Whole Genome Sequencing and Comparative Genome Analysis of a Fish-derived Pathogenic Aeromonas Hydrophila Strain XDMG

doi:10.13560/j.cnki.biotech.bull.1985.2022-1507

Abstract

Abstract:

The objectives of this study are to explore the mechanism of pathogenic Aeromonas hydrophila XDMG strain infecting fish, and to compare its genome structure, function and evolutionary relationship with the reference strain. The whole genome of A. hydrophila XDMG strain was sequenced, and the original data obtained by sequencing were assembled, spliced and annotated using Newbler, SeqMan, Glimmer and other software. Based on the constructing phylogenetic tree analysis from housekeeping genes and determining evolutionary relationships, the genomes of A. hydrophila were compared with those of four A. hydrophila strains in different regions. The complete genome sequence of A. hydrophila XDMG strain was 4.99 Mb in length and the GC content was 60.84%. A total of 4 935 coding genes were predicted, 4 137 genes with clear function were found, 99 tRNAs and 3 rRNAs were found; 4 061 genes had ortholog family classification, 3 228 genes were related to gene ontology, and 2 663 genes related to metabolic pathways; 101 tandem repeats and 14 gene islands were predicted in the genome of A. hydrophila XDMG strain, with genes related to three QS systems: AI 1, AI 2, and AI 3. Through genome comparative analysis, A. hydrophila XDMG was closely related to domestic A. hydrophila J-1 strain and US-derived A. hydrophila AL0971 strain; it was more conserved with US-derived A. hydrophila AL0971 in the comparison of virulence genes, outer membrane proteins, and O-antigen gene clusters. Comprehensive genome annotation and comparative analysis of the genome of A. hydrophila XDMG strain provide basic data for the study of functional genomics and genetic engineering vaccines of A. hydrophila.

Key words: Aeromonas hydrophila, whole genome sequencing, virulence gene, outer membrane protein, O-antigen

GUO Shao-hua, MAO Hui-li, LIU Zheng-quan, FU Mei-yuan, ZHAO Ping-yuan, MA Wen-bo, LI Xu-dong, GUAN Jian-yi. Whole Genome Sequencing and Comparative Genome Analysis of a Fish-derived Pathogenic Aeromonas Hydrophila Strain XDMG[J]. Biotechnology Bulletin, 2023, 39(8): 291-306.

