幽门螺杆菌ArsRS信号标签突变体的构建及STM文库构建方法的研究
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摘要
目前,全球约50%的人口感染幽门螺杆菌(H.pylori),我国人口感染率高于世界平均水平,为50～70%。世界卫生组织已将幽门螺杆菌列为第一类致癌因子,明确其为胃癌的危险因素。因此,抗幽门螺杆菌感染的控制和治疗是我们面临的重要医疗和社会问题。随着H pylori根除治疗中抗生素的广泛应用,H.pylori的耐药问题突出。对于耐药基因的鉴定,长期以来多以药靶基因和药物代谢相关基因的分析为主,如:H.pylori对克拉霉素的耐药性与克拉霉素的作用位点23S rRNA基因(A2142G、A2143G)突变有关;H.pylori对甲硝唑耐药是由于rdxA基因的突变失活,而rdxA基因编码NADPH硝基还原酶,能使甲硝唑还原而生成具有杀菌活性的代谢产物。但这种方法不能全面发现耐药相关基因及其机制。因此新的耐药性分析的方法和药物靶点的发现,是目前急需解决的重要课题。
信号标签突变技术(STM)能够高通量筛选病原微生物的毒力相关基因,自1995年由Holden及其同事建立起来,该技术已用于十多种病原微生物的致病基因的研究,它是通过用病原体全基因组随机插入的突变体在动物体内进行高通量筛选,从而对病原体的毒力基因进行鉴定的一种负向筛选方法。其理论基础是:如果在与病原体致病过程有关的毒力基因中插入转座子,产生的突变体就会由于毒力基因的插入失活而无法引起持续性感染,结果不能在宿主体内存活下来。通过突变体库感染宿主,然后对存活下来的突变体进行负筛选就可鉴定出病原体的毒力相关基因。这种高通量筛选方法不仅是筛选鉴定致病相关基因的有力手段,而且是筛选耐药基因以及各种特定功能相关基因的有效方法。
双组分系统是大部份细菌赋有的精密的信号传导系统,由组氨酸蛋白激酶(HPK)和效应调节蛋白(RR)两个组分组成,其功能是感知环境变化并产生适当的细胞反应,使之更好的适应外界环境变化。研究表明,H pylori ArsRS双组分系统是H pylori能够在胃黏膜内长期定植的最重要原因之一。因此,我们认为ArsRS双组分系统可能与H pylori耐药有一定关系。在ArsRS双组分系统中,酸敏感的组氨酸激酶ArsS(H pylori0165)及其反应元件ArsR(H pylori0166)调控着H pylori的大部分耐酸性基因:ureABEFGHI、α碳酸酐酶(CA)、NiKR、NixA、hypB、hypA、HspA、Fur、aspA;动力性基因:flaABEGH;毒力基因cagA等,而这些基因是H pylori生长繁殖、致病、耐药的关键,对这些基因的调控系统——ArsRS的研究有助于我们对H pylori致病及耐药机制更进一步的认识。
在本实验中,我们用带有标签的重组质粒替代转座子,通过PCR扩增ArsS、ArsR基因片段,扩增产物分别克隆于载体pID700A1中,化学法转化感受态大肠杆菌E.coli DH5α;然后将重组载体经电转化法转化幽门螺杆菌标准菌株,通过重组质粒插入失活,成功构建了ArsS和ArsR突变株。
我们通过在不同PH值的培养基上比较突变株与野生株的的生长情况,发现ArsS、ArsR基因缺失会对幽门螺杆菌生长活性产生影响:当PH值为7.0时,突变株克隆数与野生株克隆数无明显差异,随着PH值的逐渐降低,当PH在6.0、5.0和4.0时,ArsS、ArsR突变株克隆数随着PH值的逐渐降低而明显减少,而野生株克隆数的变化则不明显。我们还惊奇地发现,ArsS、ArsR突变株对药物的敏感性明显高于野生株,说明ArsSR双组分系统可能与Hp耐药具有一定关系。
在ArsS、ArsR突变株构建方法建立的基础上,我们以四种限制性内切酶酶切方法获得了Hp DNA 300-500bp片段,克隆入pID700系列载体,经电转化Hp,以抗生素筛选得到了70个幽门螺杆菌突变菌株,成功建立了Hp STM突变株文库构建方法,为进一步大规模构建H.pylori突变体文库和鉴定H.pylori耐药相关基因奠定了基础。
Among world population, 50% is infected with Helicobacter pylori (H.pylori) at present. The infected population of China is 50 ~ 70%, which is higher than the world average rate. Therefore, the anti-Helicobacter pylori infection control and treatment is an important medical and social issue we are facing with. With the increasing use of H.pylori eradication therapy antibiotics in the world, antibiotics-resistant H.pylori is prominent problem. The emergence of H.pylori drug-resistance is related to the antibiotic-related gene mutation. The molecular mechanism of H.pylori drug-resistance have been widely studied. It was demonstrated that clarithromycin-resistance is associated with two point mutations (A2142G , A2143G) in 23S rRNA of the H.pylori ,Metronidazole-resistance is associated with mutation of rdxA and rdxA gene encoding nitroreductase which can restore the generation of bactericidal activity with the metabolic products. So each drug has its own resistant gene-determining region. The point mutations and insertion loss of expression in these regions may cause abnormal changes in the structure and function of expressed proteins, resulting in the emergence of drug resistance. However,drug-resistance related genes and their mechanisms can not be fully found out by this method. Thus the new method of drug resistance analysis and drug target discovery, is the urgent need to address the important issue.
