培养后,采用MTT方法检测H. pylori dupA (2499)基因缺失株的细胞毒变化,Hoechst33258染色后显微镜下观察H. pylori dupA (2499)基因缺失株对肿瘤细胞形态变化的影响,Western blotting分析线粒体通路的凋亡相关分子标志变化,包括活化Caspase-3, PARP, Bax和Bcl-2变化。
结果：
1.经过PCR扩增后,116株H. pyloril临床分离株均携带cagA, cagL, cagM, cagT和cagX基因,而只有89.7%(104/116)和82.8%(96/116)菌株分别扩增出cagE和cagI基因。25.9%(30/116)菌株中发现cag部分缺失,其中cagPAI部分缺失株在慢性胃炎中占44.4%(24/54)显著高于胃溃疡、十二指肠溃疡和胃癌(9.7%,6/62)咖<0.001)。未发现cagPAI完全缺失的H. pylori菌株。
24株菌株cagA3'端可变区氨基酸序列可分为两大群：东亚型(91.7%)和西方型(8.3%),其中前者为EPIYA-A-B-D型,后者为EPIYA-A-B-C型。CagPAI蛋白中除了CagL蛋白外,CagI、CagM、CagT和CagX蛋白序列均为保守。系统发育树提示CagPAI蛋白也分为两个群：东亚型和西方型,大部分菌株也属于东亚型。
H. pyloril临床分离株vacA基因s区和m区的主要流行模式为s1a/m2(48.3%)和slc/m2(13.8%)。31.0%的H. pylori临床分离株携带dupA基因,其中在胃溃疡、十二指肠溃疡和胃癌携带率为40.3%,高于慢性胃炎(20.4%,p=0.02)。dupA基因序列分析显示其读码框架为2499bp,称作dupA (2499)基因。
2. H. pylori WH-21菌株携带的dupA基因全长2499bp,称作dupA (2499)基因。威海地区8株dupA碱基序列同源性为99.3%,与日本两株H. pylori菌株的dupA基因碱基序列同源性为99.25%,不同地区14株H. pylori dupA碱基序列同源性为91.65%。生物信息学分析发现DupA (2499)蛋白为稳定亲水性蛋白质,抗原性强。无信号肽且有多个跨膜区,定位于细胞内膜。a螺旋是该蛋白多肽链中主要组成结构元件,具有一个卷曲螺旋结构。三维结构预测其配体结合物质为ADP、ATP和Mg2+等。与CagE_TrbE_VirB超家族有较高的同源性,具有ATP结合位点。
3.获得了重组DupA蛋白,该蛋白ATPase活力为129.5±17.8/mgprot。成功制备了抗重组DupA蛋白多克隆抗血清,抗体效价为1：6.4x104。
4. Western blotting结果显示DupA (2499)蛋白定位于胞膜。经过免疫共沉淀实验和质谱分析,DupA (2499)蛋白的互作蛋白为脲酶和HSP60。
5.获得了H. pylori dupA (2499)基因缺失株,并通过了PCR和Western blotting的验证。
6.采用H. pylori感染GES-1细胞后,缺失株组和野生株组向胞内转运CagA蛋白无差别。CFU实验结果[(27.2±7.1)×106和(28.5±6.9)×106,p=0.772]和粘附率实验结果(81.0%和78.8%,p=0.724)均无明显差异。
7.除pH6.5-pH7.5之外,其它酸性环境下野生株组菌量显著高于缺失株组(p<0.05)。缺失株组的脲酶分泌低于野生株组,特别在酸性环境时。细菌细胞共培养后,野生株组上清液中IL-8浓度显著高于缺失株组(p<0.001)
8.与对照组比较,细菌细胞共培养12h后MKN-45细胞存活率开始降低,缺失株组MKN-45细胞存活率显著高于野生株组(p<0.05)。细菌细胞共培养12h后,Hoechst33258染色结果显示缺失株组和野生株组均可以引起MKN-45细胞凋亡,但野生株组凋亡率显著高于缺失株组(p<0.05)。6h后野生株组和缺失株组均开始出现Caspase-3和PARP的剪切活化,野生株组强于缺失株组。与缺失株组比较,野生株组Bax明显上调而Bcl-2则明显下调。
结论：
1.胃十二指肠疾病高发区(威海)H. pylori I临床分离株为强毒株,与该地区的胃十二指肠疾病高发相关。H. pylori强毒性特征与东亚型cagA基因3’端可变区、完整的cagPAI、东亚型cagFAI基因、强毒性vacA基因和dupA (2499)基因有关。
2.dupA (2499)基因保守且同源性高,适宜进行体外表达。DupA (2499)蛋白稳定且抗原性强,适合抗体的制备。
3. DupA (2499)蛋白独立于CagPAI之外,存在于H. pylori的内膜,具有ATPase活性并参与脲酶的分泌,但不参与H. pylori的CagA蛋白的转运和其粘附过程。
4. H. pylori DupA(2499)蛋白参与脲酶分泌的调节,使携带dupA (2499)基因的H. pylori菌株具有更强的耐酸特性。耐酸特性和刺激IL-8分泌的特性,与携带dupA基因H. pylori菌株引起十二指肠溃疡高发密切相关。DupA (2499)蛋白通过线粒体通路诱导肿瘤细胞凋亡,与携带dupA基因的H. pylori菌株降低胃癌发病相关。
Objectives
Helicobacter pylori (H. pylori) colonize the surface area of the gastric mucosa in the human stomach, and are associated with an increased risk for various gastroduodenal diseases. In order to estimate the virulence of H. pylori isolated from the patients in a region at high risk of gastroduodenal diseases, we investigated the prevalence of H. pylori virulence markers, including cagPAI, vac A and dupA. The function of dupA of H. pylori was analyzed to further reveal the pathogenesis of H. pylori in the study.
