Abstract
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 μg/mL and 0.5 μg/mL to 1 μg/mL and 0.125 μg/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 108 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.
Highlights
The occurrence of bacterial diseases in the aquaculture industry has led to rising demand for biocontrol agents as an alternative to antibiotics
Previous studies have shown that pathogenic virulence factors are regulated by the quorum sensing (QS) system, through which information communication between them could be blocked to reduce the pathogenicity of the bacteria [2,3]
The acyl-homoserine lactone (AHL) lactase gene YtnP were cloned from B. licheniformis T-1 genomic DNA based on the primers YtnP-F1 and YtnP-R1 (Figure 1A)
Summary
The occurrence of bacterial diseases in the aquaculture industry has led to rising demand for biocontrol agents as an alternative to antibiotics. Discovering a new type and effective antibiotic alternatives has become an emergent task for researchers. With the assistance of the virulence factor, pathogens can enter a host and break its immune defense mechanism. Previous studies have shown that pathogenic virulence factors are regulated by the quorum sensing (QS) system, through which information communication between them could be blocked to reduce the pathogenicity of the bacteria [2,3]. Quorum quenching (QQ) interferes with the quorum sensing system to stop the infection by pathogens through inhibiting the synthesis and accumulation of signal molecules or by enzymatic degradation of signal molecules. An anti-virulence strategy will become a potential approach against infections in the future
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