Structural Studies of Lymphostatin

肠道致病性大肠杆菌(EPEC)和肠出血性大肠杆菌(EHEC)变异株可引起肠道和全身疾病。EPEC是婴儿腹泻的主要原因，并在人与人之间直接传播。相比之下，人类感染EHEC经常是由于接触反胃动物的粪便引起的，通常涉及血性腹泻和危及生命的肾脏并发症。淋巴抑制素是EPEC和大多数EHEC菌株共同的关键毒力因子之一，它是迄今在大肠杆菌中发现的最大的蛋白质。淋巴抑制素似乎是一种多功能、多结构域的蛋白质，既能抑制淋巴细胞的功能，又能促进细菌与宿主细胞的附着。其中一个结构域是位于蛋白质N端的糖基转移酶(GT)结构域。在其他细菌毒素中也发现了类似的GT结构域，如黄曲霉毒素。保守的GT结构域只占淋巴抑素的一小部分，而淋巴抑素的C末端部分与革兰氏菌毒素没有同源性。我们认为淋巴抑素在感染的不同阶段通过构象转换和自身蛋白分解来激活和失活某些功能。在这里，我们建议通过电子冷冻显微镜和图像处理来确定淋巴抑素及其N-端和C-端片段的结构，以验证这一假说。这些研究将显示GT-结构域在全长蛋白中的位置，以及GT-结构域的淋巴抑素特异性延伸是否处于改变底物结合部位或不同结构域之间的结构联系的战略位置。片段和全长淋巴抑素之间的结构比较将启发关键部位的掩蔽和去掩蔽是否是自身蛋白水解酶功能转换的潜在机制。有证据表明淋巴抑素定位于细菌膜，但它如何影响这些膜尚不清楚。为了解决这个问题，我们将淋巴抑素重组为脂质体，并通过低温电子断层扫描和亚层析平均来研究蛋白质脂质体。这些研究将表明淋巴抑素是否只是修饰细胞膜，从而形成其他因子的潜在结合平台，或者它是否通过形成毛孔或病变来调节细胞膜。综上所述，我们拟议的研究将揭开淋巴抑素的整体结构及其功能模块之间的相互作用。我们期望淋巴抑素通过简单的结构变化和自身蛋白分解来激活或去激活某些功能，使其成为一种多用途工具。对这些机制的详细结构知识对于干扰淋巴抑素的作用，从而减少未来EHEC和EPEC感染的破坏性影响将是至关重要的。

Enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) variants cause intestinal and systemic diseases. EPEC are a leading cause of infant diarrhoea and transmit directly between humans. By contrast EHEC infections in humans frequently arise from contact with ruminant faeces and often involve bloody diarrhoea and life-threatening renal complications. One of the key virulence factors common to EPEC and most EHEC strains is lymphostatin, which is the largest protein yet identified in E. coli. Lymphostatin appears to be a multifunctional, multidomain protein serving both, to inhibit the function of lymphocytes and to promote bacterial attachment to host cells. One of the domains is a glycosyltransferase (GT) domain in the N-terminal half of the protein. Similar GT-domains are also found in other bacterial toxins such as the Chlostridial toxins. The conserved GT-domain accounts for only a small fraction of lymphostatin, while the C-terminal part of lymphostatin shows no homology to the Chlostridial toxins. We think that lymphostatin activates and deactivates certain functionalities by conformational switching and by auto-proteolysis during different stages of infection.Here we propose to test this hypothesis by structure determination of lymphostatin and its N- and C-terminal fragments with and without bound substrate by electron cryo microscopy and image processing. These studies will show where the GT-domain is located in the full-length protein and whether the lymphostatin-specific extensions of the GT-domain are at strategic positions to either modify the substrate binding site or alternatively the structural communication between different domains. Structure comparison between fragments and full-length lymphostatin will enlighten if masking and unmasking of key sites are potential mechanisms of functional switching by auto-proteolysis.There is evidence that lymphostatin is localized at the bacterial membranes but it is unknown how it affects these membranes. To address this question, we will reconstitute lymphostatin into liposomes and study the proteoliposomes by cryo electron tomography and subtomogram averaging. These investigations will show whether lymphostatin simply decorates the membranes and thus forms a potential binding platform for other factors or whether it modulates the membranes, e.g. by forming pores or lesions. Taken together, our proposed investigations will unravel the overall architecture of lymphostatin and the interplay between its functional modules. We expect that lymphostatin is modulated by simple structural changes and auto-proteolysis to activate or deactivate certain functions making it a multi-purpose tool. A detailed structural knowledge on these mechanisms will be essential for interfering with the action of lymphostatin and thus reducing the devastating effects of EHEC and EPEC infections in future.