Methods
1. H. pylori strains were isolated from gastric biopsies of patients in a region at high risk of gastroduodenal diseases. cagPAI genes (cagA, cagE, cagl, cagL, cagM, cagT and cagX), the cagA3'variable region, vac A, and dupA genotypes were determined by PCR. Some amplicons of the cagA3'variable region, cagPAI genes (cagl, cagL, cagM, cagT and cagX) and dupA were sequenced to compare with deposited sequences in NCBI (National Center of Biotechnology Information). The deduced amino acid sequence of proteins was obtained from the nucleotide sequence. The full-length amino acid sequences were aligned, and the phylo genetic trees were constructed by the program MEGA4.1.
2. dupA genotype of H. pylori WH-21strain was analyzed, which was isolated from the patient with duodenal ulcer. The biological functions of the deduced DupA were analyzed using the structural bioinformatics analysis methods, including the primary structure, secondary structure, tertiary structure, the prediction of protein subcellular localization and the functional domains.
3. After recombinant expression vector pET32a (+)-dupA was constructed and transformed into E. coli Rosetta (DE3), recombinant DupA fusion protein was induced by IPTG and affinity purified using Ni-nitrilotriacetic acid resin. ATPase activity of recombinant DupA fusion protein was tested using the assay based on the inorganic phosphate released by ATP hydrolysis. The polyclonal antibody to recombinant DupA fusion protein was produced by animal experiment.
4. The subcellular localization of DupA (2499) was analyzed by Western blotting. The interaction proteins of DupA (2499) were investigated using Immunoprecipitation assay and MS.
5. After the pBluescript/△dupA::KMr was constructed and introduced into H. pylori WH21strain by electroporation, an isogenic mutant of H. pylori dupA was generated by allelic exchange mutagenesis.
6. The CagA translocation activity of the dupA-deleted mutant was analyzed after GES-1cells infected with H. pylori by Western blotting. The adhesion activity of the dupA-deleted mutant was quantified by colony-forming units (CFU) assay and adhesion rate assay.
7. Susceptibility of dupA-deleted mutant to low pH was measured by the bacterial growth in the different pH liquid media. The supernatants of the different pH liquid media were collected for analyzing urease levels using Western blotting. IL-8levels in the supernatants were determined after GES-1cells co-cultured with H. pylori strains to estimate the virulence of the dupA-deleted mutant.
8. After MKN-45cells co-cultured with H. pylori strains, the death of MKN-45cells induced by H. pylori was studied in cytotoxicity assay by MTT assay, and Hoechst33258staining was performed for morphology analysis. Protein-involved mitochondria mediated apoptosis were analyzed by Western blotting, including Caspase-3, PARP, Bax and Bcl-2.
Results
1. After PCR amplification of the cagPAI genes, the results showed that100%(116/116) of the isolates were positive for cagA, cagL, cagM, cagT and cagX, whereas89.7%(104/116) and82.8%(96/116)strains had cagE and cagl, respectively. Isolates with partial cagPAI were found in25.9%(30/116) strains, which present at a higher frequency in chronic gastritis (44.4%,24/54) than that of gastric ulcer, duodenal ulcer and gastric cancer (9.7%,6/62)(p<0.001). None of the isolates completely lacked the cagPAI.
cagA3'variable regions of24strains fell into two types:East Asian-type (91.7%) classified as EPIYA-A-B-D and Western-type (28.3%) classified as EPIYA-A-B-C. The proteins encoded by cagPAI genes (CagI、CagM、CagT、CagX) were conserved with high homology except CagL.The phylogenetic trees of the full-length CagPAI proteins (CagI, CagL, CagM, CagT and CagX) demonstrated that all CagPAI proteins in the isolates could be placed into two major groups (East Asian-group and Western-group), and most of them were included in the East Asian-group.
