哈氏弧菌345Ⅲ型分泌系统诱导鱼类细胞凋亡和变圆

Type Ⅲ secretion system of Vibrio harveyi induces rapid apoptosis and cell rounding of fish cells

【背景】哈氏弧菌(Vibrio harveyi)是一种革兰氏阴性细菌,也是海洋动物的重要条件致病菌之一。Ⅲ型分泌系统(typeⅢsecretionsystem,T3SS)是革兰氏阴性致病菌中保守存在的毒力装置,已被公认在细菌致病过程中起着关键作用。【目的】探索哈氏弧菌T3SS诱导鱼类细胞死亡的机制。【方法】采用基因敲除和回补手段获得了T3SS功能缺失的哈氏弧菌345的缺失突变和回补株。利用4’,6-二脒基-2-苯基吲哚(4′,6-diamidino-2-phenylindole,DAPI)染色、原位末端转移酶标记技术(TdT-mediated dUTP nick-end labeling, TUNEL)、含半胱氨酸的天冬氨酸蛋白水解酶(cysteinyl aspartatespecific proteinase, Caspase)活性测定和lactate dehydrogenase (LDH)释放测定等技术揭示哈氏弧菌T3SS导致的细胞死亡特征。【结果】哈氏弧菌345依赖T3SS能迅速导致鱼类细胞变圆并脱落,细胞核皱缩,部分细胞呈现DNA片段化、形成凋亡小体,细胞凋亡关键分子Caspase-3被激活;此外,哈氏弧菌T3SS还导致鱼类细胞的细胞膜通透性改变,LDH等内容物被释放,并且抑制凋亡途径的激活并不能阻止细胞内容物的释放和细胞变圆。【结论】哈氏弧菌利用T3SS体外诱导鱼类细胞发生细胞凋亡途径的激活和细胞变圆,最终导致细胞发生死亡。

[Background]Vibrio harveyi is a Gram-negative marine bacterium that has been recognized as an opportunistic pathogen for marine animals.The type Ⅲ secretion system(T3SS)is a highly-conserved virulence apparatus used by Gram-negative bacterial pathogens to cause infections.[Objective]To decipher the mechanism of T3SS of V.harveyi in inducing the death of fish cells.[Methods]The T3SS disfunctional mutant was established by the in-frame deletion of vcrD in V.harveyi strain 345,and then its complemented mutant was constructed.The wild type and mutant strains were used to infect FHM cells,and then DAPI staining,in situ detection of fragmented DNA(TUNEL assay),Caspase activity assay,and lactate dehydrogenase(LDH)release assay were carried out to reveal the features of cell death.[Results]Dying cells exhibited the features such as cell rounding,nuclear condensation,and DNA fragmentation of apoptotic cells.Caspase-3 was activated by the T3SS,which confirmed that the infection with V.harveyi rapidly induced T3SS-dependent apoptosis in fish cells.Furthermore,the infection with V.harveyi strain 345 led to release of cellular contents including LDH from infected fish cells.Importantly,the inhibition of apoptosis did not prevent cells from releasing cellular contents or rounding.[Conclusion]V.harveyi uses T3SS to induce the activation of apoptosis pathways and the rounding of fish cells,ultimately leading to cell death.