Attenuation of Aeromonas hydrophila Infection in Carassius auratus by YtnP, a N-acyl Homoserine Lactonase from Bacillus licheniformis T-1

In this experiment, the quorum quenching gene ytnP of Bacillus licheniformis T-1 was cloned and expressed, and the effect against infection of Aeromonas hydrophila ATCC 7966 was evaluated in vitro and vivo. The BLAST results revealed a 99% sequence identity between the ytnP gene of T-1 and its homolog in B. subtilis sub sp. BSP1, and the dendroGram showed that the similarity in the YtnP protein in T-1 was 100% in comparison with B. subtilis 3610, which was categorized as the Aidc cluster of the MBL family. The AHL lactonase activity of the purified YtnP was detected as 1.097 +/- 0.7 U/mL with C6-HSL as the substrate. Otherwise, purified YtnP protein could significantly inhibit the biofilm formation of A. hydrophila ATCC 7966 with an inhibition rate of 68%. The MIC of thiamphenicol and doxycycline hydrochloride against A. hydrophila reduced from 4 mu g/mL and 0.5 mu g/mL to 1 mu g/mL and 0.125 mu g/mL, respectively, in the presence of YtnP. In addition, YtnP significantly inhibited the expression of five virulence factors hem, ahyB, ast, ep, aerA of A. hydrophila ATCC 7966 as well (p < 0.05). The results of inhibition on virulence showed a time-dependence tendency, while the strongest anti-virulence effects were within 4-24 h. In vivo, when the YtnP protein was co-injected intraperitoneally with A. hydrophila ATCC 7966, it attenuated the pathogenicity of A. hydrophila and the accumulated mortality was 27 +/- 4.14% at 96 h, which was significantly lower than the average mortality of 78 +/- 2.57% of the Carassius auratus injected with 10(8) CFU/mL of A. hydrophila ATCC 7966 only (p < 0.001). In conclusion, the AHL lactonase in B. licheniformis T-1 was proven to be YtnP protein and could be developed into an agent against infection of A. hydrophila in aquaculture.

Automated summary

The AHL lactonase YtnP protein from Bacillus licheniformis T-1 showed significant anti-virulence effects against Aeromonas hydrophila ATCC 7966 by inhibiting biofilm formation and reducing pathogenicity in vivo. The purified YtnP protein also decreased the expression of virulence factors and lowered mortality rates in infected fish, suggesting its potential as a therapeutic agent in aquaculture settings.