聚羟基脂肪酸影响铜绿假单胞菌PAO1绿脓菌素合成的调控机理研究

Many pathogenic bacteria existing in the natural environment are able to produce polyhydroxyalkanoates as carbon and energy storage material in their cells. The opportunistic pathogen Pseudomonas aeruginosa PAO1 has the ability to accumulate PHA. Our preliminary data indicate that the mutant strain deficient in accumulating PHA has been found to also have a decreased level of pyocyanin (PYO) secreted to the culture media. The biosynthesis of pyocyanin is dependent on four independent gene clusters. The two redundant gene clusters phz1 and phz2 encode proteins with little difference in protein sequences but bear distinct promoter regions. The study of regulation on phzM and phzS is also limited. In this study, it is proposed to investigate how the deficiency in PHA impacts the production of pyocyanin in the PHA mutant strain, and further elucidate the molecular mechanism of PHA regulating the gene clusters involving pyocyanin production and reveal the biochemical and physiological role of PHA in opportunistic pathogens. The study on how PHA influence the physiological and pathogenic function will not only broaden the scope of the metabolism in pathogenic bacteria, but also provide possible approach for the control and treatment of PAO1 infections.

存在于自然环境中的许多病原菌都能合成聚羟基脂肪酸(PHA)作为碳源与能源储存物积累于胞内，PHA在这些病原菌中生理功能和与致病性的关系尚未见研究。铜绿假单胞菌PAO1是重要的机会性致病菌，同时也具有合成PHA的能力。我们的前期实验证明，当PHA合成基因缺失时，PAO1菌株中重要的毒力因子绿脓菌素的水平显著降低。绿脓菌素的合成途径依赖于四个基因簇，且都与PHA合成基因簇相隔XXkb，排除了发生顺式突变的可能。其中有两个重复的基因簇phz1和phz2，虽然编码的蛋白几乎完全一致，但其操纵子完全不同。另外两个基因簇phzM和phzS的调控机理也尚未阐明。本项目拟研究在PAO1菌株中，绿脓菌素的合成是如何受到PHA影响的，揭示PHA影响绿脓菌素合成的调控机理，以阐明PHA在机会性致病菌中的生理及病理作用。该研究不仅丰富了对病原菌生理代谢的认识，也为预放与治疗病原菌PAO1感染提供了新的可能途径。

本项目研究了在机会致病菌铜绿假单胞菌PAO1菌株中，碳源与能源储存物质聚羟基脂肪酸（PHA）的合成如何影响毒力因子绿脓菌素（PYO）合成，并对该机制进行了初步挖掘。研究发现，当敲除PAO1菌株中的PHA聚合酶基因后，该菌生长速率没有明显改变，但PYO的分泌量有显著的下降（下调约70%左右），回补相关基因后能恢复该表型。通过鉴定PYO合成基因簇的转录水平，发现在敲除菌株中，phzA1B1C1D1E1F1G1（以下简称phz1）和phzA2B2C2D2E2F2G2（以下简称phz2）两个独立的基因簇转录水平与野生型相比有明显差异，确定了PYO水平的差异来自于基因转录调控。进一步通过比较转录组测序的分析，鉴定到了在敲除菌株中转录水平与野生型差异显著的基因多达700多个，其中包含了同时卷入PHA代谢和PYO合成途径的转录调控因子的基因rsmA，并对其转录水平进行了验证。最后我们尝试了对rsmA基因进行敲除，同时制备了抗RsmA的兔多克隆抗体，进行了免疫共沉淀等试验验证其与phz1或phz2启动子的相互作用。