Molecular mechanisms of enterobacterial resistance to complement

补体（C'）由30多种蛋白质组成，主要位于血液室中，有助于保护宿主免受微生物入侵。C’能够杀死许多革兰氏阴性菌，包括目前在医院和社区构成严重健康威胁的抗生素耐药菌株。细菌表面上的C'途径被表面结合抗体或对细菌表面外源性质的识别激活，导致形成称为C5b-9复合物的多蛋白组合，其插入细菌细胞壁的最外层，即外膜（OM）。C5b-9复合物的稳定插入破坏了OM双分子层的完整性，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GNB已经找到了一种抵抗C‘攻击的方法，要么通过阻止C’激活，要么通过确保在C5b-9复合物形成之前C'组分的降解，要么通过精心设计一种阻止C5b-9稳定插入到OM的脂质区域的OM。我们对C'耐药基础的大部分知识来自30多年前的观察，主要集中在鉴定细菌表面的大分子结构，如多糖胶囊和脂多糖o侧链，这些结构有助于耐药。这些研究几乎没有尝试提供细菌表面形貌的综合图像，这可能解释为什么C5b-9复合物不能以稳定的方式插入细菌OM。尽管有新的见解从根本上改变了我们对革兰氏阴性OM的组织结构的理解，但最近在澄清C耐药基础方面没有取得重大进展。我们首次提出采用最先进的结构，生物物理和基于显微镜的方法来详细确定定义C'耐药病原体（所谓的耐药表型）的细菌表面形貌。由于GNB已经进化出多种机制来阻止有效的C‘攻击，我们将采用一些临床分离的大肠杆菌和肺炎克雷伯菌，它们表达各种已知的表面大分子，以不同的方式影响C’活化和C5b-9沉积。我们将通过筛选由转座子定向插入测序（TraDIS）技术产生的大量突变体来确定导致C'耐药的细菌成分，然后生成一系列在候选耐药决定因子合成中有缺陷的突变体，以确定它们对耐药表型的贡献。我们将检查临床菌株及其突变体阻止C‘活化和结合可能阻止C5b-9形成的C’抑制剂蛋白的能力。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'易感GNB进行比较。我们将把这些数据整合到等高线图中，以提供细菌表面的详细地形。最后，缺乏表面结构的突变体的能力，我们已经确定有助于抵抗，将在我们内部开发的细菌感染啮齿动物模型中进行检查；这种体内工作将确定主要的C'耐药决定因素对细菌引起致命感染的能力的贡献。

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.