铜绿假单胞菌中GGDEF-EAL结构域基因PA0861的生物学功能及作用机制研究

Pseudomonas aeruginosa is Gram negative bacteria that can colonize in diversified environments. In human, the versatility enables this opportunistic pathogen to infect patients with impaired immunity. Due to the increasing antibiotics resistance, infections of Pseudomonas aeruginosa in hospitals are rising, with certain cases fatal. .Recently, it has been established that, bis-(3'-5')-cyclic dimeric guanosine monophosphate (cylic-di-GMP), a novel secondary messenger, is playing a pivotal role in regulation of various cellular processes, including virulence, quorum sensing, cell cycle and development, in bacteria. Especially, c-di-GMP controls the bacteria lifestyle transition from a free-living, motile manner to a biofilm one, which may account for many cases of antibiotic-refractory, chronic and recurring infection..Furthermore, it has been disclosed that, GGDEF and EAL domain-containing proteins are of key importance in c-di-GMP signaling pathway. Named after the conservative amino acid residues of Gly-Gly-(Asp/Glu)-Glu-Phe and Glu-Ala-Lue, GGDEF and EAL domain-containing proteins are found not only to be involved in synthesis and degradation of c-di-GMP, but also to bind to c-di-GMP and sense the changes of the cellular concentration of this small molecule. They are thus engaged in the complicated c-di-GMP signaling cascade..There are 38 GGDEF and/or EAL domain-containing proteins in P. aeruginosa PAO1, with 16 containing both GGDEF and EAL domains. Some of these GGDEF-EAL domain concatenated-proteins are identified and characterized, such as FimX, BifA and MorA; while the rest are so-called 'hypothetical proteins' and have not been analyzed till now. .From our preliminary studies, PA0861, a membrane protein from P. aeruginosa, is composed of PASPAC, GGDEF and EAL domains. Interestingly, the transposon insertion mutant of this gene shows the phenotype of decreased biofilm formation. .In current application, we aim to employ biochemical, cell biological and structural approaches to fully characterize PA0861, both functionally and structurally. We plan to identify its catalytic activity; decide its cellular localization; analyze its biological effects in biofilm formation; and attempt to understand the relevant molecular mechanism in signaling. Hopefully our work could thus help to control the infection of P.aeruginosa in the future.

铜绿假单胞菌(Pseudomonas aeruginosa)属革兰氏阴性菌,分布广泛，在临床上具显著危害。目前已知，含GGDEF及EAL结构域的蛋白与小分子第二信使环鸟苷二磷酸（C-di-GMP）的代谢及其细胞信号转导过程有密切关系；而环鸟苷二磷酸在细菌的运动性、耐药性、细菌毒性，尤其在生物膜合成等生理过程中有重要作用。铜绿假单胞菌PAOl基因组中共有38个含GGDEF及EAL结构域的基因，仅有部分得到了深入研究。我们的前期工作中，发现尚未经分析鉴定的基因PA0861由PASPAC、GGDEF及EAL结构域组成，且其转座子突变体导致细菌的生物膜合成减少。在本项申请中，我们计划从催化活性、细胞定位、其影响生物膜合成的具体信号通路及分子机制等方面，对铜绿假单胞菌PAOl的GGDEF-EAL结构域基因PA0861作出分析，以期加深对环鸟苷二磷酸作用规律的认识，并能够利于未来控制铜绿假单胞菌感染。

在本项目执行过程中，我们表达了PA0861重组蛋白；对PA0861突变菌株进行了表型分析；利用表达谱比较了野生型PAO1及PA0861突变菌株在基因表达水平上的差异。我们发现，一些涉及铁元素摄入的铁载体基因在PA0861突变菌株中表达上调。作为环鸟苷二磷酸代谢蛋白，PA0861影响环鸟苷二磷酸的胞内水平；由此初步推断，PA0861可能通过小分子第二信使环鸟苷二磷酸，调控铁载体的合成，进而影响细菌铁的摄入，最终使得生物膜合成发生改变。通过本项研究，为环鸟苷二磷酸的生物学功能、作用机制及可能的信号通路提供了新的数据及资料。