幽门螺旋杆菌亚精胺合成的替代路径研究

Spermidine and other polyamines are small molecules important for bacterial pathogenicity. Although Helicobacter pylori has spermidine synthetase SpeE in the classical pathway that is commonly used for spermidine synthesis, it lacks other enzymes in this pathway. Our previous work shows that H. pylori SpeE binds to flagellar motor protein and regulates the mobility of flagella. However, it does not show detectable activity for spermidine synthesis. Interestingly, H. pylori possesses all the enzymes in an alternative pathway including carboxyspermidine decarboxylase CASDC. Our preliminary result shows that CASDC catalyzes the production of spermidine in vitro. In this study, we will employ biochemistry, biophysics and molecular genetics approaches to further confirm and characterize the role of CASDC in H. pylori spermidine synthesis and in its pathophysiology. Specifically, we will prepare CASDC and characterize its enzymatic properties, study its oligomerization states under different pH and determine their structures. Meanwhile, we will also construct casdc-null strain, determine its spermidine content and study its role in H. pylori acidic adaptation and biofilm formation. Our research will reveal new mechanism of spermidine synthesis in H. pylori and its roles in pathogenicity.

亚精胺等多胺类小分子对于病原微生物致病性有重要作用。绝大多数生物利用经典路径中的亚精胺合成酶SpeE合成亚精胺。幽门螺旋杆菌虽然拥有SpeE，却缺乏此路径中其他酶类。我们之前的工作表明幽门螺旋杆菌SpeE可与鞭毛马达蛋白结合并调节鞭毛的运动性，但它却没有亚精胺合成酶的活性。同时，幽门螺旋杆菌拥有包括羧基亚精胺脱羧酶CASDC在内的替代路径中的所有酶类。我们的初步结果显示CASDC可在体外催化生成亚精胺。在此项研究中，我们将利用生物化学、生物物理学及分子遗传学等方法进一步验证及表征CASDC在幽门螺旋杆菌亚精胺合成及致病生理中的功能。具体而言，我们将制备CASDC并表征其酶学特性、研究它在不同pH下的聚合状态并测定其结构。同时，我们将构建幽门螺旋杆菌casdc-null突变株、测定其体内亚精胺含量并研究它在酸适应性及生物被膜形成等方面的能力。我们的研究将揭示幽门螺旋杆菌合成亚精胺的全新机制。

幽门螺杆菌与胃炎、胃癌等疾病的发生发展密切相关，了解其致病机制将有助于相关疾病的预防与治疗。在本项目中，我们以幽门螺杆菌中亚精胺代谢替代路径中的羧基亚精胺脱羧酶CASDC为研究对象，深入探究其结构及功能特性。主要研究结果如下：1，通过分子克隆、蛋白质工程等手段制备了野生型及其突变体CASDC。2，通过X射线晶体学手段测定了CASDC的高分辨率结构。3，通过结构分析鉴定了底物结合位点及催化位点等关键氨基酸。4，通过负染色、高效液相色谱等技术表征了CASDC在不同pH下的形态变化及功能活性。5，通过分子对接技术筛选了可调控CASDC功能的小分子化合物，并分析了其可能的作用方式。本研究的成果有助于理解幽门螺杆菌在亚精胺代谢上的生理机制，并为幽门螺杆菌的预防及治疗提供参考。