Figures/Tables 18

References 61

[1]	Awan F, Dong YH, Wang NN, et al. The fight for invincibility: environmental stress response mechanisms and Aeromonas hydrophila[J]. Microb Pathog, 2018, 116: 135-145. doi: 10.1016/j.micpath.2018.01.023 URL
[2]	Park SM, Kim HW, Choi C, et al. Pathogenicity and seasonal variation of Aeromonas hydrophila isolated from seafood and ready-to-eat sushi in South Korea[J]. Food Res Int, 2021, 147: 110484. doi: 10.1016/j.foodres.2021.110484 URL
[3]	Dong J, Zhang LS, Liu YT, et al. Luteolin decreases the pathogenicity of Aeromonas hydrophila via inhibiting the activity of aerolysin[J]. Virulence, 2021, 12(1): 165-176. doi: 10.1080/21505594.2020.1867455 URL
[4]	Parker JL, Shaw JG. Aeromonas spp. clinical microbiology and disease[J]. J Infect, 2011, 62(2): 109-118. doi: 10.1016/j.jinf.2010.12.003 URL
[5]	Sharpless M, Sharma R. Aeromonas hydrophila endogenous endophthalmitis in a patient with underlying adenocarcinoma of the colon[J]. BMJ Case Rep, 2021, 14(4): e241317. doi: 10.1136/bcr-2020-241317 URL
[6]	Zhou J, Zhao H, Zhang L, et al. MiRNA-seq analysis of spleen and head kidney tissue from aquacultured largemouth bass(Micropterus salmoides)in response to Aeromonas hydrophila infection[J]. Funct Integr Genomics, 2021, 21(1): 101-111. doi: 10.1007/s10142-020-00763-8
[7]	Zhang XJ, Yang WM, Wu H. et al. Multilocus sequence typing revealed a clonal lineage of Aeromonas hydrophila caused motile Aeromonas septicemia outbreaks in pond-cultured cyprinid fish in an epidemic area in central China[J]. Aquaculture, 2014, 432: 1-6. doi: 10.1016/j.aquaculture.2014.04.017 URL
[8]	贺文旭, 毛会丽, 杨利敏, 等. 豫北地区主要淡水鱼类感染嗜水气单胞菌的流行病学调查[J]. 水产科学, 2016, 35(3): 278-283.
	He WX, Mao HL, Yang LM, et al. Epidemiological investigation of Aeromonas hydrophila infection isolated in major fresh-water fishes from northern regions in Henan, China[J]. Fish Sci, 2016, 35(3): 278-283.
[9]	关建义, 刘涌涛, 毛会丽, 等. 一种嗜水气单胞菌及维氏气单胞菌二联灭活疫苗及制备方法: CN105031636B[P]. 2018-06-19.
	Guan JY, Liu YT, Mao HL, et al. Bi-combined inactivate vaccine of Aeromonas hydrophila and Aeromonas veronii and preparation method thereof: CN105031636B[P]. 2018-06-19.
[10]	储卫华, 陆承平. 嗜水气单胞菌J-1株丝氨酸蛋白酶基因克隆与序列分析[J]. 水产学报, 2004, 28(1): 84-88.
	Chu WH, Lu CP. Cloning and sequence analysis of an extracellular serine-protease gene of Aeromonas hydrophila J-1[J]. J Fish China, 2004, 28(1): 84-88.
[11]	Pridgeon JW, Zhang DH, Zhang LE. Complete genome sequence of the highly virulent Aeromonas hydrophila AL09-71 isolated from diseased channel catfish in west Alabama[J]. Genome Announc, 2014, 2(3): e00450-e00414.
[12]	张国亮, 王浩, 张也, 等. 嗜水气单胞菌AH10(CCTCC AB2014155)的全基因组测序及比较分析[J]. 中国水产科学, 2016, 23(5): 995-1005.
	Zhang GL, Wang H, Zhang Y, et al. Whole genome sequencing and comparative analysis of Aeromonas hydrophila AH10(CCTCC AB2014155)[J]. Chinese Aquatic Science, 2016, 23(5): 995-1005.
[13]	Seshadri R, Joseph SW, Chopra AK, et al. Genome sequence of Aeromonas hydrophila ATCC 7966T: jack of all trades[J]. J Bacteriol, 2006, 188(23): 8272-8282. pmid: 16980456
[14]	Nederbragt AJ. On the middle ground between open source and commercial software - the case of the Newbler program[J]. Genome Biol, 2014, 15(4): 113. pmid: 25180324
[15]	Swindell SR, Plasterer TN. SEQMAN. contig assembly[J]. Methods Mol Biol, 1997, 70: 75-89. pmid: 9089604
[16]	Delcher AL, Bratke KA, Powers EC, et al. Identifying bacterial genes and endosymbiont DNA with Glimmer[J]. Bioinformatics, 2007, 23(6): 673-679. doi: 10.1093/bioinformatics/btm009 pmid: 17237039
[17]	Bairoch A, Apweiler R. The SWISS-PROT protein sequence database and its supplement TrEMBL in 2000[J]. Nucleic Acids Res, 2000, 28(1): 45-48. doi: 10.1093/nar/28.1.45 pmid: 10592178
[18]	Finn RD, Bateman A, Clements J, et al. Pfam: the protein families database[J]. Nucleic Acids Res, 2014, 42(Database issue): D222-D230.
[19]	Ogata H, Goto S, Sato K, et al. KEGG: Kyoto encyclopedia of genes and genomes[J]. Nucleic Acids Res, 1999, 27(1): 29-34. doi: 10.1093/nar/27.1.29 pmid: 9847135
[20]	Lombard V, Golaconda Ramulu H, Drula E, et al. The carbohydrate-active enzymes database(CAZy)in 2013[J]. Nucleic Acids Res, 2014, 42(D1): D490-D495. doi: 10.1093/nar/gkt1178 URL
[21]	Chen LH, Zheng DD, Liu B, et al. VFDB 2016: hierarchical and refined dataset for big data analysis—10 years on[J]. Nucleic Acids Res, 2016, 44(D1): D694-D697. doi: 10.1093/nar/gkv1239 URL