Signature-tagged mutagenesis study of microbial pathogens is the most widely used methods in recent years. Herein, the gene in question is inactivated by insertional mutation; a transposon is used which inserts itself into the gene sequence. When that gene is transcribed and translated into a protein, the insertion of the transposon affects the protein structure and prevents it from functioning. In STM, mutants are created by random transposon insertion and each transposon contains a different 'tag' sequence that uniquely identifies it. If an insertional mutant bacterium exhibits a phenotype of interest , such as susceptability to an antibiotic it was previously resistant to, then we will sequence its genome and run a search for any of the tags used in the experiment. When a tag is located, the gene that it disrupts is also thus located. At present, the application of this technology has been applied in more than 10 kinds of pathogenic microorganisms for virulence factors discovery. It also has been proved to be an effective way to resistant gene screening and a variety of specific functions in related genes.
Two-component system is in the most of bacteria .Its sophisticated signal transduction system histidine protein kinase (HPK) and the effect of regulatory protein (RR) composed of two components, the fuction is adapt to the change of environment and to generate appropriate cell reaction, Study shows that, H pylori ArsRS two-component system is the most important reasons why H pylori can colonize in gastric mucosal in a long-term . Therefore, we believe that ArsRS two-component system may be associated with H pylori resistance to some extent. In the two-component system,ArsS and ArsR regulate the majority of H pylori acid resistance genes, ureABEFGHI、CA、NiKR、NixA、hypB、hypA、HspA、Fur、aspA;dynamic gene,flaABEGH and virulence gene cagA . These genes are the key of H pylori growth、pathogenicity and drug resistance.Study the regulation System of these genes will help us to further understand the pathogenicity and the mechanism of resistance of H pylori.
In this study we firstly choose the main control system , ArsRS two-component system of H.pylori, as the mutation target. The fragments of ArsR and ArsS were amplified using PCR from total DNA of standard H.pylori strains,and then cloned into vector pID700A1 containing chloramphenicol-resistant gene by transforming into E.coli DH5α. After identification by restriction enzyme digestion and DNA sequencing,the correct recombinant vectors pID700A1-ArsS and pID700A1-ArsR were electrotransfected into standard H.pylori strain. Finally, the chloramphenicol -resistant clones were collected, and gene mutations were detected using PCR. We found that the growth rates of ArsS and ArsR deleted mutants were significant lower than that of wild type. To our surprise, the mutants also showed sensitive to clarithromycin and metronidazole. The results verified that the growth activity of H.pylori was close with ArsRS, and for the first time indicated that ArsRS was associated with drug sensitivity.
