Identification of novel virulence-related genes in Aeromonas hydrophila by screening transposon mutants in a Tetrahymena infection model

- Aeromonas-Tetrahymena co-culture model could be used for high-throughput screening of A. hydrophila virulence factors.
- Fourteen novel virulence-related genes were identified by screening a mutant library comprising 1030 mutant strains.
- The genes chiA and trkH play important roles in the virulence of A. hydrophila.

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.