[22]	Jia BF, Raphenya AR, Alcock B, et al. CARD 2017: expansion and model-centric curation of the comprehensive antibiotic resistance database[J]. Nucleic Acids Res, 2017, 45(D1): D566-D573. doi: 10.1093/nar/gkw1004 URL
[23]	Urban M, Cuzick A, Rutherford K, et al. PHI-base: a new interface and further additions for the multi-species pathogen-host interactions database[J]. Nucleic Acids Res, 2017, 45(D1): D604-D610. doi: 10.1093/nar/gkw1089 URL
[24]	Jensen LJ, Julien P, Kuhn M, et al. eggNOG: automated construction and annotation of orthologous groups of genes[J]. Nucleic Acids Res, 2008, 36(Database issue): D250-D254.
[25]	Saier MH Jr, Reddy VS, Tsu BV, et al. The Transporter Classification Database(TCDB): recent advances[J]. Nucleic Acids Res, 2016, 44(D1): D372-D379. doi: 10.1093/nar/gkv1103 URL
[26]	Saier MH Jr, Tran CV, Barabote RD. TCDB: the Transporter Classification Database for membrane transport protein analyses and information[J]. Nucleic Acids Res, 2006, 34(Database issue): D181-D186.
[27]	Krogh A, Larsson B, von Heijne G, et al. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes[J]. J Mol Biol, 2001, 305(3): 567-580. doi: 10.1006/jmbi.2000.4315 pmid: 11152613
[28]	Chan PP, Lowe TM. tRNAscan-SE: searching for tRNA genes in genomic sequences[J]. Methods Mol Biol, 2019, 1962: 1-14. doi: 10.1007/978-1-4939-9173-0_1 pmid: 31020551
[29]	Benson G. Tandem repeats finder: a program to analyze DNA sequences[J]. Nucleic Acids Res, 1999, 27(2): 573-580. doi: 10.1093/nar/27.2.573 pmid: 9862982
[30]	Bertelli C, Laird MR, Williams KP, et al. IslandViewer 4: expanded prediction of genomic islands for larger-scale datasets[J]. Nucleic Acids Res, 2017, 45(W1): W30-W35. doi: 10.1093/nar/gkx343 URL
[31]	Bland C, Ramsey TL, Sabree F, et al. CRISPR recognition tool(CRT): a tool for automatic detection of clustered regularly interspaced palindromic repeats[J]. BMC Bioinformatics, 2007, 8: 209. doi: 10.1186/1471-2105-8-209
[32]	孙静, 万晓媛, 杨倩, 等. 病例研究:未知病因的凡纳滨对虾溞状幼体的病原和微生物组分析[J]. 渔业科学进展, 2019, 40(5): 134-144.
	Sun J, Wan XY, Yang Q, et al. Case studies: pathogenic agent and microbiome analysis for zoea of litopenaeus vannamei suffering from an unknown disease[J]. Prog Fish Sci, 2019, 40(5): 134-144.
[33]	Yu ZH, Geng Y, Wang KY, et al. Complete genome sequence of Vibrio mimicus strain SCCF01 with potential application in fish vaccine development[J]. Virulence, 2017, 8(6): 1028-1030. doi: 10.1080/21505594.2016.1250996 URL
[34]	Chen F, Liu XF, Hu NW, et al. Aeromonas hydrophila Ssp1: a secretory serine protease that disrupts tight junction integrity and is essential for host infection[J]. Fish Shellfish Immunol, 2022, 127: 530-541. doi: 10.1016/j.fsi.2022.06.068 URL
[35]	Tseng TT, Tyler BM, Setubal JC. Protein secretion systems in bacterial-host associations, and their description in the Gene Ontology[J]. BMC Microbiol, 2009, 9(S 1): 1-9. doi: 10.1186/1471-2180-9-1
[36]	Rasmussen-Ivey CR, Figueras MJ, McGarey D, et al. Virulence factors of Aeromonas hydrophila: in the wake of reclassification[J]. Front Microbiol, 2016, 7: 1337. doi: 10.3389/fmicb.2016.01337 pmid: 27610107
[37]	Vilches S, Urgell C, Merino S, et al. Complete type III secretion system of a mesophilic Aeromonas hydrophila strain[J]. Appl Environ Microbiol, 2004, 70(11): 6914-6919. doi: 10.1128/AEM.70.11.6914-6919.2004 URL
[38]	Barger PC, Liles MR, Newton JC. Type II secretion is essential for virulence of the emerging fish pathogen, hypervirulent Aeromonas hydrophila[J]. Front Vet Sci, 2020, 7: 574113. doi: 10.3389/fvets.2020.574113 URL
[39]	Jin L, Chen Y, Yang WG, et al. Complete genome sequence of fish-pathogenic Aeromonas hydrophila HX-3 and a comparative analysis: insights into virulence factors and quorum sensing[J]. Sci Rep, 2020, 10: 15479. doi: 10.1038/s41598-020-72484-8
[40]	Pissaridou P, Allsopp LP, Wettstadt S, et al. The Pseudomonas aeruginosa T6SS-VgrG1b spike is topped by a PAAR protein eliciting DNA damage to bacterial competitors[J]. Proc Natl Acad Sci USA, 2018, 115(49): 12519-12524. doi: 10.1073/pnas.1814181115 pmid: 30455305
[41]	Khajanchi BK, Kozlova EV, Sha J, et al. The two-component QseBC signalling system regulates in vitro and in vivo virulence of Aeromonas hydrophila[J]. Microbiology(Reading), 2012, 158(Pt 1): 259-271.
[42]	Abisado RG, Benomar S, Klaus JR, et al. Bacterial quorum sensing and microbial community interactions[J]. mBio, 2018, 9(3): e02331-e02317.
[43]	Sun B, Luo HZ, Jiang H, et al. Inhibition of quorum sensing and biofilm formation of esculetin on Aeromonas hydrophila[J]. Front Microbiol, 2021, 12: 737626. doi: 10.3389/fmicb.2021.737626 URL