Using this vector-based but not transposon-based STM method, we digested total DNA of Helicobacter pylori with four restriction enzyme, and recovered the 300-500bp DNA fragments after electrophoresis. After cloning these fragments into the vector and transferring into the standard strain of H.pylori, we obtained 70 mutants with chloramphenicol-resistance. This work lays the foundation for further large-scale construction of mutant library and identification of drug-resistance associated genes of H.pylori.
信号标签突变技术(STM)能够高通量筛选病原微生物的毒力相关基因,自1995年由Holden及其同事建立起来,该技术已用于十多种病原微生物的致病基因的研究,它是通过用病原体全基因组随机插入的突变体在动物体内进行高通量筛选,从而对病原体的毒力基因进行鉴定的一种负向筛选方法。其理论基础是:如果在与病原体致病过程有关的毒力基因中插入转座子,产生的突变体就会由于毒力基因的插入失活而无法引起持续性感染,结果不能在宿主体内存活下来。通过突变体库感染宿主,然后对存活下来的突变体进行负筛选就可鉴定出病原体的毒力相关基因。这种高通量筛选方法不仅是筛选鉴定致病相关基因的有力手段,而且是筛选耐药基因以及各种特定功能相关基因的有效方法。
双组分系统是大部份细菌赋有的精密的信号传导系统,由组氨酸蛋白激酶(HPK)和效应调节蛋白(RR)两个组分组成,其功能是感知环境变化并产生适当的细胞反应,使之更好的适应外界环境变化。研究表明,H pylori ArsRS双组分系统是H pylori能够在胃黏膜内长期定植的最重要原因之一。因此,我们认为ArsRS双组分系统可能与H pylori耐药有一定关系。在ArsRS双组分系统中,酸敏感的组氨酸激酶ArsS(H pylori0165)及其反应元件ArsR(H pylori0166)调控着H pylori的大部分耐酸性基因:ureABEFGHI、α碳酸酐酶(CA)、NiKR、NixA、hypB、hypA、HspA、Fur、aspA;动力性基因:flaABEGH;毒力基因cagA等,而这些基因是H pylori生长繁殖、致病、耐药的关键,对这些基因的调控系统——ArsRS的研究有助于我们对H pylori致病及耐药机制更进一步的认识。
在本实验中,我们用带有标签的重组质粒替代转座子,通过PCR扩增ArsS、ArsR基因片段,扩增产物分别克隆于载体pID700A1中,化学法转化感受态大肠杆菌E.coli DH5α;然后将重组载体经电转化法转化幽门螺杆菌标准菌株,通过重组质粒插入失活,成功构建了ArsS和ArsR突变株。
我们通过在不同PH值的培养基上比较突变株与野生株的的生长情况,发现ArsS、ArsR基因缺失会对幽门螺杆菌生长活性产生影响:当PH值为7.0时,突变株克隆数与野生株克隆数无明显差异,随着PH值的逐渐降低,当PH在6.0、5.0和4.0时,ArsS、ArsR突变株克隆数随着PH值的逐渐降低而明显减少,而野生株克隆数的变化则不明显。我们还惊奇地发现,ArsS、ArsR突变株对药物的敏感性明显高于野生株,说明ArsSR双组分系统可能与Hp耐药具有一定关系。
在ArsS、ArsR突变株构建方法建立的基础上,我们以四种限制性内切酶酶切方法获得了Hp DNA 300-500bp片段,克隆入pID700系列载体,经电转化Hp,以抗生素筛选得到了70个幽门螺杆菌突变菌株,成功建立了Hp STM突变株文库构建方法,为进一步大规模构建H.pylori突变体文库和鉴定H.pylori耐药相关基因奠定了基础。
Among world population, 50% is infected with Helicobacter pylori (H.pylori) at present. The infected population of China is 50 ~ 70%, which is higher than the world average rate. Therefore, the anti-Helicobacter pylori infection control and treatment is an important medical and social issue we are facing with. With the increasing use of H.pylori eradication therapy antibiotics in the world, antibiotics-resistant H.pylori is prominent problem. The emergence of H.pylori drug-resistance is related to the antibiotic-related gene mutation. The molecular mechanism of H.pylori drug-resistance have been widely studied. It was demonstrated that clarithromycin-resistance is associated with two point mutations (A2142G , A2143G) in 23S rRNA of the H.pylori ,Metronidazole-resistance is associated with mutation of rdxA and rdxA gene encoding nitroreductase which can restore the generation of bactericidal activity with the metabolic products. So each drug has its own resistant gene-determining region. The point mutations and insertion loss of expression in these regions may cause abnormal changes in the structure and function of expressed proteins, resulting in the emergence of drug resistance. However,drug-resistance related genes and their mechanisms can not be fully found out by this method. Thus the new method of drug resistance analysis and drug target discovery, is the urgent need to address the important issue.
