Molecular mechanisms of enterobacterial resistance to complement

补体（C '）包括主要位于血液隔室中的三十多种蛋白质，其帮助保护宿主免受微生物入侵。C'能够杀死许多革兰氏阴性细菌（GNB），包括目前在医院和社区中构成严重健康威胁的抗生素耐药菌株。通过表面结合的抗体或通过识别细菌表面的外来性质，在细菌表面上激活C'途径，导致形成多蛋白质聚集体，称为C5 b-9复合物，其插入细菌细胞壁的最外层，外膜（OM）。C5 b-9复合物的稳定插入破坏了OM双层的完整性，导致内膜、细胞质膜的扰动和细菌细胞死亡，其机制尚不完全清楚。不幸的是，许多巨人银行已经找到了抵抗C'攻击的方法，要么通过阻止C'激活，通过确保在形成C5 b-9复合物之前降解C'组分或通过制备防止C5 b-9稳定插入的OM，我们对C'抗性基础的大部分知识来自三十多年前的观察，并专注于鉴定大分子细菌表面的结构，如多糖胶囊和脂多糖O-侧链，有助于抵抗。这些研究几乎没有试图提供一个完整的图片的细菌表面形貌，可能解释为什么C5 b-9复合物不插入到细菌OM稳定的方式。尽管有新的见解从根本上改变了我们对革兰氏阴性OM组织的理解，但最近在澄清C'抗性的基础方面没有重大进展。我们建议，第一次，采用最先进的结构，生物物理学和显微镜为基础的方法来确定，在细节，细菌表面形貌，定义C '-耐药病原体（所谓的耐药表型）。由于GNB已经进化出多种防止有效C'攻击的机制，我们将采用大肠杆菌和肺炎克雷伯氏菌的许多临床分离株，其表达已知以不同方式影响C'活化和C5 b-9沉积的多种表面大分子。我们将通过筛选由称为转座子定向插入测序或TraDIS的技术产生的突变体的大型文库来确定负责C'抗性的细菌组分，然后产生一系列在候选抗性决定簇的合成中有缺陷的突变体，以确定它们对抗性表型的贡献。我们将检查临床菌株及其突变体阻止C'激活和结合可能阻止C5 b-9形成的C'抑制蛋白的能力。我们将使用内部定制的分子探针以及市售抗体，通过荧光显微镜观察细菌表面的主要成分，并将其分布和丰度与突变体和C '-敏感GNB的分布和丰度进行比较。我们将把这些数据合并成等高线图，以提供细菌表面的详细地形图。最后，将在我们内部开发的细菌感染的啮齿动物模型中检查缺乏我们已经鉴定为有助于抗性的表面结构的突变体的能力;这种体内工作将确定主要C'抗性决定簇对细菌引起致死性感染的能力的贡献。

Complement (C') comprises over thirty proteins located predominantly in the blood compartment that help defend the host against microbial invaders. C' is able to kill many Gram-negative bacteria (GNB), including antibiotic resistant strains that currently pose a severe health threat in hospitals and in the community. C' pathways are activated on bacterial surfaces, either by surface-bound antibodies or by recognition of the foreign nature of the bacterial surface, leading to the formation of multi-protein assemblages, termed C5b-9 complexes, that insert into the outermost layer of the bacterial cell wall, the outer membrane (OM). Stable insertion of C5b-9 complexes disrupts the integrity of the OM bilayer leading to perturbation of the inner, cytoplasmic membrane and bacterial cell death by mechanisms that are not completely understood. Unfortunately, many GNB have found a way to resist C' attack, either by preventing C' activation, by ensuring degradation of C' components prior to formation of C5b-9 complexes or by elaboration of an OM that prevents stable insertion of C5b-9 into lipid regions of the OM.Much of our knowledge of the basis of C' resistance comes from observations made over thirty years ago and focused on identifying the macromolecular structures at the bacterial surface, such as polysaccharide capsules and lipolysaccharide O-side chains, that contribute to resistance. These studies made little attempt to provide an integrated picture of bacterial surface topography that might explain why C5b-9 complexes do not insert in stable fashion into the bacterial OM. There has been no significant recent progress towards clarifying the basis of C' resistance in spite of new insights that have fundamentally changed our understanding of the organisation of the Gram-negative OM. We propose, for the first time, to employ state-of-the-art structural, biophysical and microscopy-based approaches to determine, in fine detail, the bacterial surface topography that defines C'-resistant pathogens (the so-called resistance phenotype). As GNB have evolved a variety of mechanisms that prevent effective C' attack, we will employ a number of clinical isolates of Escherichia coli and Klebsiella pneumoniae that express a variety of surface macromolecules known to affect C' activation and C5b-9 deposition in different ways. We will determine the bacterial components responsible for C' resistance by screening large libraries of mutants generated by a technique known as Transposon Directed Insertion Sequencing, or TraDIS, and then generate a range of mutants defective in the synthesis of candidate resistance determinants to define their contribution to the resistant phenotype. We will examine the capacity of the clinical strains and their mutants to prevent C' activation and to bind C' inhibitor proteins that might prevent C5b-9 formation. We will use molecular probes that have been tailored in-house, as well as commercially available antibodies, to visualise by fluorescence microscopy the principal components of the bacterial surface and compare their distribution and abundance with that of the mutants and C'-susceptible GNB. We will consolidate this data into contour maps to provide a detailed topography of the bacterial surface. Finally, the capacity of mutants lacking surface structures that we have identified as contributing to resistance will be examined in rodent models of bacterial infection that we have developed in-house; such in vivo work will define the contribution of major C' resistance determinants to the capacity of the bacteria to cause lethal infection.