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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 To study the role of density-sensitive signaling system (QS) in pulmonary infection induced by Pseudomonas aeruginosa (PA) in rats. Methods The PA alginate inclusions were made into PA. Pulmonary infection rat model was established. 258 male Sprague-Dawley rats were randomly divided into three groups. PAO1 and PAO1-JP2 gene deletion mutant strains of PAO1 and QS gene were compared Bacteriological and pathological differences were observed in the mouse lung infection model to evaluate the pathogenicity of the two strains in lung infection in rats. The activity of the two strains of bacterial elastase was measured by congo red method. The expression of exotoxin A was detected by Western blotting. Results The colony counts of PAO1-JP2 lung homogenates were (9.6 ± 3.3) lgCFU / g and (4.2 ± 3.1) lgCFU / g on the 14th day and the 28th day after infection, respectively, which were significantly lower than (11.3 ± 2.8) lgCFU / g and (9.1 ± 1.5) lgCFU / g in the PAO1 group (P <0.05). On the 7th, 14th and 28th day after infection, the pathological index of lungs, the gross pathological score of lung tissue, the average diameter of lung abscess / granuloma and the microscopic pathological score of lung tissue of PAO1-JP2 infection group were significantly lower than those of PAO1 group. The results of in vitro experiments showed that the elastase activity (absorbance value) of PAO1-JP2 strain was 0.35 ± 0.03, higher than that of PAO1 strain of 0.02 ± 0.00. Western blotting showed PAO1 showed a brown band at 73 000 relative molecular mass, while PAO1-JP2 showed no brown band. Conclusions The QS system can not express exogenous toxins because of certain virulence factors such as elastase, which leads to the decrease of pulmonary infection.