Signature-tagged mutagenesis study of microbial pathogens is the most widely used methods in recent years. Herein, the gene in question is inactivated by insertional mutation; a transposon is used which inserts itself into the gene sequence. When that gene is transcribed and translated into a protein, the insertion of the transposon affects the protein structure and prevents it from functioning. In STM, mutants are created by random transposon insertion and each transposon contains a different 'tag' sequence that uniquely identifies it. If an insertional mutant bacterium exhibits a phenotype of interest , such as susceptability to an antibiotic it was previously resistant to, then we will sequence its genome and run a search for any of the tags used in the experiment. When a tag is located, the gene that it disrupts is also thus located. At present, the application of this technology has been applied in more than 10 kinds of pathogenic microorganisms for virulence factors discovery. It also has been proved to be an effective way to resistant gene screening and a variety of specific functions in related genes.
Two-component system is in the most of bacteria .Its sophisticated signal transduction system histidine protein kinase (HPK) and the effect of regulatory protein (RR) composed of two components, the fuction is adapt to the change of environment and to generate appropriate cell reaction, Study shows that, H pylori ArsRS two-component system is the most important reasons why H pylori can colonize in gastric mucosal in a long-term . Therefore, we believe that ArsRS two-component system may be associated with H pylori resistance to some extent. In the two-component system,ArsS and ArsR regulate the majority of H pylori acid resistance genes, ureABEFGHI、CA、NiKR、NixA、hypB、hypA、HspA、Fur、aspA;dynamic gene,flaABEGH and virulence gene cagA . These genes are the key of H pylori growth、pathogenicity and drug resistance.Study the regulation System of these genes will help us to further understand the pathogenicity and the mechanism of resistance of H pylori.
In this study we firstly choose the main control system , ArsRS two-component system of H.pylori, as the mutation target. The fragments of ArsR and ArsS were amplified using PCR from total DNA of standard H.pylori strains,and then cloned into vector pID700A1 containing chloramphenicol-resistant gene by transforming into E.coli DH5α. After identification by restriction enzyme digestion and DNA sequencing,the correct recombinant vectors pID700A1-ArsS and pID700A1-ArsR were electrotransfected into standard H.pylori strain. Finally, the chloramphenicol -resistant clones were collected, and gene mutations were detected using PCR. We found that the growth rates of ArsS and ArsR deleted mutants were significant lower than that of wild type. To our surprise, the mutants also showed sensitive to clarithromycin and metronidazole. The results verified that the growth activity of H.pylori was close with ArsRS, and for the first time indicated that ArsRS was associated with drug sensitivity.
Using this vector-based but not transposon-based STM method, we digested total DNA of Helicobacter pylori with four restriction enzyme, and recovered the 300-500bp DNA fragments after electrophoresis. After cloning these fragments into the vector and transferring into the standard strain of H.pylori, we obtained 70 mutants with chloramphenicol-resistance. This work lays the foundation for further large-scale construction of mutant library and identification of drug-resistance associated genes of H.pylori.
引文
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39 Mobley HL , Island MD , Hausinger RP. Molecular biologyof microbial urease [ J] . Microbiol Rev , 1995 ,59 (3) :451.
40 Mehta N, Benoita S, Maier RJ. Roles of conserved nucleo tide binding domains in accessory proteins, HypB and UreG,in the maturation of nickelenzymes required for efficient Helicobacter pylori colonization [J]. Microb Pathog , 2003 ,35 :229.
41 Benoit S , Maier RJ . Dependence of Helicobacter pylori Urease Activity on the Nickel Sequestering Ability of the UreE Accessory Protein [J].J Bacterio , 2003 ,185 (16) :4787.
