Peng Mengfan, Tong Wentao, Zhao Zhen, Xiao Ling, Wang Zhaoyue, Liu Xuanming, He Xuanhao, Song Zengfu
National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China.
National Pathogen Collection for Aquatic Animals, Shanghai Ocean University, Shanghai 201306, China.
Antibiotics (Basel). 2021 May 26;10(6):631. doi: 10.3390/antibiotics10060631.
In this experiment, the quorum quenching gene of T-1 was cloned and expressed, and the effect against infection of ATCC 7966 was evaluated in vitro and vivo. The BLAST results revealed a 99% sequence identity between the gene of T-1 and its homolog in sub sp. BSP1, and the dendroGram showed that the similarity in the YtnP protein in T-1 was 100% in comparison with 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 ATCC 7966 with an inhibition rate of 68%. The MIC of thiamphenicol and doxycycline hydrochloride against 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 of ATCC 7966 as well ( < 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 ATCC 7966, it attenuated the pathogenicity of 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 injected with 10 CFU/mL of ATCC 7966 only ( < 0.001). In conclusion, the AHL lactonase in T-1 was proven to be YtnP protein and could be developed into an agent against infection of in aquaculture.
在本实验中，克隆并表达了T-1的群体感应淬灭基因，并在体外和体内评估了其对ATCC 7966感染的作用。BLAST结果显示，T-1的基因与其在亚种BSP1中的同源基因序列同一性为99%，系统发育树表明，T-1中YtnP蛋白与3610的相似性为100%，3610属于MBL家族的Aidc簇。以C6-HSL为底物时，纯化后的YtnP的AHL内酯酶活性检测为1.097±0.7 U/mL。此外，纯化后的YtnP蛋白可显著抑制ATCC 7966生物膜的形成，抑制率为68%。在YtnP存在的情况下，甲砜霉素和盐酸多西环素对其的最低抑菌浓度分别从4 μg/mL和0.5 μg/mL降至1 μg/mL和0.125 μg/mL。此外，YtnP也显著抑制了ATCC 7966的五种毒力因子的表达（P<0.05）。对毒力的抑制结果呈现时间依赖性趋势，最强的抗毒力作用在4-24小时内。在体内，当YtnP蛋白与ATCC 7966腹腔联合注射时，它减弱了其致病性，96小时时累计死亡率为27±4.14%，显著低于仅注射10 CFU/mL ATCC 7966的平均死亡率78±2.57%（P<0.001）。总之，T-1中的AHL内酯酶被证明是YtnP蛋白，可开发成为水产养殖中防治感染的制剂。
