Structure and function of the Pseudomonas aeruginosa type VI secretion system: On the bacteriophage trail

细菌病原体通过各种方式感染人类。它们会产生急性感染，在短时间内对宿主致命，但它们也会产生慢性感染，并在宿主体内持续一生。这就是囊性纤维化（CF）患者肺部感染的例子。在感染的早期阶段，可以鉴定出多种细菌，包括金黄色葡萄球菌、洋葱伯克霍尔德菌或铜绿假单胞菌。在CF患者生命的后期，留在肺部的唯一微生物是铜绿假单胞菌，它是牢固而长期建立的，并将导致患者死亡。铜绿假单胞菌是一个强有力的病原体参与许多感染，不仅在CF患者。它是第三大最常被隔离的医院病原体，占医院获得性感染的10%，英国每年有10,000例病例。感染是危及生命的，感染的范围很广，包括尿路、呼吸系统、皮炎、软组织、菌血症、骨骼和关节、肠道以及各种全身感染，特别是免疫抑制患者。铜绿假单胞菌感染的治疗涉及联合治疗，例如β -内酰胺加氨基糖苷或氟喹诺酮，但效果不如预期。替代药物（如粘菌素）对多重耐药菌株是有用的，但未来的创新治疗选择仍然很少，而开发疫苗的尝试也没有成功。为了开发新的治疗方法，确定分子靶点是很重要的，这些靶点一旦被抑制就会损害铜绿假单胞菌的成功感染。选择的目标通常是一个主要的毒力因素。分泌系统是细菌用来释放酶和毒素的分子装置，这将有助于定植，宿主组织降解，持久性或免疫系统逃逸。这些分泌系统是嵌在细菌包膜中的分子复合物，由许多蛋白质成分组成。我们研究的系统被称为VI型分泌系统（T6SS），由13个核心成分组成。我们选择这个系统是因为它是铜绿假单胞菌建立慢性感染所必需的，并且它在重要的细菌病原体中是保守的。我们选择研究7种T6SS成分（HsiABCEFGH），我们怀疑它们构成了分泌机制的核心。我们有证据表明，这些成分与细菌病毒（即噬菌体）的成分有相似之处。由于我们对噬菌体的工作原理了解很多，我们希望了解T6SS和噬菌体之间的关系将有助于了解T6SS是如何工作的。噬菌体和T6SS同源物的初级序列已经分化到如此程度，以至于很难确定共同的祖先。虽然进化可以导致同源蛋白的不同的初级序列，结构往往是保守的。我们的目标是通过使用多学科方法，如x射线晶体学，核磁共振和冷冻电子显微镜来确定T6SS蛋白的结构。我们将解决单个组件和子复合物的结构。这些结构将提供有关T6SS机器如何组织的分子见解，并将为旨在评估T6SS功能和组装的定点诱变研究提供信息。T6SS机器的扰动将通过一套独特的测试进行评估，使我们能够探测机器是否正确组装，是否能够运输毒素，以及是否能够将毒素输送到目标细胞中。这些知识随后将用于设计潜在的小分子抑制剂，以T6SS为靶点，从而改变T6SS的功能，防止铜绿假单胞菌的定植和毒力。由于T6SS在其他细菌性病原体中是保守的，我们的研究将适用于其他细菌性疾病的潜在预防。

Bacterial pathogens infect humans using various strategies. They develop acute infections, which turn fatal to the host in a short period of time, but they also establish chronic infections and persist within the host over a lifetime. That is for example the case for lung infections in cystic fibrosis (CF) patients. At early stages of infection, diverse bacteria can be identified, including Staphylococcus aureus, Burkholderia cepacia or Pseudomonas aeruginosa. At later stages in the CF patient life the sole microorganism left in the lung is P. aeruginosa, which is firmly and chronically established and will lead to patient death.P. aeruginosa is a potent pathogen involved in many infections, not only in CF patients. It is the 3rd most commonly-isolated nosocomial pathogen accounting for 10% of hospital-acquired infections, with 10,000 cases each year in UK. Infection is life threatening and the range of infection is broad, including urinary tract, respiratory system, dermatitis, soft tissue, bacteremia, bone and joint, intestine and a variety of systemic infections, particularly in immuno-suppressed patients. Treatment of P. aeruginosa infections involves combination therapies, e.g. a beta-lactam plus an aminoglycoside or a fluoroquinolone, but are not as efficient as desired. Alternative drugs (e.g., colistin) are useful against multi-resistant strains, but innovative therapeutic options for the future remain scarce, while attempts to develop vaccines have been unsuccessful.To develop new therapies it is important to identify molecular targets, which once inhibited will compromise successful infection by P. aeruginosa. The target of choice is usually a major virulence factor. Secretion systems are molecular devices that bacteria use to release enzymes and toxins, which will contribute to colonization, host tissues degradation, persistence or immune system escape. These secretion systems are molecular complexes embedded in the bacterial envelope and are made up of a number of protein components.The system we study is called the type VI secretion system (T6SS) comprising 13 core components. We selected this system because it is essential for P. aeruginosa to establish chronic infections and it is conserved in important bacterial pathogens. We chose to study 7 of the T6SS components (HsiABCEFGH), which we suspect form the core of the secretion machine. We have evidence that these components share similarities with components of bacterial viruses, i.e. bacteriophages. Since a lot is known on how bacteriophages work, we hope that understanding the correlation between T6SS and bacteriophage will shed light on how the T6SS works. The primary sequences for the phage and T6SS homologues have diverged to such an extent, that it is difficult to identify common ancestors. Although evolution can result in divergent primary sequences for homologous proteins, the structures are often conserved. Our goal is to determine the structures of T6SS proteins by using multidisciplinary approaches such as X-ray crystallography, NMR and cryo-electron microscopy. We will solve the structures of individual components and sub-complexes. The structures will provide molecular insights into how the T6SS machine is organised and will inform site directed mutagenesis studies aimed at assessing T6SS function and assembly. Perturbation in the T6SS machine will be assessed by a unique set of tests allowing us to probe whether the machine is properly assembled, able to transport toxins, and able to deliver toxins into target cells. This knowledge will later be used to design potential small molecule inhibitors that target the T6SS machine and by doing so alter T6SS function and prevent P. aeruginosa colonization and virulence. Since the T6SS is conserved in other bacterial pathogens our research will be applicable in the potential prevention of other bacterial diseases.