基于RecA-LexA介导的类凋亡程序性死亡研究光动力非热灭活铜绿假单胞菌的作用机制

As a conditional pathogenic bacterium frequently isolated from frozen aquatic food products, pseudomonas aeruginosa may cause severe or even fatal infection to humans. Our previous studies have demonstrated that LED-activated curcumin-mediated non-thermal photodynamic action could efficiently sterilize Pseudomonas aeruginosa by inducing the increase of RecA expression, bacteria DNA damage and membrane depolarization, which suggested RecA-LexA mediated bacterial apoptosis-like programmed cell death (ALD) may be one of the underlying mechanisms by which the photodynamic action kills Pseudomonas aeruginosa. In order to further explore this mechanism, we proposed to further analyze the effects of photodynamic treatment on the expression of RecA, the degradation of LexA as well as the expression of downstream Edin genes to confirm the activation of RecA-LexA pathway. Furthermore, the characteristic apoptosis-like programmed cell death (ALD) events including the decrease in the oxidation respiratory chain activity, the membrane depolarization and phosphatidylserine exposure, the condensation and fragmentation of bacterial genome DNA and the degradation of rRNA will be evaluated in Pseudomonas aeruginosa after the photodynamic treatment to confirm the bacterial apoptosis-like programmed cell death (ALD) can be activated by curcumin-mediated photodynamic treatment. Finally, the role of RecA-LexA mediated bacterial apoptosis-like death (ALD) in curcumin-mediated photodynamic action against Pseudomonas aeruginosa will be further confirm by RecA-knockout Pseudomonas aeruginosa strain. This study may lead to a better understanding towards the mechanism of non-thermal antimicrobial photodynamic inactivation.

作为冷藏水产品中常见的条件致病菌，铜绿假单胞菌可引起严重甚至致死性感染。本课题前期研究发现姜黄素介导的光动力作用高效灭活铜绿假单胞菌过程中，可诱导RecA表达上调、大量DNA损伤及膜电位去极化。基于此，我们拟进一步研究RecA-LexA介导的细菌类凋亡程序性死亡（ALD）参与光动力作用灭活铜绿假单胞菌的作用机制；首先测定光动力作用对假单胞菌RecA表达，LexA裂解及下游Edin基因表达的影响，探究光动力作用对假单胞菌RecA-LexA途径的激活；并进一步检测光动力作用降低假单胞菌氧化呼吸链活力，诱导细胞膜去极化和磷脂酰丝氨酸外翻，引起细菌基因组DNA凝聚化和片段化，及rRNA降解等细菌程序性死亡事件；结合RecA缺失菌株的构建；从而明确RecA-LexA介导的细菌类凋亡程序性死亡在姜黄素光动力非热灭活假单胞菌中所发挥的作用机制。该研究为阐明光动力非热灭活食源有害菌的作用机制提供理论基础。

作为冷藏水产品中常见的条件致病菌，铜绿假单胞菌可引起严重甚至致死性感染。本课题研究发现光动力减菌技术对铜绿假单胞菌具有显著的灭活效果，姜黄素浓度100μM条件下受蓝光LED激活30分钟，其杀菌率达到99.99%。进一步的作用机理研究证实经姜黄素介导光动力处理后，铜绿假单胞菌菌液OD260、OD280及电导率明显上升，结合扫描电镜和透射电镜证实光动力处理可引起铜绿假单胞菌细胞膜明显破坏，提示存在细菌细胞质内容物大量泄露。通过DiBAC4(3)荧光探针检测细菌胞内活性氧（Reactive oxygen species，ROS）水平发现，光动力处理组细菌荧光强度显著性大于对照组；且随着光照时间的增加，铜绿假单胞菌产活性氧水平逐渐增加。光动力引起的过量活性氧累积可能使破坏细胞膜结构的重要机制，并且可进一步通过氧化损伤引起铜绿假单胞菌细菌基因组DNA断裂和蛋白广泛降解（少量蛋白条带反而增多或基本未降解）。通过label free蛋白质组学和Wesrern Blot实验对这些光动力处理后增多及未明显降解的蛋白进行研究表明，光动力处理可以引起铜绿假单胞菌细菌细胞RecA蛋白的表达量上调，诱导LexA蛋白裂解，揭示其可能激活RecA-LexA细菌细胞程序性类凋亡途径。为了进一步确认RecA-LexA凋亡途径参与了光动力灭活铜绿假单胞菌过程，通过构建RecA缺失突变株；并采用Annexin V-FITC细胞凋亡试剂盒检测光动力处理对野生型和缺失型铜绿假单胞菌细胞膜内侧磷脂酰丝氨酸外翻的影响。结果表明：光动力处理可引起野生型铜绿假单胞菌发生细胞膜外翻的比率达到64.3%，而突变型铜绿假单胞菌中出现细胞膜外翻的比率仅有26.8%。提示RecA蛋白的缺失会阻碍光动力诱导铜绿假单胞菌进入类凋亡程序性死亡过程，而直接进入晚期凋亡或坏死阶段，从而明确了RecA-LexA介导的细菌类凋亡程序性死亡在姜黄素介导光动力非热灭活假单胞菌中所发挥的作用机制。该研究为阐明光动力非热灭活食源有害菌的作用机制提供新的证据和理论基础。