肠出血性大肠杆菌O157：H7 z3672基因的敲除与毒力评价
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摘要
病原微生物的流行对人类健康构成了重大威胁,研究病原微生物与宿主相互作用的分子基础,对预防与治疗感染性疾病、改善人类健康具有重要意义,它也一直是医学界面临的重要课题之一。生物信息学作为一门新兴学科已广泛地渗透到生命科学的各个研究领域,发挥着巨大作用。随着越来越多的全基因组序列的测定完成,对基因组序列进行比较分析,探索序列结构与病原微生物致病能力间关系的比较基因组学也应运而生,为病原微生物学的研究注入了新的活力。在前期研究中,我们应用生物信息学技术预测发现了一个新的蛋白家族(FlxA-like proteins),分析表明该蛋白家族可能参与细菌与宿主相互作用,并且可能是具有III型分泌系统的致病菌的重要效应蛋白(毒力蛋白)。
肠出血性大肠杆菌(Enterohemorrhagic E. coli,EHEC)O157:H7是常见的肠道致病菌,感染该菌可引起腹泻、出血性结肠炎,溶血性尿毒综合征及血栓形成性血小板减少性紫癜等严重并发症,严重者可导致死亡,致死率达5%～10%。1982年,Riley等报道了由O157:H7引起的出血性结肠炎的暴发流行,这也是把O157:H7确认为严重致病菌的首次报道。随后在加拿大、日本、英国、澳大利亚等地发生了多起该菌的感染流行。1999年,在我国安徽、江苏两省曾暴发流行O157感染性腹泻,患者超过2万人,死亡177人,流行时间7个月,可能是迄今为止世界上流行规模最大的一次。我国已将EHEC列为21世纪可能对国人卫生健康有重大影响的12种病原微生物之一。由于O157:H7的感染呈现暴发流行趋势,感染后对人体有强烈的致病性与致死性,而且抗生素治疗可能会加剧病情,因此,它已经成为全球性的公共卫生问题。O157培养容易、传播途径多样,感染性很强,<100 CFU(菌落形成单位)即可致病,且多数病症较为严重。它也被美国疾病控制中心(CDC)列为B类生物恐怖病原体严加防范。通过近三十多年的研究,我们对O157:H7的致病机制已经有了初步认识,但尚未完全阐明,了解感染的致病机理对预防和治疗由该菌引起的流行性疾病具有重要的理论指导意义。
我们应用生物信息学技术预测发现的新蛋白家族(FlxA-like proteins)成员中存在Z3672蛋白,它是一个来源于O157:H7的假想蛋白,根据预测信息我们推测它很可能是具有Ⅲ型分泌系统的致病菌重要的效应蛋白(毒力蛋白)。因此,通过对O157:H7 Z3672蛋白的研究,有望发现一个新的Ⅲ型分泌系统效应分子,从而推进O157:H7致病的分子基础研究工作,为进一步了解FlxA-like蛋白家族在细菌与宿主相互作用过程中所发挥的功能奠定基础。
本研究通过Red重组系统成功构建了O157:H7 z3672缺失突变株。首先经过PCR、酶切、连接等步骤,构建了两端同源序列分别约500bp的z3672基因长同源臂打靶片段,该片段连接在pET-24a载体上。然后通过pKOBEG质粒介导的Red重组,使电击转化入菌体内部的同源臂打靶片段与O157:H7基因组序列进行了同源重组,经过抗性筛选、PCR鉴定以及测序分析,成功获得了O157:H7 z3672缺失突变体。通过Real-Time PCR实验,针对z3672基因在野生株是否转录进行了检测,比较O157:H7野生株与O157:H7 z3672缺失突变株中z3672基因的转录差异,并且证明了在O157:H7野生株中z3672基因存在转录活性。
在获得了O157:H7 z3672缺失突变株后,主要对z3672基因的功能进行了初步研究。一方面,利用双向电泳以及蛋白质谱鉴定的方法,对O157:H7野生株和O157:H7 z3672缺失突变株进行了蛋白质组学分析。研究表明,在O157:H7 z3672缺失突变株中,外膜蛋白A(OmpA)的表达量升高。通过Real-Time PCR实验从基因的转录水平对以上实验结果进行了验证。结果证明,Real-Time PCR与双向电泳结果一致,因此,在O157:H7中,z3672基因与ompA(外膜蛋白A)基因存在某种联系,敲除z3672基因可以提高外膜蛋白A的表达水平。另一方面,通过体外细胞实验,比较O157:H7野生株与O157:H7 z3672缺失突变株对真核细胞(HeLa)造成的A/E损伤是否存在差异。利用细胞骨架蛋白的免疫荧光实验,观察O157:H7野生株与O157:H7 z3672缺失突变株分别粘附HeLa细胞后,细胞骨架蛋白Actin,Keratin,Tubulin改变差异。实验表明,敲除z3672基因前后, O157:H7对细胞骨架蛋白Actin,Keratin,Tubulin改变差异并不明显。这也说明,Z3672蛋白不是O157:H7中影响真核细胞骨架改变的核心因子。
在细胞骨架肌动蛋白荧光染色实验中发现:利用含10% FBS的DMEM培养的O157:H7感染HeLa细胞后可以形成明显的肌动蛋白聚集现象。于是对这一现象进行了初步探索。利用不同的培养基条件(LB,DMEM,DMEM含10% FBS,DMEM含终浓度为25m mol/L HEPES)培养O157:H7并对其生长状态,粘附HeLa细胞,HeLa细胞骨架肌动蛋白聚集状况以及蛋白表达情况进行了初步分析。结果表明,虽然在DMEM含10% FBS中生长最慢,但是O157:H7粘附性以及对肌动蛋白聚集的能力是最强的。由于O157:H7对真核细胞的A/E损伤主要表现在两个方面,一是其对真核细胞的粘附作用;二是其对真核细胞的细胞骨架肌动蛋白聚集能力。因此,以上的实验结果也说明了利用含10% FBS的DMEM培养基培养的O157:H7对真核细胞的A/E损伤能力增强,进而其致病能力也增强了。
本研究构建了O157:H7 z3672缺失突变株,证明了z3672基因的缺失可以上调外膜蛋白A的表达,并且证明该基因不是影响真核细胞骨架改变的核心因子,这些为进一步研究Z3672蛋白及FlxA-like蛋白家族在细菌与宿主相互作用过程中所发挥的功能奠定了基础。同时还发现,利用DMEM(10% FBS)培养的O157:H7与宿主细胞体外作用,可以使O157:H7对宿主细胞A/E损伤的能力增强,但具体的作用机制仍需要进一步研究,这也为更全面地了解O157:H7的致病机制提供了新思路。
