信号系统在大鼠肺部铜绿假单胞菌感染中的作用及与致病因子的关系

The role of the quorum sensing system in a rat model of Pseudomonas aeruginosa pulmonary infection and its relationship with the expression of virulence factors

目的研究密度感应信号系统(QS)在铜绿假单胞菌(PA)致大鼠肺部感染中的作用。方法将PA制成PA藻酸盐包裹体,建立大鼠肺部感染模型,SD大鼠258只,随机分为3组;比较野生型PA菌株PAO1及QS基因缺失的PA变异株PAO1-JP2大鼠肺部感染模型中细菌学、病理学的不同,从而评价2株菌在大鼠肺部感染中的致病性差异。用刚果红法测定该2株细菌弹性蛋白酶的活性;用免疫印迹法检测外毒素A的表达。结果感染后第14天、28天,PAO1-JP2肺组织匀浆菌落计数分别为(9．6±3．3)lgCFU／g及(4．2±3．1)lgCFU／g,显著低于PAO1组的(11．3±2．8)lgCFU／g及(9．1±1．5)lgCFU／g(P<0．05)。感染后第7、14及28天,PAO1-JP2感染组大鼠肺部病理变化指数及肺组织大体病理评分、肺部脓肿／肉芽肿的平均直径及肺组织显微病理评分均显著低于PAO1组。体外实验结果表明,PAO1-JP2菌株弹性蛋白酶活性(吸光度值)为0．35±0．03,高于PAO1菌株的0．02±0．00。免疫印迹实验可见PAO1株在相对分子质量73 000处显示棕色条带,PAO1-JP2株未见棕色条带。结论QS系统缺损后由于某些致病因子如弹性蛋白酶、外毒素不能表达,其导致的肺部感染程度有所减低。

Objective A variety of in vitro experiments indicated that most of the extracellular virulence factors of Pseudomonas aeruginosa （PA） were controlled by the quorum sensing system. The aim of this study was to explore the role of the quorum sensing system in a rat model of PA pulmonary infection. Methods Rats were infected intratracheally with PA strain PAO1 or PAO1-JP2 （lasI rhlI double mutant） which was embedded in seaweed alginate bead. The difference of bacteriology and pathology between the two groups was compared. The in vitro activity of elastase was measured by the Congo Red method and the expression of exotoxin A was detected by Western blot. Results At the 14th and 28th days after challenge, the bacterial colony counts in the PAO1-JP2 group were （9. 6 ± 3.3 ） lgCFU/g and （4. 2 ± 3.1 ） lgCFU/g respectively; however, those of the PAO1 group were （ 11.3 ± 2. 8 ） lgCFU/g and （ 9. 1 ± 1.5 ） lgCFU/g respectively,which showed significant difference （P 〈 0. 05）. The lung index of macroscopic pathology （LIMP） and the score of macroscopic pathology of the PAO1-JP2 group were significantly lower than those of the PAO1 group at 7th,14th and 28th days after challenge. The size of lung abscesses or granuloma in the lung under the microscope and the score of microscopic pathology of the PAO1-JP2 group were also significantly lower at the 7th, 14th and 28th days. The in vitro activity of elastase of the PAO1-JP2 strain was 0. 02 ± 0. 00 and that of the PAO1 strain was 0. 35 ± 0. 03, which showed significant difference （P 〈 0. 05）. The expression of exotoxin A could be detected by Western blot in the PAO1 strain but not in the PAO1-JP2 strain. Conclusion When the quorum sensing system of PA was deficient the expression of some virulence factors,such as elastase and exotoxin A,were decreased,which resulted in milder pulmonary infection.