title: Identification of novel virulence-related genes in Aeromonas hydrophila by screening transposon mutants in a Tetrahymena infection model.
abstract: Outbreaks of motile Aeromonad septicemia (MAS) in fish caused by sequence type (ST) 251 Aeromonas hydrophila have become a prominent problem for the aquaculture industry. The pathogenesis of A. hydrophila is very complicated, and some virulence factors remain to be identified. In this study, to identify novel virulence-related factors, ST251 A. hydrophila strain NJ-35 was used as the parental strain to construct a mutant library comprising 1030 mutant strains by transposon insertion mutagenesis. Subsequently, 33 virulence-attenuated transposon insertion mutants were identified using Tetrahymena and zebrafish as model hosts in sequence. Thermal asymmetric interlaced (Tail)-PCR and Southern blot analysis identified 21 single transposon insertion sites. Seven of the insertion sites are located in non-coding regions, whereas the other 14 insertion sites are located in genes, including aroA, rmlA, rtxA, chiA and plc. All insertion mutants exhibited attenuated virulence in Tetrahymena and zebrafish. Furthermore, the relationship of two genes, chiA and trkH, to virulence was confirmed by gene inactivation and subsequent restoration assays. This study provides new information about the genetic determinants of A. hydrophila pathogenicity and validates the Aeromonas-Tetrahymena co-culture model for high-throughput screening of A. hydrophila virulence factors.
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China; Key Lab of Food Quality and Safety of Jiangsu Province-State Key Laboratory Breeding Base, Institute of Food Safety, Jiangsu Academy of Agricultural Sciences, Nanjing, China.
authors.forename: Maoda
authors.initials: M
authors.lastname: Pang
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
authors.forename: Xing
authors.initials: X
authors.lastname: Xie
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
authors.forename: Yuhao
authors.initials: Y
authors.lastname: Dong
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
authors.forename: Hechao
authors.initials: H
authors.lastname: Du
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
authors.forename: Nannan
authors.initials: N
authors.lastname: Wang
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
authors.forename: Chengping
authors.initials: C
authors.lastname: Lu
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China. Electronic address: liuyongjie@njau.edu.cn.
authors.forename: Yongjie
authors.initials: Y
authors.lastname: Liu
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics; D016980:Aeromonas hydrophila / Q000235:genetics* / Q000472:pathogenicity*; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D015139:Blotting, Southern; D002688:Chitinases / Q000235:genetics; D018920:Coculture Techniques / Q000592:standards; D004251:DNA Transposable Elements / Q000235:genetics; D020868:Gene Silencing; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D013768:Tetrahymena / Q000382:microbiology*; D037521:Virulence Factors / Q000235:genetics*; D015027:Zebrafish / Q000382:microbiology
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D037521:Virulence Factors; D002688:Chitinases
keywords: Aeromonas hydrophila; Tetrahymena; Transposon; Virulence-related genes