猪源致病性大肠杆菌基因组比较与毒力因子分析

doi:10.13560/j.cnki.biotech.bull.1985.2023-0578

摘要/Abstract

摘要：

不同菌株猪源致病性大肠杆菌的致病力有较大差异，通过着重研究菌株基因组中携带的毒力因子种类和数量，以解析猪源大肠杆菌致病的分子机制。利用二代高通量DNA测序技术测定猪源致病性大肠杆菌的基因组DNA序列，通过生物信息学方法，分析不同毒力菌株的基因组结构特征、进化类型、携带的毒力因子基因及其碱基变异，采用PCR方法对候选毒力因子基因进行验证。猪源致病性大肠杆菌不同毒力菌株的基因组全长范围为4.62-5.3 Mb，含有3 364-3 557个编码基因，菌株的系统进化群和多位点序列分型多属于A群和ST10克隆复合群。6株菌携带112-280个毒力因子基因，强毒菌株的毒力因子基因尤其是毒素基因多于弱毒菌株，且各菌株分泌系统基因的数量变异较大。此外，毒力因子基因中检测到插入缺失、移码突变和无义突变等高影响碱基变异。采用PCR验证了代表性毒力因子基因及其变异。猪源致病性大肠杆菌的毒力与基因组长度、编码基因的数量和变异相关，与GC含量、前噬菌体和CRISPR序列没有必然联系。

关键词: 猪源致病性大肠杆菌, 致病力, 全基因组, 基因分型, 毒力因子基因

Abstract:

The pathogenicity of different strains of porcine-derived Escherichia coli varies greatly. By focusing on the types and quantities of virulence factors carried in the genomes of the strains, we can analyze the molecular mechanism of porcine-derived E. coli causing disease. Next-generation high-throughput DNA sequencing technology was used to determine the genomic DNA sequence of porcine-derived pathogenic E. coli, and bioinformatics methods were adapted to analyze the genome structural characteristics, evolutionary types, virulence factor genes and base variation of different virulent strains. PCR method was to verify the candidate virulence factor genes. The full lengths of the genomes from different virulent strains of porcine pathogenic E. coli ranged from 4.62 Mb to 5.3 Mb, containing 3 364 to 3 557 coding genes. Most phylogenetic groups and multi-locus sequence typing of strains belonged to group A and ST10 clonal complex groups. Six virulent strains carried 112 to 280 virulence factor genes. Virulence factor genes, especially toxin genes, in strong virulent strains were more than that in the weak virulent strains, and the number of genes in the secretion system of each strain varied greatly. In addition, there were high-impact variations（e.g., insertion deletion, frameshift mutation, and nonsense mutation）in virulence factor genes. And representative virulence factor genes and their mutations were confirmed by PCR method. The virulence of porcine-derived pathogenic E. coli was correlated with genome size and number and variation in coding genes, and was not necessarily related to the GC content, prophage, and CRISPR sequence in genome.

Key words: porcine-derived pathogenic Escherichia coli, virulence, genome-wide, genotyping, virulence factor genes

吴莉丹, 冉雪琴, 牛熙, 黄世会, 李升, 王嘉福. 猪源致病性大肠杆菌基因组比较与毒力因子分析[J]. 生物技术通报, 2023, 39(12): 287-299.

WU Li-dan, RAN Xue-qin, NIU Xi, HUANG Shi-hui, LI Sheng, WANG Jia-fu. Genome Comparison and Virulence Factor Analysis of Pathogenic Escherichia coli from Porcine[J]. Biotechnology Bulletin, 2023, 39(12): 287-299.