42 Rektorschek M , Buhmann A , Weeks DL , et al . Acid resistance of Helicobacter pylori depends on the UreI membrane protein and an inner membrane proton barrier [ J] . Mol Microbiol , 2000 ,36 (1) :141.
43 Weeks DL , Eskandari S , Scot t DR , et al . A H +2 gated ureachannel : the link between Helicobacter pylori urease and gastric colonization[J]. Science , 2000 ,287 (5452) :482.
44 Clyne M , Labigne A , Drumm B. Helicobacter pylori requires an acidic environ ment to survive in t he presence of urea [J] . Infec Immun, 1995 ,63 (5) :1669.
45 Skouloubris S , Thiberge JM , Labigne A , et al . The Helicobacter pylori UreI protein is not involved in urease activity but is essential for bacterial survival in vivo [ J] . Infec Immun , 1998 ,66 (9) :4517.
46 Fulkerson J F , Garner RM , Mobley HL. Conserved Residues and Motifs inthe NixA Protein of Helicobacter pylori Are Critical for the High Affinity Transport of Nickel Ions [ J] . JBio Chem , 1998 ,273 (1) :235.
47 Ernst FD , Kuipers EJ , Heijens A , et al . The Nickel Responsive Regulator NikR Controls Activation and Repression of Gene Transcription in Helicobacter pylori [ J] . Infec Immu , 2005 ,73 (11) :7252.
48 Mehta N , Olson JW, Maier RJ . Characterization of Helicobacter pylori Nickel Metabolism Accessory Proteins Needed for Maturation of both Urease and Hydrogenase [ J] . J Bacterio , 2003 ,185 (3) :726.
49 Marcus EA , Moshfegh AP , Sachs G, et al . The PeriplasmicαCarbonic Anhydrase Activity of Helicobacter pylori Is Essential for Acid Acclimation [ J] . J B acterio , 2005 ,187 (2) :729.
50 Hensel M,Shea J E, Gleeson C, et al.Simultaneous identification ofbacterial virulence genes by negative selection [J] .Science ,1995 ,269 :400～403.
51 Armelle M ,Claudio B ,Cantet F. Characterization of the genes rdxA andfrxA involved in metronidazole resistance in Helicobacter pylori. Research in Microbiology. 2003 ,154 (2) :1372144.
52 Michael Pflock,Nadja Finsterer,Biju Joseph,et al.Characterization of the ArsRS Regulon of Helicobacter pylori Involved in Acid Adaptation [J].J Bacteriol 2006,188(10): 3449–3462.
53 Dufour M,Manson JM,et a1.Characterization of monolaurin resistance in Enterococcus faecalis[J]. AppLEnviron Microbiol.2007,3(17):5507-15.
54中华医学会消化病学分会.对幽门螺杆菌若干问题的共识意见(2003·中国) [J] .中华医学杂志,2004,84 (6) :522-523.
55 Holger Kavermann, Brendan P.Burns,Katrin Angermuller,et a1. Identification and Characterization of Helicobacter pylori Genes Essential for Gastric Colonization[J]. Exp.Med.1997:813-822.
56 Armelle M ,Claudio B ,Cantet F. Characterization of the genes rdxA andfrxA involved in metronidazole resistance in Helicobacter pylori. Re2search in Microbiology. 2003 ,154 (2) :1372144.
57 Dufour M,Manson JM,Bremer PJ,DufourJP,Cook GM,Simmonds RS.Characterization of monolaur in resistance in Enterococcus faecalis.Appl Environ Microbiol,2007;73(17): 5507-15 .
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37 Michael Pflock,Simone Kennard, Nadja Finsterer, et a1. Acid-responsive gene regulation in the human pathogen Helicobacter pylori[J] . J Biotechnol,2006, 126(1): 52–60.
38 Scott DR, Weeks D , Hong C , et al . The role of internal urease in acid resistance of Hel icobacter p y lori [ J] . Gast roenterology , 1998 ,114 (1) :58.
