ABSTRACT

MvaT and MvaU are global transcriptional regulators belonging to the H-NS family, and pyocyanin is an important virulence factor produced by Pseudomonas aeruginosa. mvaT mvaU double knockout mutant of P. aeruginosa PAO1 demonstrated pyocyanin abolishment in the previous study. Here, we further explored the mechanism. Two main directions were studied: pyocyanin biosynthesis pathway and QS system. The effect on the expression of the pyocyanin biosynthesis genes was evaluated by promoter strength determination and Real-Time PCR assay, and significant changes leading to low pyocyanin production were found. The effect on the QS system was studied by signal molecule quantification using LC-MS/MS and related gene expression measurements using Real-Time PCR. In mvaT mvaU double knockout, the production of 3-oxo-C12-HSL obviously increased, while those of C4-HSL and PQS obviously decreased, and the changes can be recovered by mvaT or mvaU complementation. The expressions of transcriptional activator genes binding with QS system signal molecules were all decreased, resulting in decreased formation of signal-transcriptional activator complexes. And the decreased expression of rhlR and pqsE also led to the lower expression of phzA1 and phzA2. Further exploration found that QS system downregulation may be related to QsrO, a QS system repressor, which was highly upregulated with mvaT mvaU double knockout. Hence, the synthesis of pyocyanin was suffocated and the biofilm formation ability was decreased. These results were also confirmed by transcriptome analysis, which demonstrated similar gene expression changes of the aforementioned genes together with decreased expression of other virulence factor genes regulated by QS system.

摘要

MvaT和MvaU是属于H-NS家族的全局转录调节因子，而花青素是铜绿假单胞菌产生的重要毒力因子。铜绿假单胞菌PAO1的mvaT mvaU双敲除突变体在先前的研究中证明了花青素的消除。在这里，我们进一步探讨了该机制。研究了两个主要方向：绿脓素的生物合成途径和QS系统。通过启动子强度测定和实时PCR测定评价了对绿脓素生物合成基因表达的影响，发现了导致绿脓素产量低的显着变化。通过使用LC-MS / MS进行信号分子定量分析以及使用实时PCR进行相关基因表达测量来研究对QS系统的影响。在移动中两次敲除，3-oxo-C12-HSL的产量明显增加，而C4-HSL和PQS的产量明显减少，并且可以通过mvaT或mvaU互补来恢复其变化。与QS系统信号分子结合的转录激活基因的表达均降低，导致信号转录激活复合物的形成减少。rhlR和pqsE的表达降低也导致phzA1和phzA2的表达降低。进一步的研究发现，QS系统下调可能与QsrO（QS系统阻遏物）有关，而QsrO被mvaT mvaU上调双重淘汰赛。因此，脓余素的合成被窒息并且生物膜形成能力降低。转录组分析也证实了这些结果，该转录组分析证明了上述基因的相似基因表达变化以及受QS系统调节的其他毒力因子基因的表达降低。