Deletion of AS87_03730 gene changed the bacterial virulence and gene expression of Riemerella anatipestifer

Riemerella anatipestifer is an important pathogen of waterfowl, which causes septicemia anserum exsudativa in ducks. In this study, an AS87_03730 gene deletion R. anatipestifer mutant Yb2ΔAS87_03730 was constructed to investigate the role of AS87_03730 on R. anatipestifer virulence and gene regulation. By deleting a 708-bp fragment from AS87_03730, the mutant Yb2ΔAS87_03730 showed a significant decreased growth rate in TSB and invasion capacity in Vero cells, compared to wild-type strain Yb2. Moreover, the median lethal dose (LD(50)) of Yb2ΔAS87_03730 was 1.24 × 10(7) colony forming units (CFU), which is about 80-fold attenuated than that of Yb2 (LD(50) = 1.53 × 10(5) CFU). Furthermore, RNA-Seq analysis and Real-time PCR indicated 19 up-regulated and two down-regulated genes in Yb2ΔAS87_03730. Functional analysis revealed that 12 up-regulated genes were related to “Translation, ribosomal structure and biogenesis”, two were classified into “Cell envelope biogenesis, outer membrane”, one was involved in “Amino acid transport and metabolism”, and the other four had unknown functions. Polymerase chain reaction and sequence analysis indicated that the AS87_03730 gene is highly conserved among R. anatipestifer strains, as the percent sequence identity was over 93.5%. This study presents evidence that AS87_03730 gene is involved in bacterial virulence and gene regulation of R. anatipestifer.