The prevalence of pathogenic microorganisms is a major threat to human health. Elucidating the molecular and cellular mechanisms underlying bacteria-host interactions and understanding the origination and evolution of these mechanisms are crucial for understanding the relationship between humans and microbes, as well as for preventing and treating infectious diseases and improving human health. As a new subject, bioinformatics which is widely used in all fields of life science researches, is playing a significant role. Comparative genomics have emerged to study the sequences of pathogenic microorganisms that are related with pathogenesis with more complete genome sequences are known, so that the study of pathogenic microorganisms are more effectively. In the previous study, we discovered a new protein family (FlxA-like proteins) in bioinformatics methods. The proteins of this family are suspected to involve in the interactions between pathogens and their hosts, and may be important effectors (virulence proteins) of pathogenic bacteria with type III secretion system.
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is highly adapted enteropathogens that successfully colonize their host’s gastrointestinal tract via the formation of attachingand effacing (A/E) lesions. It causes serious illness known as diarrhea, hemorrhagic colitis, hemolytic uremic syndrome and thrombotic thrombo- cytopenic purpura, and severe cases can lead to death, with the death rate of 5% to 10%. In 1982, Riley reported an outbreak of hemorrhagic colitis caused by the O157: H7. This was the first time that O157: H7 is recognized as a serious pathogen. And prevalence in Canada, Japan, Britain, Australia are reported from then. In 1999, O157 broke out in Jiangsu and Anhui Province and lasted for 7 months. More than 20,000 people were infected, and 177 patients died. Therefore, EHEC is known as one of the 12 pathogenic bacteria which may cause serious illness in China. O157: H7 which can cause significant morbidity and mortality worldwide, has become a global problem of public health. O157 which cultured easily, spread via many different ways, and cause many serious disease. Less than 100 CFU (colony forming units) can cause infections. Thus, O157 is classified as B class bioterrorism pathogens by Centers for Disease Control (CDC). In the last 30 years, much of the mechanism of O157: H7 are characterized but is still not clearly understood. It is important to improve our ability of preventing and treating the outbreak of disease caused by O157: H7.
