Form and function in the ESX-1 secretion system; elucidation of mechanism and structural determinants of bacterial virulence.

几乎所有的细菌都是通过产生毒素而致病的，这些毒素会分泌到宿主体内。这些毒素中有许多是蛋白质分子，细菌的细胞膜上有专门的机器，可以控制这些毒素进入体外。我们正在研究其中一台机器；最近发现的ESX-1系统试图了解微生物学的一个核心方面，即蛋白质分泌机器是如何工作的。该ESX-1系统已被证明对重要病原体（结核分枝杆菌、炭疽杆菌和金黄色葡萄球菌）的毒力至关重要，因此该基础研究具有生物医学和农业方面的意义。我们已经确定，金黄色葡萄球菌ESX-1似乎是最易于处理的结构-功能研究。金黄色葡萄球菌是一种共生的革兰氏阳性细菌，导致人类和动物的许多疾病，从轻微的皮肤感染（如丘疹和脓肿）、中毒性休克综合征（TSS）到奶牛群和猪的败血症和乳腺炎。然而，最广为人知的是它是医院获得性感染的主要原因，也是术后伤口感染的常见原因。一些金黄色葡萄球菌菌株对许多抗生素具有耐药性（例如耐甲氧西林金黄色葡萄球菌），使这种情况更加恶化，使其成为一个严重且难以治疗的问题。金黄色葡萄球菌感染导致的死亡人数与获得性免疫缺陷综合征导致的死亡人数相当。如果我们要设计新的治疗方法来预防或治愈感染，在分子水平上理解金黄色葡萄球菌的生物学和发病机制是必不可少的。ESX-1转运系统驱动金黄色葡萄球菌至少三种不同蛋白质的分泌，已知当该系统失活时，金黄色葡萄球菌引起感染的能力急剧下降。我们寻求这种独特的细菌分泌机器的结构和功能的知识，并鉴定其货物。ESX-1系统由5-8种不同的蛋白质组分组成。我们将研究这些蛋白质如何相互作用来组装分泌机器，以及机器如何工作的分子基础。为了实现这一目标，我们必须推导出每个组件的结构和功能，阐明组件如何相互作用并量化关联，确定货物识别的分子基础，以及动力的产生。我们已经取得了重大进展，其中六种蛋白质被克隆、表达和纯化，两种被结晶。我们将利用单晶x射线衍射方法来获得精确的分子结构和一系列生物物理技术来表征蛋白质-蛋白质相互作用。了解蛋白质成分的结构以及它们之间的相互作用是很重要的，因为从长远来看，这可能为直接设计或发现化合物的研究奠定基础，这些化合物可以阻止这些蛋白质相互作用，或者阻止原动力被用来分泌出建立和延长感染的蛋白质。有关分泌的蛋白质和在细菌表面发现的结构的信息也可能为疫苗设计提供机会。

Almost all bacteria cause disease by producing toxins that they secrete into the host. Many of these toxins are protein molecules, and the bacterium has specialised machines in the cell membrane that allows the controlled passage of these toxins to the outside. We are working on one of these machines; the recently discovered ESX-1 system seeking to understand a central aspect of microbiology, namely how protein secretion machines work. This ESX-1 system has been shown to be essential for the virulence of important pathogens (M. tuberculosis, B. anthracis and S. aureus) and so there is a biomedical and agricultural aspect to this basic research. We have identified that the Staphylococcus aureus ESX-1 appears to be the most tractable for structure-function studies. S. aureus is a commensal Gram positive bacterium responsible for a number of illnesses in humans and animals that range from minor skin infections, such as pimples and abscesses, Toxic shock syndrome (TSS) through to septicaemia and mastitis in dairy herds and pigs. However, it is most widely known as a major cause of hospital acquired infections, and is a frequent cause of post-surgical wound infections. This situation is exacerbated by the fact that some strains of S. aureus are resistant to many antibiotics (e.g. Methicillin Resistant Staphylococcus aureus), making it a severe and difficult to treat problem. The number of deaths attributed to S. aureus infection is comparable to that attributed to acquired immune deficiency syndrome. An understanding, at the molecular level, of S. aureus biology and pathogenesis is essential if we are to design new treatments to prevent or cure infection. The ESX-1 transport system drives the secretion of at least three different proteins from S. aureus, and it is known that when this system is inactivated S. aureus shows a dramatic reduction in its ability to cause infection. We seek knowledge of the architecture and function of this distinctive bacterial secretion machine and identification of its cargo. The ESX-1 system consists of 5-8 different protein components. We will investigate how these proteins interact to assemble the secretion machine, and the molecular basis for how the machine works. To attain this goal we must derive the structure and function of each component, elucidate how the components interact and quantify the association, determine the molecular basis for cargo recognition, and the generation of the motive force. We have already made significant progress with six of these proteins cloned, expressed and purified and two crystallised. We will exploit single crystal X-ray diffraction methods to derive accurate molecular structures and a battery of biophysical techniques to characterise the protein-protein interactions. Knowing the structures of the protein components and how they interact with each other is important because this might, in the longer term, lay the foundation for studies directed to the design or discovery of compounds that will prevent these proteins from working with each other or prevent the motive force from being used to secrete out the proteins that establish and prolong infection. Information on proteins that are secreted and of the structures found on the surface of the bacterium may also provide opportunities for vaccine design.