图/表 11

参考文献 48

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基因类型 Gene type	检测基因 Gene	引物序列 Primer sequence（5'-3'）	退火温度 Annealing temperature/℃	产物长度 Product size/bp
二型分泌系统 Type II secretory system	gspD	F：TGGTGAAGGCAGCGACAACT	65	273
	gspD	R：TCCGAAGCAGAGGCGTTATCC	65	273
	etpD	F：GCCAGACTTGTTACGGAACTGA	62	421
	etpD	R：GGACCTGAGGACGACGAATATC	62	421
	gspE	F：TGCTGCGACTGCTGGACAA	65	334
	gspE	R：TCACCGACCATCACCACATCC	65	334
三型分泌系统 Type III secretory system	eae	F：ACCAGGCTTCGTCACAGTTG	63	573
	eae	R：CCATCGTCACCAGAGGAATCG	63	573
	eprK	F：TGAGCAGCGGTTAGAGCAATC	62	370
	eprK	R：CTTCCATAACAGCCAGCAAGTC	62	370
	eivF	F：TTGCTGATGCCTTGCCCTTT	62	377
	eivF	R：GTGTGATGAGGACGAGTAACCA	62	377
	espX1	F：GGTATCTCAACTTCGCCACATT	59	323
	espX1	R：ACATTCGCTTGTTCTGTCATCA	59	323
	espO1-2	F：TTTCAGGACACACTTGGCATCA	62	213
	espO1-2	R：TTGACATCCATGCGACTTCTGA	62	213
六型分泌系统 Type VI secretory system	aec16	F：AATGCTGCCAACCACTGAACTT	63	218
	aec16	R：CGGAGGTGCCAGATTTGACG	63	218
	hcp1	F：TTGCTCAACGCTTGATTCTGTC	61	336
	hcp1	R：CTCTCCTGTGGTTCCAGTTCTT	61	336
耐热肠毒素类似物 Heat-stabile enterotoxin analogues	east1	F：TGCCATCAACACAGTATATCCG	60	95
耐热肠毒素类似物 Heat-stabile enterotoxin analogues	east1	R：TGTAGTCCTTCCATGACACGAA	60	95
溶血素Hemolysin	hlyA	F：CAGTGACGCACATACAGGAACA	62	362
溶血素Hemolysin	hlyA	R：CCGCAGATACAGAACTCAGGAC	62	362
铁载体转运系统 Ferric transport system	sitA	F：GCACAACTGATTCTCGCCAATG	62	345
铁载体转运系统 Ferric transport system	sitA	R：GGTGACCATCCATCGCTGATT	62	345
菌毛Pilus	cfaB	F：AGGCTCCGCATTGGCAGTA	63	233
菌毛Pilus	cfaB	R：CATCCAGGCTTTGAACCAGTTG	63	233
黏附素Adhesin	eaeH	F：TGTTCCGCAAGACACTAATGGT	63	400
	eaeH	R：CGCCGTAGAGCCGAGAGTAA	63	400
	aatB	F：GAATGCAGAGCAAGCGTTCAAT	63	402
	aatB	R：GCCGACTTTAGCCCTCCCATA	63	402
	ehaA	F：TGACAGACACGCCATCCATACT	64	241
	ehaA	R：CAGCCGCAAGCACAGGTTAC	64	241
	ehaB	F：CGGCATCATGGTCGGTGTT	59	213
	ehaB	R：AGGTCGTTGTTGAAGTGAGAGT	59	213
志贺样毒素 Shiga-toxin	stxB	F：CGACGCCTGATTGTGTAACTG	61	131
	stxB	R：CGCACTGAGAAGAAGAGACTGA	61	131
	stx2A	F：TCCATGACAACGGACAGCAG	62	191
	stx2A	R：CGTAAGGCTTCTGCTGTGAC	62	191
耐热肠毒素 Heat-stabile enterotoxin	st	F：TTTCACCTTTCGCTCAGGATG	60	165
耐热肠毒素 Heat-stabile enterotoxin	st	R：CGGTACAAGCAGGATTACAACA	60	165
热敏肠毒素 Heat-labile enterotoxin	lt	F：CGGGACTTCGACCTGAAATGTT	62	472
	lt	R：CGGCAGAGGATGGTTACAGATT	62	472
	ltB	F：GCTCCCCAGACTATTACAG	53	312
	ltB	R：CTAGTTTTTCATACTGATTGC	53	312
具有高影响变异的毒力因子基因Virulence factor genes with high impact variation	espY1	F：CTGGCGGTCACTTCTTTGAG	59	1 116
	espY1	R：TTGTGAAATCATCCCACGCT	59	1 116
	espL4	F1：CTTTTACGGCTGCATTATGAGA	58	1 980
		R1：ATTATTTGCCAGGAGGAGGG	58	1 980
		F2：CTGAACGCCTTACCTAAACTGG	60	909
		R2：GCGCACTACAAAACCGCATA	60	909
	espX4	F：CAGCAGTGTAAAAGGCAAGGG	59	1 957
	espX4	R：CAACAATCGCATGAAGTGGTAA	59	1 957
	fimD	F1：CTCCAATGGGCGAAAGCA	62	1 281
		R1：GCGGGACTGACGAATAGGGT	62	1 281
		F2：CGTGGTACTGCACAGGTCACT	59	1 960
		R2：GTGACAGCAAAATACGAAATGG	59	1 960
	fimI	F：CTAATGCGGATGCGACCTT	60	740
	fimI	R：ACTGCCAGCAAGCAGAATGT	60	740
	rpoS	F：ACCCGCTGCGTTATTTGC	63	1 308
	rpoS	R：CAGGGTTCTGGATTGTGACCAA	63	1 308
	allB	F：GTTTGTCGGCGTCATCGTC	59	1 723
	allB	R：AAAGCATATTGCGTGAGCGT	59	1 723