In the previous study, we discovered a new protein family (FlxA-like proteins) in bioinformatics methods. The O157: H7 Z3672 protein is a member of this family. Being a hypothetical protein, Z3672 is suspected to involve in the interactions between pathogens and their hosts and secret by type III secretion system. Therefore, O157: H7 is expected to be a new effector of typeⅢsecretion system. It may be helpful to clarify the molecular mechanisms underlying the host-microbe interactions with understanding of the function of EHEC O157: H7 Z3672 protein. Also, it will be useful for our further understanding of FlxA-like protein family in host-microbe interaction.
To examine the function of Z3672, we constructed z3672 deletion mutant strain O157:H7Δz3672. Mutation in z3672 gene of O157:H7 was constructed by the PCR one-stepλred recombinant system. First of all, we amplified z3672 gene homologous sequences from O157:H7 genome, and then inserted them into pET-24a. We get a long-arm homologous sequence of z3672, which had 500bp homologous sequence in both ends. The homologous sequences were electroporated into O157:H7 recipient strains carrying the Red system expression plasmid pKOBEG, and mutants were selected on LB plates with kanamycin. The strain of z3672 deletion mutant was verified by PCR and sequencing. At last, the z3672 deletion mutant strain O157:H7Δz3672 was constructed. The Real-Time PCR assay was applied to determine whether z3672 is transcribed in O157: H7 wide type. We are sure of that z3672 is transcribed in O157: H7 wide type.
On one hand, to characterize the Z3672 function, a z3672 mutant and the wild-type O157:H7 strains were assessed for two dimensional (2D) electrophoresis and LC/MS/MS to characterize the Z3672 function. In O157: H7 z3672 mutant, the expression of a outer membrane protein (OmpA) is increased. Then, Real-Time PCR assay was applied to determine the transcription level of OmpA in both z3672 mutant and the wild-type O157:H7 strains. It also demonstrated that the transcription of OmpA is increased in z3672 mutant. So, z3672 gene may affect in the expression of OmpA (outer membrane protein A). On the other hand, a z3672 mutant and the wild-type O157:H7 strains were assessed for A/E lesion formation in vitro. Infecting HeLa cells with the mutant and the wild-type strains and employing the fluorescence stain test revealed actin-rich pedestals、Keratin、Tublin under adherent O157: H7Δz3672 were indistinguishable from those formed by the wild-type strains, indicating thatΔz3672 is not required for this activity in vitro.
In FAS(fluorescence actin staining) assay, we found an interesting phenomenon: the ability of induce actin polymerization may enhance if O157:H7 is cultured in DMEM (10% FBS) rather than in LB. After cultured in different media conditions (LB, DMEM, DMEM containing 10% FBS, DMEM containing 25m mol/L HEPES), O157: H7 were test in the following aspects: the growth rates, the ability of adhesion to HeLa cells, actin accumulation and the expression of proteins. O157: H7 grew slowest when cultured in DMEM containing 10% FBS, but the adhesion to HeLa cells and actin accumulation is the strongest. Attaching and effacing (A/E) lesions are characterized by the localized destruction (effacement) of intestinal epithelial microvilli, an intimate attachment between the bacterium and the host cell apical membrane, and the formation of pedestal-like structures containing high concentrations of actin and intermediate filaments directly beneath sites of bacterial attachment. Thus, it may enhance the ability of A/E lesion for O157: H7 when cultured in DMEM (10% FBS).