It showed that sla/m2(48.3%) and s1c/m2(13.8%) strains were the most prevalent H. pylori strains in this region. Thirty-one percent of strains (31.0%) possessed dupA, and the prevalence of dupA was significantly higher in strains from patients with gastric ulcer, duodenal ulcer and gastric cancer (40.3%) than that from chronic gastritis (20.4%, p=0.02). Sequencing of dupA revealed an ORF of2,449-bp, which was called as dupA (2499).
2. Sequencing of dupA in H. pylori WH-21revealed an ORF of2,449-bp, which was dupA (2499). There was homology among dupA sequences of the nine strains from Weihai, approximately99.3%. The homology among strains from Weihai and two strains from Japan was99.25%, while it was91.65%among fourteen strians isolated from different regions. Using the bioinformatics software, DupA (2499) was classified as stable one with high hydrophilicity and antigenicity. There was transmembrane helices but no signal peptide in DupA (2499), which located in membrane. The major structural element of polypeptide chain was α-helix, and one coiled coil was predicted in DupA (2499). It contained combined sites of ADP、ATP and Mg2+in the tertiary structure. The protein was predicted to have high homology with the CagE_TrbE_VirB superfamily with ATP binding site.
3. The expressed recombination DupA fusion protein was obtained, and the protein exhibited a clear ATPase activity (129.5±17.8U/mgproft. The polyclonal antibody to recombinant DupA fusion protein was produced in the study, and the antibody titer was1:6.4×104.
4. DupA (2499) was located in bacterial membrane using Western blotting, which interacted urease and HSP60by IP and MS.
5. The isogenic dupA mutant of H. pylori was obtained and identified by PCR using one pair of primers (P1and P4) and Western blotting using anti-DupA antibody.
6. After used H. pylori to infect GES-1cells, the quantities of CagA protein translocated into the cells were similar between the wild type strain and the dupA-deleted mutant. No Significantly decreased numbers of the dupA-deleted mutant bound to GES-1cells were compared with the wild type strain both in CFU assay [(27.2±7.1)×106and (28.5±6.9)×106, p=0.772] and adhesion rate assay (81.0%and78.8%,p=0.724).
7. The result revealed that the wild type strain had a stronger growth than the dupA-deleted mutant in low pH (p<0.05). Reduced urease secretion levels were found in dup A-deleted mutant especially in low pH comparing to the wild type strain.
After the two strains infected GES-1cells, IL-8levels were significantly higher in supernatants co-cultured with the wild type strain than in those with its dupA-deleted mutant (p<0.001)
8. The amounts of vital cells were decreased which were infected with wild type strain, compared to those for the mutant-infected cells after12h (p<0.05). After12h, apoptosis was observed in MKN-45cells treated both with the wild type strain and the deleted-dupA mutant. The number of apoptotic cells resulting from treatment with the wild type strain was significantly higher than in cells treated with the deleted-dupA mutant (p<0.05). The increase of cleaved Caspase-3, cleaved PARP and Bax in MKN-45cells exposed to the wild type strain was significantly higher than that exposed to the deleted-dupA mutant, while the decrease of Bcl-2in the wild type strain was more obvious from6h.
Conclusions
1. H. pylori isolated from patients are highly virulent strains, which associates with the high risk of gastroduodenal diseases in the region at high risk of gastroduodenal diseases. The virulent characteristics of H. pylori involve the more active East Asian-type cagA, intact cagPAI, East Asian-type cagPAI, highly virulent vacA genotypes, and dupA (2499).
2. dupA (2499) is conserved and the homology is high, which is suitable for cloning and expression in vitro. DupA (2499) is classified as stability and has strong antigenicity, which is suitable for inducing antibody.
3. DupA (2499) locates in membrane independently of CagPAI to involved the urease secretion as ATPase, which is not involved the adhesion and CagA delivery to host cells
4. DupA interacted and involved the urease secretion in H. pylori cells, and the characteristic makes dupA-positive strains resistant to high acid conditions. dupA-positive strain is resistant to high acid conditions and induces IL-8secretion, and the characteristics associated with duodenal ulcer development. DupA inhibits tumor cell growth, and leads to mitochondria mediated apoptosis to reduce the risk for gastric cancer.
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