基因类型 Gene type	检测基因 Gene	引物序列 Primer sequence（5'-3'）	退火温度 Annealing temperature/℃	产物长度 Product size/bp
二型分泌系统 Type II secretory system	gspD	F：TGGTGAAGGCAGCGACAACT	65	273
	gspD	R：TCCGAAGCAGAGGCGTTATCC	65	273
	etpD	F：GCCAGACTTGTTACGGAACTGA	62	421
	etpD	R：GGACCTGAGGACGACGAATATC	62	421
	gspE	F：TGCTGCGACTGCTGGACAA	65	334
	gspE	R：TCACCGACCATCACCACATCC	65	334
三型分泌系统 Type III secretory system	eae	F：ACCAGGCTTCGTCACAGTTG	63	573
	eae	R：CCATCGTCACCAGAGGAATCG	63	573
	eprK	F：TGAGCAGCGGTTAGAGCAATC	62	370
	eprK	R：CTTCCATAACAGCCAGCAAGTC	62	370
	eivF	F：TTGCTGATGCCTTGCCCTTT	62	377
	eivF	R：GTGTGATGAGGACGAGTAACCA	62	377
	espX1	F：GGTATCTCAACTTCGCCACATT	59	323
	espX1	R：ACATTCGCTTGTTCTGTCATCA	59	323
	espO1-2	F：TTTCAGGACACACTTGGCATCA	62	213
	espO1-2	R：TTGACATCCATGCGACTTCTGA	62	213
六型分泌系统 Type VI secretory system	aec16	F：AATGCTGCCAACCACTGAACTT	63	218
	aec16	R：CGGAGGTGCCAGATTTGACG	63	218
	hcp1	F：TTGCTCAACGCTTGATTCTGTC	61	336
	hcp1	R：CTCTCCTGTGGTTCCAGTTCTT	61	336
耐热肠毒素类似物 Heat-stabile enterotoxin analogues	east1	F：TGCCATCAACACAGTATATCCG	60	95
耐热肠毒素类似物 Heat-stabile enterotoxin analogues	east1	R：TGTAGTCCTTCCATGACACGAA	60	95
溶血素Hemolysin	hlyA	F：CAGTGACGCACATACAGGAACA	62	362
溶血素Hemolysin	hlyA	R：CCGCAGATACAGAACTCAGGAC	62	362
铁载体转运系统 Ferric transport system	sitA	F：GCACAACTGATTCTCGCCAATG	62	345
铁载体转运系统 Ferric transport system	sitA	R：GGTGACCATCCATCGCTGATT	62	345
菌毛Pilus	cfaB	F：AGGCTCCGCATTGGCAGTA	63	233
菌毛Pilus	cfaB	R：CATCCAGGCTTTGAACCAGTTG	63	233
黏附素Adhesin	eaeH	F：TGTTCCGCAAGACACTAATGGT	63	400
	eaeH	R：CGCCGTAGAGCCGAGAGTAA	63	400
	aatB	F：GAATGCAGAGCAAGCGTTCAAT	63	402
	aatB	R：GCCGACTTTAGCCCTCCCATA	63	402
	ehaA	F：TGACAGACACGCCATCCATACT	64	241
	ehaA	R：CAGCCGCAAGCACAGGTTAC	64	241
	ehaB	F：CGGCATCATGGTCGGTGTT	59	213
	ehaB	R：AGGTCGTTGTTGAAGTGAGAGT	59	213
志贺样毒素 Shiga-toxin	stxB	F：CGACGCCTGATTGTGTAACTG	61	131
	stxB	R：CGCACTGAGAAGAAGAGACTGA	61	131
	stx2A	F：TCCATGACAACGGACAGCAG	62	191
	stx2A	R：CGTAAGGCTTCTGCTGTGAC	62	191
耐热肠毒素 Heat-stabile enterotoxin	st	F：TTTCACCTTTCGCTCAGGATG	60	165
耐热肠毒素 Heat-stabile enterotoxin	st	R：CGGTACAAGCAGGATTACAACA	60	165
热敏肠毒素 Heat-labile enterotoxin	lt	F：CGGGACTTCGACCTGAAATGTT	62	472
	lt	R：CGGCAGAGGATGGTTACAGATT	62	472
	ltB	F：GCTCCCCAGACTATTACAG	53	312
	ltB	R：CTAGTTTTTCATACTGATTGC	53	312
具有高影响变异的毒力因子基因Virulence factor genes with high impact variation	espY1	F：CTGGCGGTCACTTCTTTGAG	59	1 116
	espY1	R：TTGTGAAATCATCCCACGCT	59	1 116
	espL4	F1：CTTTTACGGCTGCATTATGAGA	58	1 980
		R1：ATTATTTGCCAGGAGGAGGG	58	1 980
		F2：CTGAACGCCTTACCTAAACTGG	60	909
		R2：GCGCACTACAAAACCGCATA	60	909
	espX4	F：CAGCAGTGTAAAAGGCAAGGG	59	1 957
	espX4	R：CAACAATCGCATGAAGTGGTAA	59	1 957
	fimD	F1：CTCCAATGGGCGAAAGCA	62	1 281
		R1：GCGGGACTGACGAATAGGGT	62	1 281
		F2：CGTGGTACTGCACAGGTCACT	59	1 960
		R2：GTGACAGCAAAATACGAAATGG	59	1 960
	fimI	F：CTAATGCGGATGCGACCTT	60	740
	fimI	R：ACTGCCAGCAAGCAGAATGT	60	740
	rpoS	F：ACCCGCTGCGTTATTTGC	63	1 308
	rpoS	R：CAGGGTTCTGGATTGTGACCAA	63	1 308
	allB	F：GTTTGTCGGCGTCATCGTC	59	1 723
	allB	R：AAAGCATATTGCGTGAGCGT	59	1 723