In this study, we constructed O157: H7Δz3672 strain, and illuminated z3672 deletion mutant strain can increase the expression level of outer membrane protein A. It is a basal study for further research of Z3672 protein and FlxA-like protein family. O157: H7 may do more damage to infected HeLa cell in vitro if cultured in DMEM (10% FBS). O157: H7 may respond properly to surrounding environment (containing FBS) to corordinate virulence gene expression, but further study is still needed to understand the pathogenic mechanism.
肠出血性大肠杆菌(Enterohemorrhagic E. coli,EHEC)O157:H7是常见的肠道致病菌,感染该菌可引起腹泻、出血性结肠炎,溶血性尿毒综合征及血栓形成性血小板减少性紫癜等严重并发症,严重者可导致死亡,致死率达5%～10%。1982年,Riley等报道了由O157:H7引起的出血性结肠炎的暴发流行,这也是把O157:H7确认为严重致病菌的首次报道。随后在加拿大、日本、英国、澳大利亚等地发生了多起该菌的感染流行。1999年,在我国安徽、江苏两省曾暴发流行O157感染性腹泻,患者超过2万人,死亡177人,流行时间7个月,可能是迄今为止世界上流行规模最大的一次。我国已将EHEC列为21世纪可能对国人卫生健康有重大影响的12种病原微生物之一。由于O157:H7的感染呈现暴发流行趋势,感染后对人体有强烈的致病性与致死性,而且抗生素治疗可能会加剧病情,因此,它已经成为全球性的公共卫生问题。O157培养容易、传播途径多样,感染性很强,<100 CFU(菌落形成单位)即可致病,且多数病症较为严重。它也被美国疾病控制中心(CDC)列为B类生物恐怖病原体严加防范。通过近三十多年的研究,我们对O157:H7的致病机制已经有了初步认识,但尚未完全阐明,了解感染的致病机理对预防和治疗由该菌引起的流行性疾病具有重要的理论指导意义。
我们应用生物信息学技术预测发现的新蛋白家族(FlxA-like proteins)成员中存在Z3672蛋白,它是一个来源于O157:H7的假想蛋白,根据预测信息我们推测它很可能是具有Ⅲ型分泌系统的致病菌重要的效应蛋白(毒力蛋白)。因此,通过对O157:H7 Z3672蛋白的研究,有望发现一个新的Ⅲ型分泌系统效应分子,从而推进O157:H7致病的分子基础研究工作,为进一步了解FlxA-like蛋白家族在细菌与宿主相互作用过程中所发挥的功能奠定基础。
本研究通过Red重组系统成功构建了O157:H7 z3672缺失突变株。首先经过PCR、酶切、连接等步骤,构建了两端同源序列分别约500bp的z3672基因长同源臂打靶片段,该片段连接在pET-24a载体上。然后通过pKOBEG质粒介导的Red重组,使电击转化入菌体内部的同源臂打靶片段与O157:H7基因组序列进行了同源重组,经过抗性筛选、PCR鉴定以及测序分析,成功获得了O157:H7 z3672缺失突变体。通过Real-Time PCR实验,针对z3672基因在野生株是否转录进行了检测,比较O157:H7野生株与O157:H7 z3672缺失突变株中z3672基因的转录差异,并且证明了在O157:H7野生株中z3672基因存在转录活性。
在获得了O157:H7 z3672缺失突变株后,主要对z3672基因的功能进行了初步研究。一方面,利用双向电泳以及蛋白质谱鉴定的方法,对O157:H7野生株和O157:H7 z3672缺失突变株进行了蛋白质组学分析。研究表明,在O157:H7 z3672缺失突变株中,外膜蛋白A(OmpA)的表达量升高。通过Real-Time PCR实验从基因的转录水平对以上实验结果进行了验证。结果证明,Real-Time PCR与双向电泳结果一致,因此,在O157:H7中,z3672基因与ompA(外膜蛋白A)基因存在某种联系,敲除z3672基因可以提高外膜蛋白A的表达水平。另一方面,通过体外细胞实验,比较O157:H7野生株与O157:H7 z3672缺失突变株对真核细胞(HeLa)造成的A/E损伤是否存在差异。利用细胞骨架蛋白的免疫荧光实验,观察O157:H7野生株与O157:H7 z3672缺失突变株分别粘附HeLa细胞后,细胞骨架蛋白Actin,Keratin,Tubulin改变差异。实验表明,敲除z3672基因前后, O157:H7对细胞骨架蛋白Actin,Keratin,Tubulin改变差异并不明显。这也说明,Z3672蛋白不是O157:H7中影响真核细胞骨架改变的核心因子。
在细胞骨架肌动蛋白荧光染色实验中发现:利用含10% FBS的DMEM培养的O157:H7感染HeLa细胞后可以形成明显的肌动蛋白聚集现象。于是对这一现象进行了初步探索。利用不同的培养基条件(LB,DMEM,DMEM含10% FBS,DMEM含终浓度为25m mol/L HEPES)培养O157:H7并对其生长状态,粘附HeLa细胞,HeLa细胞骨架肌动蛋白聚集状况以及蛋白表达情况进行了初步分析。结果表明,虽然在DMEM含10% FBS中生长最慢,但是O157:H7粘附性以及对肌动蛋白聚集的能力是最强的。由于O157:H7对真核细胞的A/E损伤主要表现在两个方面,一是其对真核细胞的粘附作用;二是其对真核细胞的细胞骨架肌动蛋白聚集能力。因此,以上的实验结果也说明了利用含10% FBS的DMEM培养基培养的O157:H7对真核细胞的A/E损伤能力增强,进而其致病能力也增强了。
