pVA质粒中II型分泌系统同源基因对副溶血弧菌生物学特性及PirAB表达和分泌的影响

Two T2SS homologous genes of pVA plasmid affect biological characteristics and PirAB expression and secretion of Vibrio parahaemolyticus

摘要:

【背景】副溶血弧菌(Vibrio parahaemolyticus)是引起养殖对虾急性肝胰腺坏死病(acute hepatopancreatic necrosis disease, AHPND)的病原之一，毒素蛋白PirAB是该病原的致病因子，由病原携带的pVA质粒上的pirA和pirB编码。PirAB的分泌途径尚未明确。【目的】探究pVA质粒中Ⅱ型分泌系统同源基因epsG2和epsE2对副溶血弧菌生物学特性及PirAB表达和分泌的影响。【方法】通过生物信息学分析发现副溶血弧菌VP220218株pVA质粒上存在2个Ⅱ型分泌系统(type Ⅱ secretion system, T2SS)的同源基因epsG2和epsE2；利用框内缺失方法构建了突变株ΔepsG2和ΔepsE2，检测其生长、生物被膜形成、运动力和胞外蛋白酶活力的变化，进一步用RT-qPCR和Western blotting分析了PirAB表达和分泌的变化。【结果】epsG2和epsE2能够抑制VP220218的胞外蛋白酶活力(P<0.001)、在对数生长期抑制细菌的生长(P<0.05)；此外，epsG2能够促进细菌的运动(P<0.05)、在平台期抑制VP220218的生物膜形成(P<0.05)，epsE2能够抑制细菌的运动、在对数生长中后期促进VP220218的生物膜形成(P<0.05)。RT-qPCR结果显示，epsE2对pirA和pirB的表达无影响；epsG2在对数生长初期和中期抑制pirA和pirB的表达，在对数生长后期和平台期则促进pirA和pirB的表达。Western blotting结果显示，epsG2对PirAB的合成和分泌无影响，epsE2能够促进PirAB的合成和分泌。【结论】epsG2和epsE2参与了副溶血弧菌VP220218的生理活动，且epsE2影响PirAB的合成和分泌。研究结果为了解致AHPND菌PirAB毒素的合成和分泌途径提供了参考。

Abstract:

[Background] Vibrio parahaemolyticus is one of the pathogens causing acute hepatopancreatic necrosis disease (AHPND) in cultured shrimp. PirAB, encoded by pirA and pirB located on the plasmid pVA, acts as the virulence factor of AHPND. However, the secretion pathway of PirABremains unclear. [Objective] To explore the effect of two T2SS homologous genes epsG2 and epsE2 of pVA on biological characteristics and PirAB expression and secretion of Vibrio parahaemolyticus. [Methods] The bioinformatics analysis revealed two type II secretion system (T2SS) homologous genes epsG2 and epsE2 on the pVA plasmid in V. parahaemolyticus VP220218. The mutants ΔepsG2 and ΔepsE2 were constructed by in-frame deletion. The growth, biofilm formation, swimming ability, and extracellular protease activity were compared between VP220218 and mutants. RT-qPCR and Western blotting were performed to compare the expression and secretion of PirAB between VP220218 and mutants.[Results] epsG2 and epsE2 suppressed the extracellular protease activity (P<0.001) and the growth during the logarithmic stage (P<0.05). In addition, epsG2 promoted the swimming (P<0.05) and inhibited the biofilm formation of VP220218 at the plateau stage (P<0.05); epsE2 restrained the swimming ability and promoted the biofilm formation of VP220218 at the middle and late logarithmic stage (P<0.05). The results of RT-qPCR showed that epsE2 had no effect on the expression of pirA or pirB; epsG2 inhibited the expression of pirA and pirB at the early and middle logarithmic stage, while it promoted the expression of pirA and pirBat the late logarithmic stage and plateau stage. The results of Western blotting showed that epsE2 promoted the synthesis and secretion of PirAB while epsG2 had no significant effect on the synthesis or secretion of PirAB. [Conclusion] The results indicated that epsG2 and epsE2 were involved in the physiological activities of V. parahaemolyticus VP220218. Moreover, epsE2 affected the synthesis and secretion of PirAB. The findings provided a reference for understanding the synthetic and secretory pathways of PirAB in AHPND-causing bacteria.