39 Mobley HL , Island MD , Hausinger RP. Molecular biologyof microbial urease [ J] . Microbiol Rev , 1995 ,59 (3) :451.
40 Mehta N, Benoita S, Maier RJ. Roles of conserved nucleo tide binding domains in accessory proteins, HypB and UreG,in the maturation of nickelenzymes required for efficient Helicobacter pylori colonization [J]. Microb Pathog , 2003 ,35 :229.
41 Benoit S , Maier RJ . Dependence of Helicobacter pylori Urease Activity on the Nickel Sequestering Ability of the UreE Accessory Protein [J].J Bacterio , 2003 ,185 (16) :4787.
42 Rektorschek M , Buhmann A , Weeks DL , et al . Acid resistance of Helicobacter pylori depends on the UreI membrane protein and an inner membrane proton barrier [ J] . Mol Microbiol , 2000 ,36 (1) :141.
43 Weeks DL , Eskandari S , Scot t DR , et al . A H +2 gated ureachannel : the link between Helicobacter pylori urease and gastric colonization[J]. Science , 2000 ,287 (5452) :482.
44 Clyne M , Labigne A , Drumm B. Helicobacter pylori requires an acidic environ ment to survive in t he presence of urea [J] . Infec Immun, 1995 ,63 (5) :1669.
45 Skouloubris S , Thiberge JM , Labigne A , et al . The Helicobacter pylori UreI protein is not involved in urease activity but is essential for bacterial survival in vivo [ J] . Infec Immun , 1998 ,66 (9) :4517.
46 Fulkerson J F , Garner RM , Mobley HL. Conserved Residues and Motifs inthe NixA Protein of Helicobacter pylori Are Critical for the High Affinity Transport of Nickel Ions [ J] . JBio Chem , 1998 ,273 (1) :235.
47 Ernst FD , Kuipers EJ , Heijens A , et al . The Nickel Responsive Regulator NikR Controls Activation and Repression of Gene Transcription in Helicobacter pylori [ J] . Infec Immu , 2005 ,73 (11) :7252.
48 Mehta N , Olson JW, Maier RJ . Characterization of Helicobacter pylori Nickel Metabolism Accessory Proteins Needed for Maturation of both Urease and Hydrogenase [ J] . J Bacterio , 2003 ,185 (3) :726.
49 Marcus EA , Moshfegh AP , Sachs G, et al . The PeriplasmicαCarbonic Anhydrase Activity of Helicobacter pylori Is Essential for Acid Acclimation [ J] . J B acterio , 2005 ,187 (2) :729.
50 Hensel M,Shea J E, Gleeson C, et al.Simultaneous identification ofbacterial virulence genes by negative selection [J] .Science ,1995 ,269 :400～403.
51 Armelle M ,Claudio B ,Cantet F. Characterization of the genes rdxA andfrxA involved in metronidazole resistance in Helicobacter pylori. Research in Microbiology. 2003 ,154 (2) :1372144.
52 Michael Pflock,Nadja Finsterer,Biju Joseph,et al.Characterization of the ArsRS Regulon of Helicobacter pylori Involved in Acid Adaptation [J].J Bacteriol 2006,188(10): 3449–3462.
53 Dufour M,Manson JM,et a1.Characterization of monolaurin resistance in Enterococcus faecalis[J]. AppLEnviron Microbiol.2007,3(17):5507-15.
54中华医学会消化病学分会.对幽门螺杆菌若干问题的共识意见(2003·中国) [J] .中华医学杂志,2004,84 (6) :522-523.
55 Holger Kavermann, Brendan P.Burns,Katrin Angermuller,et a1. Identification and Characterization of Helicobacter pylori Genes Essential for Gastric Colonization[J]. Exp.Med.1997:813-822.
56 Armelle M ,Claudio B ,Cantet F. Characterization of the genes rdxA andfrxA involved in metronidazole resistance in Helicobacter pylori. Re2search in Microbiology. 2003 ,154 (2) :1372144.
57 Dufour M,Manson JM,Bremer PJ,DufourJP,Cook GM,Simmonds RS.Characterization of monolaur in resistance in Enterococcus faecalis.Appl Environ Microbiol,2007;73(17): 5507-15 .