本研究构建了O157:H7 z3672缺失突变株,证明了z3672基因的缺失可以上调外膜蛋白A的表达,并且证明该基因不是影响真核细胞骨架改变的核心因子,这些为进一步研究Z3672蛋白及FlxA-like蛋白家族在细菌与宿主相互作用过程中所发挥的功能奠定了基础。同时还发现,利用DMEM(10% FBS)培养的O157:H7与宿主细胞体外作用,可以使O157:H7对宿主细胞A/E损伤的能力增强,但具体的作用机制仍需要进一步研究,这也为更全面地了解O157:H7的致病机制提供了新思路。
The prevalence of pathogenic microorganisms is a major threat to human health. Elucidating the molecular and cellular mechanisms underlying bacteria-host interactions and understanding the origination and evolution of these mechanisms are crucial for understanding the relationship between humans and microbes, as well as for preventing and treating infectious diseases and improving human health. As a new subject, bioinformatics which is widely used in all fields of life science researches, is playing a significant role. Comparative genomics have emerged to study the sequences of pathogenic microorganisms that are related with pathogenesis with more complete genome sequences are known, so that the study of pathogenic microorganisms are more effectively. In the previous study, we discovered a new protein family (FlxA-like proteins) in bioinformatics methods. The proteins of this family are suspected to involve in the interactions between pathogens and their hosts, and may be important effectors (virulence proteins) of pathogenic bacteria with type III secretion system.
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is highly adapted enteropathogens that successfully colonize their host’s gastrointestinal tract via the formation of attachingand effacing (A/E) lesions. It causes serious illness known as diarrhea, hemorrhagic colitis, hemolytic uremic syndrome and thrombotic thrombo- cytopenic purpura, and severe cases can lead to death, with the death rate of 5% to 10%. In 1982, Riley reported an outbreak of hemorrhagic colitis caused by the O157: H7. This was the first time that O157: H7 is recognized as a serious pathogen. And prevalence in Canada, Japan, Britain, Australia are reported from then. In 1999, O157 broke out in Jiangsu and Anhui Province and lasted for 7 months. More than 20,000 people were infected, and 177 patients died. Therefore, EHEC is known as one of the 12 pathogenic bacteria which may cause serious illness in China. O157: H7 which can cause significant morbidity and mortality worldwide, has become a global problem of public health. O157 which cultured easily, spread via many different ways, and cause many serious disease. Less than 100 CFU (colony forming units) can cause infections. Thus, O157 is classified as B class bioterrorism pathogens by Centers for Disease Control (CDC). In the last 30 years, much of the mechanism of O157: H7 are characterized but is still not clearly understood. It is important to improve our ability of preventing and treating the outbreak of disease caused by O157: H7.