[44]	Swift S, Lynch MJ, Fish L, et al. Quorum sensing-dependent regulation and blockade of exoprotease production in Aeromonas hydrophila[J]. Infect Immun, 1999, 67(10): 5192-5199. doi: 10.1128/IAI.67.10.5192-5199.1999 pmid: 10496895
[45]	Merino S, Rubires X, Aguilar A, et al. The role of flagella and motility in the adherence and invasion to fish cell lines by Aeromonas hydrophila serogroup O: 34 strains[J]. FEMS Microbiol Lett, 1997, 151(2): 213-217. pmid: 9228756
[46]	Saleh A, Elkenany R, Younis G. Virulent and multiple antimicrobial resistance Aeromonas hydrophila isolated from diseased Nile Tilapia fish(Oreochromis niloticus)in Egypt with sequencing of some virulence-associated genes[J]. Biocontrol Sci, 2021, 26(3): 167-176. doi: 10.4265/bio.26.167 URL
[47]	Zhao XL, Wu G, Chen H, et al. Analysis of virulence and immunogenic factors in Aeromonas hydrophila: towards the development of live vaccines[J]. J Fish Dis, 2020, 43(7): 747-755. doi: 10.1111/jfd.v43.7 URL
[48]	El-Bahar HM, Ali NG, Aboyadak IM, et al. Virulence genes contributing to Aeromonas hydrophila pathogenicity in Oreochromis niloticus[J]. Int Microbiol, 2019, 22(4): 479-490. doi: 10.1007/s10123-019-00075-3 pmid: 30989358
[49]	Lange MD, Abernathy J, Shoemaker CA, et al. Proteome analysis of virulent Aeromonas hydrophila reveals the upregulation of iron acquisition systems in the presence of a xenosiderophore[J]. FEMS Microbiol Lett, 2020, 367(20): fnaa169. doi: 10.1093/femsle/fnaa169 URL
[50]	Teng T, Xi BW, Chen K, et al. Comparative transcriptomic and proteomic analyses reveal upregulated expression of virulence and iron transport factors of Aeromonas hydrophila under iron limitation[J]. BMC Microbiol, 2018, 18(1): 52. doi: 10.1186/s12866-018-1178-8 pmid: 29866030
[51]	Zhu W, Zhou S, Chu W. Comparative proteomic analysis of sensitive and multi-drug resistant Aeromonas hydrophila isolated from diseased fish[J]. Microb Pathog, 2020, 139: 103930. doi: 10.1016/j.micpath.2019.103930 URL
[52]	Roges EM, Gonçalves VD, Cardoso MD, et al. Virulence-associated genes and antimicrobial resistance of Aeromonas hydrophila isolates from animal, food, and human sources in Brazil[J]. Biomed Res Int, 2020, 2020: 1052607.
[53]	Xu GY, Tang XP, Shang XY, et al. Identification of immunogenic outer membrane proteins and evaluation of their protective efficacy against Stenotrophomonas maltophilia[J]. BMC Infect Dis, 2018, 18(1): 347. doi: 10.1186/s12879-018-3258-7
[54]	Rekha MK, Biswajit M, Malathi S, et al. Recombinant Aeromonas hydrophila outer membrane protein 48(Omp48)induces a protective immune response against Aeromonas hydrophila and Edwardsiella tarda[J]. Res Microbiol, 2012, 163(4): 286-291.
[55]	Yadav SK, Meena JK, Sharma M, et al. Recombinant outer membrane protein C of Aeromonas hydrophila elicits mixed immune response and generates agglutinating antibodies[J]. Immunol Res, 2016, 64(4): 1087-1099. doi: 10.1007/s12026-016-8807-9 URL
[56]	Yadav SK, Dash P, Sahoo PK, et al. Recombinant outer membrane protein OmpC induces protective immunity against Aeromonas hydrophila infection in Labeo rohita[J]. Microb Pathog, 2021, 150: 104727. doi: 10.1016/j.micpath.2020.104727 URL
[57]	He L, Wu L, Tang Y, et al. Immunization of a novel outer membrane protein from Aeromonas hydrophila simultaneously resisting A. hydrophila and Edwardsiella anguillarum infection in European eels(Angullia angullia)[J]. Fish Shellfish Immunol, 2020, 97: 300-312. doi: 10.1016/j.fsi.2019.12.060 URL
[58]	Yadav SK, Dash P, Sahoo PK, et al. Modulation of immune response and protective efficacy of recombinant outer-membrane protein F(rOmpF)of Aeromonas hydrophila in Labeo rohita[J]. Fish Shellfish Immunol, 2018, 80: 563-572. doi: 10.1016/j.fsi.2018.06.041 URL
[59]	Heiss C, Wang Z, Thurlow CM, et al. Structure of the capsule and lipopolysaccharide O-antigen from the channel catfish pathogen, Aeromonas hydrophila[J]. Carbohydr Res, 2019, 486: 107858. doi: 10.1016/j.carres.2019.107858 URL
[60]	Cao HC, Wang M, Wang Q, et al. Identifying genetic diversity of O antigens in Aeromonas hydrophila for molecular serotype detection[J]. PLoS One, 2018, 13(9): e0203445. doi: 10.1371/journal.pone.0203445 URL
[61]	Dworaczek K, Kurzylewska M, Karaś MA, et al. A unique sugar l-perosamine(4-amino-4, 6-dideoxy-l-mannose)is a compound building two O-chain polysaccharides in the lipopolysaccharide of Aeromonas hydrophila strain JCM 3968, serogroup O6[J]. Mar Drugs, 2019, 17(5): 254. doi: 10.3390/md17050254 URL

