幽门螺杆菌katA基因功能及其在耐受氧化损伤中的作用分析

doi:10.13560/j.cnki.biotech.bull.1985.2023-1114

生物技术通报 ›› 2024, Vol. 40 ›› Issue (5): 310-320.doi: 10.13560/j.cnki.biotech.bull.1985.2023-1114

幽门螺杆菌katA基因功能及其在耐受氧化损伤中的作用分析

王鑫鑫¹^,²(), 管玉祝¹^,², 李晓苇¹^,², 洪伟¹^,³, 吴道艳¹^,², 康颖倩¹^,², 刘永畅¹^,², 陈峥宏¹^,²(), 崔古贞¹^,²()

1.贵州医科大学基础医学院微生物学教研室，贵阳 550000
2.贵州省病原生物学特色重点实验室，贵阳 550000
3.贵州省分子生物学重点实验室，贵阳 550000

收稿日期:2023-11-27 出版日期:2024-05-26 发布日期:2024-03-28
通讯作者: 崔古贞，男，博士，教授，研究方向：医学微生物学；E-mail: cuiguzhen@gmc.edu.cn；
陈峥宏，女，博士，教授，研究方向：医学微生物学；E-mail: chenzhenghong@gmc.edu.cn
作者简介:王鑫鑫，女，硕士研究生，研究方向：医学微生物学；E-mail: wxx_999w@163.com
基金资助:
国家自然科学基金项目(32160015);国家自然科学基金项目(32170134);贵州省高层次留学人才项目(2022-08);贵州省科技计划项目([2020]1Z067);贵州省高层次创新人才团队及人才基地项目(GCC[2022]036-1);贵州省高层次创新人才团队及人才基地项目([RCJD2018-22]);高等学校学科创新引智计划(111 计划);高等学校学科创新引智计划(D20009);贵州省大学生创新训练计划项目(S202310660078);贵州省病原生物学重点实验室(QJJ[2022]019)

Function of katA in Helicobacter pylori and Its Role in the Tolerance to Oxidative Damage

WANG Xin-xin¹^,²(), GUAN Yu-zhu¹^,², LI Xiao-wei¹^,², HONG Wei¹^,³, WU Dao-yan¹^,², KANG Ying-qian¹^,², LIU Yong-chang¹^,², CHEN Zheng-hong¹^,²(), CUI Gu-zhen¹^,²()

1. Department of Microbiology, College of Basic Medicine, Guizhou Medical University, Guiyang 550000
2. Pathogenic Biology Characteristic Laboratory of Guizhou Province, Guiyang 550000
3. Key Laboratory of Molecular Biology of Guizhou Province, Guiyang 550000

Received:2023-11-27 Published:2024-05-26 Online:2024-03-28

摘要/Abstract

摘要：

【目的】过氧化氢酶（KatA）是幽门螺杆菌编码的重要毒力因子，在抵抗宿主免疫杀伤和疫苗研发等方面具有重要功能。为了鉴定幽门螺杆菌 KatA 的酶学特性，并分析其在幽门螺杆菌氧化耐受中的功能。【方法】首先，从幽门螺杆菌临床耐药菌株 Hp_G272 基因组中分离 katA 基因，并在大肠杆菌中克隆表达、分离纯化KatA^G272 蛋白；然后，利用 CAT 检测试剂盒体外分析 KatA^G272 的过氧化氢酶活性；其次，利用同源重组技术构建 katA 基因工程菌株，包括敲除菌株和回补菌株；最后，通过比较野生菌株与工程菌株生长表型差异、分解过氧化氢能力差异及耐受过氧化氢能力差异，阐述 katA 基因的功能及其在幽门螺杆菌耐受氧化损伤中的作用。【结果】在大肠杆菌中成功克隆表达并分离纯化 KatA^G272，体外酶学分析表明，KatA^G272 是一类嗜酸性过氧化氢酶，基因敲除分析表明，敲除该基因幽门螺杆菌丧失分解和耐受过氧化氢的能力，而回补该基因幽门螺杆菌回复分解和耐受过氧化氢的能力。【结论】katA 基因是幽门螺杆菌分解和耐受过氧化氢的唯一功能基因，在幽门螺杆菌氧化耐受中具有重要功能。

关键词: 幽门螺杆菌, 过氧化氢酶, katA, 耐受性, 克隆表达, 基因敲除

Abstract:

【Objective】Catalase（KatA）is an important virulence factor encoded by Helicobacter pylori, and plays an important role in bacterial resistance to host immune killing and vaccine development. The objective of this study is to identify the enzymatic properties of H. pylori catalase and to analyze its function in H. pylori oxidation tolerance.【Method】Firstly, katA gene was isolated from the genome of clinical drug-resistant strain Hp_G272, and KatA^G272protein was cloned and expressed in Escherichia coli. Then, the catalase activity of KatA^G272 was analyzed in vitro with CAT detection kit. Secondly, katA genetic engineering strains were constructed by homologous recombination, including knockout strains and complemented strains. Finally, the function of katA gene and its role in tolerance to oxidative damage of H. pylori were analyzed by comparing the differences in growth phenotype, decomposition and tolerance of hydrogen peroxide between wild and engineered strains.【Result】KatA^G272 was successfully cloned, expressed and purified from E. coli. Enzymatic analysis showed that KatA^G272 was a class of eosinophilic catalase. Gene knockout analysis showed that H. pylori lost its ability to decompose and tolerate hydrogen peroxide, while regained its ability to decompose and tolerate hydrogen peroxide after gene complement.【Conclusion】katA is the only functional gene that decomposes and tolerates hydrogen peroxide in H. pylori, and plays an important role in the oxidative tolerance of H. pylori.

Key words: Helicobacter pylori, catalase, katA, tolerance, clone and expression, gene knockout

王鑫鑫, 管玉祝, 李晓苇, 洪伟, 吴道艳, 康颖倩, 刘永畅, 陈峥宏, 崔古贞. 幽门螺杆菌katA基因功能及其在耐受氧化损伤中的作用分析[J]. 生物技术通报, 2024, 40(5): 310-320.

WANG Xin-xin, GUAN Yu-zhu, LI Xiao-wei, HONG Wei, WU Dao-yan, KANG Ying-qian, LIU Yong-chang, CHEN Zheng-hong, CUI Gu-zhen. Function of katA in Helicobacter pylori and Its Role in the Tolerance to Oxidative Damage[J]. Biotechnology Bulletin, 2024, 40(5): 310-320.

图/表 11

表1 菌株及质粒

Table 1 Strains and plasmids used in this study

菌株及质粒Strain and plasmid	相关特征Relevant characteristics	来源Source
Strains E.coli
DH5α	Clone strain, F^-lacZ ΔM15Δ（lacZYA-aragF）relA1	This lab
DH5α::pSD	Derived fom DH5α, carrying plasmid pSD, kan^R	This lab
DH5α:: pSD-KO-katA	Derived fom DH5α, carrying plasmid pSD-KO-katA, kan^R	This work
DH5α:: pCD	Derived fom DH5α, carrying plasmid pCD, Cm^R	This work
DH5α:: pCD-EX-katA	Derived fom DH5α, carrying plasmid pCD-EX-katA, Cm^R	This work
DH5α:: pET-28a-katA	Derived fom DH5α, carrying plasmid pET-28a-katA, kan^R	This work
BL21（DE3）	Expression strain, F^-lon^-11Δ（ompT-nfrA）885Δ（galM-ybhJ）884	This lab
BL21（DE3）::pET-28a	Derived fom BL21（DE3）, carrying plasmid pET-28a, kan^R	This work
BL21（DE3）::pET-28a-katA	Derived fom BL21（DE3）, carrying plasmid pET-28a-katA, kan^R	This work
H.pylori
HpG272	Clinical strains	This lab
Hp∆katA	H.pylori strain G272, knockout gene katA	This work
Hp∆katA::katA	H.pylori strain HpΔkatA::katA, anaplerosis gene katA	This work
Plasmids
pSD	From pill570, suicide plasmid, kan^R	This lab
PCD	From pSD, suicide plasmid, Cm^R	This lab
pSD-KO-katA	Suicide plasmid, knockout gene katA, kan^R	This work
pCD-EX-katA	HpΔkatA::katA replenishing plasmid, Cm^R	This work
pET-28a	N-T7, N-His, C-His, E. coli protein expression vector	This lab
pET-28a-katA	Carrying katA gene, E. coli protein expression vector	This work

