磷酸二酯酶PA4781对铜绿假单胞菌运动能力及毒力的影响

 目的 探讨磷酸二酯酶PA4781对细菌运动能力及毒力的影响。方法 通过测量菌苔的直径检测细菌的运动能力, 利用刚果红染色和苯酚-硫酸显色法检测细菌生物膜分泌胞外多糖的能力。通过实时荧光定量PCR检测介导蹭行运动的基因PilZ、与胞外多糖合成有关的基因PelA以及控制绿脓菌素的基因PhzM的mRNA表达量。利用琼脂糖凝胶电泳检测细菌不同时间段内产生的胞外DNA量, 通过测量菌斑在牛奶平板中透明圈的直径检测胞外蛋白酶的总活性。结果 相比于PA03野生型菌株, PA4781敲除菌株的运动能力及PilZ的mRNA表达量降低, PA4781过表达菌株的运动能力及PilZ的mRNA表达量增高, 差异均有统计学意义(均P＜0.05);相比于PA03野生型菌株, PA4781敲除菌株分泌的胞外多糖及PelA的mRNA表达量增多, PA4781过表达菌株分泌的胞外多糖及PelA的mRNA表达量减少, 差异均有统计学意义(均P＜0.05); PA4781敲除菌株和PA4781过表达菌株的胞外DNA分泌量、绿脓菌素基因PhzM的表达量及胞外蛋白酶的水解能力均低于PA03野生型菌株, 差异均有统计学意义(均P＜0.05)。结论 PA4781可能通过促进基因PilZ的表达, 从而促进铜绿假单胞菌的运动能力。PA4781可能通过抑制基因PelA的表达, 抑制胞外多糖的分泌。同时, PA4781可能不直接影响胞外DNA分泌量, 毒力因子绿脓菌素和胞外蛋白酶的产生。

Effect of phosphodiesterase PA4781 on motility and virulence of Pseudomonas aeruginosa

Objective To explore the effect of phosphodiesterase PA4781 on bacterial motility and virulence. Methods Motility of bacteria was detected by measuring the diameter of bacterial lawn, the ability of bacterial biofilm to secrete extracellular polysaccharides was detected by Congo-red staining and phenol-sulphate colorimetry. The expression levels of mRNA of twitching motility-mediating gene PilZ, extracellular polysaccharide synthesis relevant gene PelA and pyocyanin-controlling gene PhzM were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Extracellular DNA produced by bacteria during different periods was detected by agarose gel electrophoresis, the overall activity of extracellular protease was detected by measuring the diameter of transparent circle of plaque in milk plate. Results Compared with PA03 wild-type strain, motility and mRNA expression of PilZ of PA4781 knockout strain decreased, motility and mRNA expression of PilZ of PA4781 overexpressed strain increased, differences were both significant (both P < 0.05); compared with PA03 wild-type strain, secretion of extracellular polysaccharide and mRNA expression of PelA of PA4781 knockout strain increased, secretion of extracellular polysaccharide and mRNA expression of PelA of PA4781 overexpressed strain decreased, diffe-rences were both significant (both P < 0.05); extracellular DNA secretion, expression of pyocyanine gene PhzM, and hydrolysis ability of extracellular protease of PA4781 knockout strain and overexpressed strain were all lower than PA03 wild-type strain, differences were all significant (all P < 0.05). Conclusion PA4781 may promote the motility of Pseudomonas aeruginosa by promoting the expression of gene PilZ. PA4781 may inhibit the secretion of extracellular polysaccharide by inhibiting the expression of gene PelA. PA4781 may not directly affect the secretion of extracellular DNA, as well as the production of virulence factor pyocyanine and extracellular protease.