In the previous study, we discovered a new protein family (FlxA-like proteins) in bioinformatics methods. The O157: H7 Z3672 protein is a member of this family. Being a hypothetical protein, Z3672 is suspected to involve in the interactions between pathogens and their hosts and secret by type III secretion system. Therefore, O157: H7 is expected to be a new effector of typeⅢsecretion system. It may be helpful to clarify the molecular mechanisms underlying the host-microbe interactions with understanding of the function of EHEC O157: H7 Z3672 protein. Also, it will be useful for our further understanding of FlxA-like protein family in host-microbe interaction.
To examine the function of Z3672, we constructed z3672 deletion mutant strain O157:H7Δz3672. Mutation in z3672 gene of O157:H7 was constructed by the PCR one-stepλred recombinant system. First of all, we amplified z3672 gene homologous sequences from O157:H7 genome, and then inserted them into pET-24a. We get a long-arm homologous sequence of z3672, which had 500bp homologous sequence in both ends. The homologous sequences were electroporated into O157:H7 recipient strains carrying the Red system expression plasmid pKOBEG, and mutants were selected on LB plates with kanamycin. The strain of z3672 deletion mutant was verified by PCR and sequencing. At last, the z3672 deletion mutant strain O157:H7Δz3672 was constructed. The Real-Time PCR assay was applied to determine whether z3672 is transcribed in O157: H7 wide type. We are sure of that z3672 is transcribed in O157: H7 wide type.
On one hand, to characterize the Z3672 function, a z3672 mutant and the wild-type O157:H7 strains were assessed for two dimensional (2D) electrophoresis and LC/MS/MS to characterize the Z3672 function. In O157: H7 z3672 mutant, the expression of a outer membrane protein (OmpA) is increased. Then, Real-Time PCR assay was applied to determine the transcription level of OmpA in both z3672 mutant and the wild-type O157:H7 strains. It also demonstrated that the transcription of OmpA is increased in z3672 mutant. So, z3672 gene may affect in the expression of OmpA (outer membrane protein A). On the other hand, a z3672 mutant and the wild-type O157:H7 strains were assessed for A/E lesion formation in vitro. Infecting HeLa cells with the mutant and the wild-type strains and employing the fluorescence stain test revealed actin-rich pedestals、Keratin、Tublin under adherent O157: H7Δz3672 were indistinguishable from those formed by the wild-type strains, indicating thatΔz3672 is not required for this activity in vitro.
In FAS(fluorescence actin staining) assay, we found an interesting phenomenon: the ability of induce actin polymerization may enhance if O157:H7 is cultured in DMEM (10% FBS) rather than in LB. After cultured in different media conditions (LB, DMEM, DMEM containing 10% FBS, DMEM containing 25m mol/L HEPES), O157: H7 were test in the following aspects: the growth rates, the ability of adhesion to HeLa cells, actin accumulation and the expression of proteins. O157: H7 grew slowest when cultured in DMEM containing 10% FBS, but the adhesion to HeLa cells and actin accumulation is the strongest. Attaching and effacing (A/E) lesions are characterized by the localized destruction (effacement) of intestinal epithelial microvilli, an intimate attachment between the bacterium and the host cell apical membrane, and the formation of pedestal-like structures containing high concentrations of actin and intermediate filaments directly beneath sites of bacterial attachment. Thus, it may enhance the ability of A/E lesion for O157: H7 when cultured in DMEM (10% FBS).
In this study, we constructed O157: H7Δz3672 strain, and illuminated z3672 deletion mutant strain can increase the expression level of outer membrane protein A. It is a basal study for further research of Z3672 protein and FlxA-like protein family. O157: H7 may do more damage to infected HeLa cell in vitro if cultured in DMEM (10% FBS). O157: H7 may respond properly to surrounding environment (containing FBS) to corordinate virulence gene expression, but further study is still needed to understand the pathogenic mechanism.
引文
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[9] Yu DG, Ellis HM, Lee EC, et al. An efficient recombination system for chromos- ome engineering in Escherichia coli [J]. Proc Natl Acad Sci USA, 2000, 97(11): 5978-5983.
[10]张影,朱力,袁静,等.弗氏2a志贺氏菌2457T株yciD基因缺失突变株的构建[J].生物技术通讯,2006,17(4):483-488.
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