表2 本研究所用引物

Table 2 Primers used in this study

引物Primer	序列Sequence（5'-3'）	注释Notes
Kan-1	GAATTCGAGCTCGGTACCCG	kan^R 基因扩增和检测引物
Kan-2	CCCGGGTCATTATTCCCTCC
Kat-1	CTGCAGGGTATAAAAACTCACCGCCCCA	敲除质粒及敲除菌株检测引物
Kat-2	ATCGATACCTAGTTTCAAGCCTTGCA	敲除质粒及敲除菌株检测引物
KatA-1	ATCGACTGCAGGGTATAAAAACTCACCGCCCCA	katA 基因敲除载体上游同源臂扩增
KatA-2	ATCGAGAATTCCGTAATTGACACTAAGCCGATTAC
KatA-3	ATCGAGGATCCCTTATTTTTTAGGAACGCTTTGTC	katA 基因敲除载体下游同源臂扩增
KatA-4	ATCGAATCGATACCTAGTTTCAAGCCTTGCA
Chl-1	TTAAAAAAATTACGCCCCGCCCTG	Cm^R 基因扩增和检测引物
Chl-2	AAATGGAGAAAAAAATCACTGGATATACCACCG
KatA -5	CCCCGGGGACCTGCAGGCCGCTTGCACGGGT	katA 基因回补载体上游同源臂
KatA -6	TTTTTTAAGGCAGTCTGCAGGCGACCAAAACCTCTCATGGAG
KatA -7	TTGACAGCTTATCATCGATGTGAGTCAGAATGTCTTTCACAACCC	katA 基因回补载体下游同源臂
KatA -8	CTCCTGAAAATCTCGGATCCGGGCGGTTTCACTGAGAAAACTT
KatA -9	CAGTGAAACCGCCCGGATCTTTATAAATTCTAAAGGGG	katA 基因回补载体Hp尿素酶启动子PureA 扩增
KatA -10	TTAGCTCCTGAAAATCTCGGATCCTTATTCTCCTATTCTTAAAGTG
KatA -11	AACACTTTAAGAATAGGAGAATAAGATGGTTAATAAAGATGTGAAACAAACTACTGC	回补载体 katA 基因扩增
KatA -12	TTAGCTCCTGAAAATCTCGTTACTTTTTCTTTTTTGTGTGGTGCATGTCT
Kat-3	TTGACAGCTTATCATCGATGTGAGTCAGAATGTCTTTCACAACCC	回补质粒及回补菌株检测引物
Kat-4	CCCCGGGGACCTGCAGGCCGCTTGCACGGGT
KatA-R	GTCGACGGAGCTCGAATTCGTTACTTTTTCTTTTTTGTGTGGT	katA 基因扩增
KatA-F	AGCAAATGGGTCGCGGATCATGGTTAATAAAGATGTGAAAC
KatA-C1	CAAAAAACCCCTCAAGACCCGTTTAG	KatA^G272 蛋白表达质粒检测引物
KatA-C2	ACCGGCATACTCTGCGACATC

图1 katA 氨基酸序列保守性分析红色：保守氨基酸；黄色：相对保守氨基酸；无色：不保守氨基酸

Fig. 1 Conservative analysis of amino acid sequence for katA Red: Conservative amino acids. Yellow: Relatively conserved amino acid. Colorless: Non conserved amino acids

图2 katA 基因 PCR 扩增及表达载体构建 A：katA 基因 PCR 扩增；B：表达质粒 PCR 鉴定（泳道1-2：阳性质粒 PCR 鉴定；泳道3：pET28a 空白质粒对照，扩增引物为 KatA-C1/KatA-C2）；C：pET28a-katA 表达质粒

Fig. 2 PCR amplification for katA and construction of expression vector A: PCR amplification for katA gene. B: Expression plasmid PCR identification (Lane 1-2: PCR identification for positive plasmid; lane 3: blank control, primers used in this identification are KatA-C1/KatA-C2). C: pET28a-katA expression plasmid

图3 KatAG272 蛋白表达及分离纯化 A：KatAG272 蛋白在大肠杆菌中的诱导表达（泳道 1、4、7、10：超声破碎前菌体沉淀，泳道 2、5、8、11：超声破碎前培养上清，泳道 3、6、9、12：超声破碎后上清，泳道 13-14：含 pET28a 载体的野生型超声破碎前菌体沉淀对照）；B：KatAG272 蛋白分离纯化（泳道 1：流穿液，泳道 2：10 mmol/L咪唑漂洗液，泳道 3-5：200 mmol/L咪唑洗脱液）