菌株名 Strain	Scaffolds数目 Number of scaffolds	基因组总长度 Genomic length/Mb	GC含量 GC content/%	N50/kb	L50	对参考基因组的覆盖度 Genomic coverage/%	重复序列占比 Duplication ratio/%
S10670	148	5.30	50.31	160.71	9	87.45	1
E24190	171	4.96	50.54	91.86	15	90.87	1.01
P555	113	4.84	50.70	125.71	13	91.55	1.01
P211	107	4.75	50.63	120.03	15	89.80	1
P32	191	4.99	50.33	158.35	10	91.98	1
P111	108	4.62	50.90	110.78	15	89.86	1

菌株名 Strain	Scaffolds数目 Number of scaffolds	基因组总长度 Genomic length/Mb	GC含量 GC content/%	N50/kb	L50	对参考基因组的覆盖度 Genomic coverage/%	重复序列占比 Duplication ratio/%
S10670	148	5.30	50.31	160.71	9	87.45	1
E24190	171	4.96	50.54	91.86	15	90.87	1.01
P555	113	4.84	50.70	125.71	13	91.55	1.01
P211	107	4.75	50.63	120.03	15	89.80	1
P32	191	4.99	50.33	158.35	10	91.98	1
P111	108	4.62	50.90	110.78	15	89.86	1

菌株名 Strain	编码基因数Number of encoded genes	CDS个数Number of CDS	GC含量 GC content/%	tRNA数目Nu-mber og tRNAs	rRNA数目Nu-mber of rRNAs	前噬菌体数目Nu-mber of prophages	CRISPR序列/可能存在的CRISPR序列数目Number of Crispr/PCrispr
K-12（Ref）	3 459	4 305	51.86	88	22	2	2/3
S10670	3 519	5 040	51.37	96	9	3	1/4
E24190	3 497	4 732	51.63	85	11	0	2/6
P555	3 428	4 531	51.71	85	11	0	2/8
P211	3 434	4 483	51.70	83	11	2	4/4
P32	3 557	4 837	51.39	98	14	2	1/4
P111	3 364	4 308	51.93	85	12	1	3/8