Post-translational regulation of the exopolysaccharide alginate in Pseudomonas aeruginosa

机会致病菌铜绿假单胞菌引起遗传性疾病囊性纤维化（CF）患者危及生命的肺部感染。在患者肺部的黏液分泌物中，铜绿假单胞菌形成富含海藻酸盐的生物膜，这是导致慢性感染的原因。P. aeruginosa在常氧条件下不表达藻酸盐外多糖，但在CF粘液中存在的低氧条件下，其表达量大幅增加。该项目的中心是阐明一种新的共价酶修饰机制，我们已经发现并差异调节P. aeruginosa中海藻酸盐的产生。遵循两种互补的工作策略。首先，通过形成第二信使环双-(3-5)-二胍酸酯单磷酸（c-di-GMP）激活海藻酸盐表达的二胍酸酯环化酶（DGC） PA4332被纯化为甘露糖结合蛋白加合物（PA4332- mbp），并分别与纯化的羟化酶/还原酶PA4331和脱水酶PA4330孵育。P4332-MBP中Pro-410共价修饰的证据已经获得（羟脯氨酸，脱氢脯氨酸）。这些修饰将在体外用高效液相色谱和质谱分析；将研究反应的动力学和必要的辅助因素，并评估反应伙伴的物理相互作用。其次，在铜绿假单胞菌（P. aeruginosa）菌株PAO1及其等基因突变体PA4331和PA4330中，PA4332- MBP将被过表达、分离并使用相同的方法进行共价蛋白修饰研究。最后，我们研究了DGC PA4332与靶蛋白Alg44的物理相互作用，并询问其他细菌物种的DGCs是否以类似的翻译后方式受到调节。新的共价酶修饰机制为铜绿假单胞菌快速适应不断变化的环境条件提供了一个重要的已知现象。

The opportunistic pathogen Pseudomonas aeruginosa causes life threatening lung infections in patients with the hereditary disease cystic fibrosis (CF). In the viscous mucus secretions of the patients’ lungs, P. aeruginosa forms alginate-rich biofilms, which are held responsible for the chronicity of the infections. P. aeruginosa does not express the exopolysaccharide alginate under normoxic conditions, however its expression is highly increased under low oxygen conditions which are present in the CF mucus. In the centre of this project is the elucidation of a novel covalent enzyme modification mechanism, which we have discovered and which differentially regulates alginate production in P. aeruginosa. Two complementary work strategies are followed. Firstly, the diguanylate cyclase (DGC) PA4332 which activates alginate expression via the formation of the second messenger cyclic bis-(3-5)-diguanylate monophosphate (c-di-GMP) is purified as a mannose binding protein adduct (PA4332-MBP) and incubated with the purified hydroxylase/reductase PA4331 and the dehydratase PA4330, respectively. Evidence for covalent modifications of Pro-410 in P4332-MBP has already been obtained (hydroxyprolin, dehydroproline). These modifications will be analyzed in vitro using HPLC und mass spectroscopy; the kinetic and essential co-factors of the reactions will be investigated and the physical interaction of the reaction partners is assessed. Secondly, in the P. aeruginosa strain PAO1 and in its isogenic PA4331 and PA4330 mutants the PA4332- MBP will be over-expressed, isolated and investigated for covalent protein modifications using the same methods. Finally, we investigate the physical interaction of the DGC PA4332 with the target protein Alg44 and ask whether DGCs of other bacterial species are similarly regulated in a post-translational manner. The novel covalent enzyme modification mechanism provides an important example for the known phenomenon of the rapid adaptation of P. aeruginosa to changing environmental condi.