序列长度范围 Sequence length range/kb	基因骨架的数量 Number of scaffolds	重叠群的数量 Number of contigs
>9	24	24
8-9	0	0
7-8	0	0
6-7	1	1
5-6	1	1
4-5	1	1
3-4	0	0
2-3	3	3
1-2	4	4
0-1	13	13

序列长度范围 Sequence length range/kb	基因骨架的数量 Number of scaffolds	重叠群的数量 Number of contigs
>9	24	24
8-9	0	0
7-8	0	0
6-7	1	1
5-6	1	1
4-5	1	1
3-4	0	0
2-3	3	3
1-2	4	4
0-1	13	13

菌株 Strain	基因长度 Gene length/Mb	GC含量 GC Content/%	编码基因 Cds	rRNA	tRNA	年份 Year	来源 Source	来源地 Source location
J-1	5.00	60.9	4 268	31	110	1989	病鲤鱼Diseased Carp	中国南京Nanjing, China
AL09_71	5.02	60.8	4 297	31	111	2009	斑点叉尾鮰Channel catfish	美国USA
AH10	4.91	61.1	4 327	33	128	2012	病草鱼Diseased Ctenopharyngodon idella	中国上海Shanghai, China
ATCC_7966	4.74	61.5	4 119	31	127	不详	牛奶Milk	美国USA
XDMG	4.99	60.8	4 935	3	99	2009	病麦穗鱼Diseased Pseudorasbora parva	中国新乡Xinxiang, China

菌株 Strain	基因长度 Gene length/Mb	GC含量 GC Content/%	编码基因 Cds	rRNA	tRNA	年份 Year	来源 Source	来源地 Source location
J-1	5.00	60.9	4 268	31	110	1989	病鲤鱼Diseased Carp	中国南京Nanjing, China
AL09_71	5.02	60.8	4 297	31	111	2009	斑点叉尾鮰Channel catfish	美国USA
AH10	4.91	61.1	4 327	33	128	2012	病草鱼Diseased Ctenopharyngodon idella	中国上海Shanghai, China
ATCC_7966	4.74	61.5	4 119	31	127	不详	牛奶Milk	美国USA
XDMG	4.99	60.8	4 935	3	99	2009	病麦穗鱼Diseased Pseudorasbora parva	中国新乡Xinxiang, China

长度 Length/kb	数量 Count
0-5	1
5-10	2
10-15	5
15-20	2
20-25	3
25-30	1