Fig. 3 Protein expression, isolation and purification for KatAG272 A: The induced expression of KatAG272 protein in Escherichia coli（Lane 1, 4, 7, 10: Bacterial precipitation before ultrasound fragmentation; lane 2, 5, 8, 11: culture supernatant before ultrasound fragmentation; lane 3, 6, 9, 12: supernatant after ultrasound fragmentation; lane 13-14: control of bacterial precipitation before wild-type ultrasound fragmentation containing pET28a vector）. B: KatAG272 protein isolation and purification（Lane 1: Flow through fluid; lane 2:10 mmol/L imidazole rinse; lane 3-5: 200 mmol/L imidazole elution）

图4 过氧化氢酶活性分析及鉴定 A：KatAG272 纯酶的过氧化氢酶活性检测；B：重组大肠杆菌的 KatAG272 过氧化氢酶活性检测；C：温度对 KatAG272 活性的影响； D：pH 对KatAG272 活性的影响；E：KatAG272 蛋白温度耐受性；F：KatAG272 蛋白 pH 耐受性

Fig. 4 Analysis and identification of catalase activity A: Detection of catalase activity of KatAG272 pure enzyme. B: Detection of KatAG272 catalase activity in recombinant E. coli; C: The effect of temperature on the activity of KatAG272. D: The effect of pH on the activity of KatAG272. E: KatAG272 protein temperature tolerance. F: KatAG272 protein pH tolerance

图5 敲除菌株（HpΔkatA）构建及鉴定 A：敲除菌株（HpΔkatA）构建示意图；B：敲除菌株（HpΔkatA）鉴定（泳道 1：引物 Kan-1 和 Kan-2，敲除菌株卡那霉素抗性验证，泳道 2：引物 Kat-1 和 Kat-2，敲除菌株卡那霉素抗性片段和上下游同源臂片段验证，泳道 3：引物 Kat-1 和 Kat-2，野生型对照）

Fig. 5 Construction and identification for katA-knockout strain HpΔkatA A: Schematics of HpΔkatA construction. B: Identification of HpΔkatA（Lane 1: primer Kan-1 and Kan-2, validation of kanamycin resistance in knockout strains; lane 2: primer Kat-1 and Kat-2, validation of kanamycin resistance fragments and upstream and downstream homologous arm fragments in knockout strains; lane 3: primer Kat-1 and Kat-2, wild-type control）

图6 回补菌株（HpΔkatA::katA）构建及鉴定 A：回补菌株（HpΔkatA::katA）构建示意图；B：回补菌株（HpΔkatA::katA）鉴定（泳道 1：引物 Chl-1 和 Chl-2，回补菌株氯霉素抗性验证，泳道 2：引物 Kat-3 和 Kat-4，回补菌株氯霉素抗性片段和上下游同源臂片段验证）

Fig. 6 Construction and identification for katA-complemented strain HpΔkatA::katA A: Schematics of HpΔkatA::katA construction; B: Identification HpΔkatA::katA（lane 1: primer Chl-1 and Chl-2, validation of chloramphenicol resistance of the complementary strain; lane 2: primer Kat-3 and Kat-4, validation of chloramphenicol resistance fragments and upstream and downstream homologous arm fragments of the complementary strain）

图7 幽门螺杆菌生长曲线

Fig. 7 Growth curve of Hp（Helicobacter pylori）

图8 幽门螺杆菌分解过氧化氢特性分析

Fig. 8 Hydrogen peroxide decomposing characteristics analysis of Hp

表3 Hp 对过氧化氢耐受性分析

Table 3 Analysis of tolerance of Hp to hydrogen peroxide

菌株Strain	浓度Concentration/（mmol·L^-1）
菌株Strain	0	3	6	10	20	40	60	80	100
Hp_G272	+	+	+	+	+	+	-	-	-
HpΔkatA	+	+	+	-	-	-	-	-	-
HpΔkatA::katA	+	+	+	+	+	+	-	-	-

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幽门螺杆菌katA基因功能及其在耐受氧化损伤中的作用分析

Function of katA in Helicobacter pylori and Its Role in the Tolerance to Oxidative Damage

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