脂多糖是内毒素吗?

脂多糖是革兰阴性菌细胞壁上的一种重要结构,同时也和细菌的致病作用紧密联系,但长期以来直接将脂多糖定义为细菌内毒素,形成了医学细菌学教学中的一个重大误区,可能对细菌致病作用的理解形成学习上的障碍,故就此提出一些见解,以供探讨。     

细菌类脂A结构与功能研究进展

类脂A分布在革兰氏阴性细菌的外表层,它是脂多糖分子的疏水基团.脂多糖,俗名内毒素,可以引起致命的脓毒症、内毒素血症和多器官功能障碍综合症等疾病.近年来的研究发现:脂多糖分子中只有类脂A部分具有内毒素的活性.当细菌侵入人体后,其表面的类脂A可以刺激宿主细胞表面的Toll样受体-4,在细胞内引起一连串的反应,产生一系列的细胞因子.本文根据近年来内毒素领域的国际研究进展,系统综述了类脂A的结构特征、合成途径和致病机理,并在此基础上分析了内毒素在疫苗开发领域的应用前景.     

LPS和抗LPS治疗的研究及应用进展

细菌脂多糖LPS是革兰阴性菌致病的关键因子,可作为抗感染药物治疗的作用靶位。本文主要对LPS结构和功能及致病机制和抗LPS治疗策略的研究和应用进行综述,为临床上内毒素介导的感染性疾病的防治提供一定的依据。     

甘蔗宿根矮化病病原菌Leifsonia xyli subsp.xyli 基因组学研究进展

综述甘蔗宿根矮化病病原细菌Leifsonia xyli subsp.xyli(Lxx)基因组组成特征、基因组进化、基因组中致病相关基因与寄主适应性等方面的研究进展,并对该病原茵、引起番茄细菌性萎蔫和溃疡的病原细菌、马铃薯环腐病痛原细菌的基因组和致病相关基因进行比较,发现它们存在同源致病基因,如celA、pat1基因,其致病机理可能有相似性.     

致病菌基因组岛的研究进展

细菌基因组岛是细菌基因组上的特定区域,和水平基因转移相关,具有一定的结构特点,常携带致病、耐药及与适应性等功能相关的基因。通过基因组岛在细菌间的移动,可以造成相关基因在细菌间的传播,在细菌生存和致病等过程中具有重要作用。目前已经可通过生物信息和分子生物学实验等方法对基因组岛进行预测和验证。通过对致病菌基因组岛的研究,可以阐释细菌致病性和耐药等重要功能的获得,对疾病进行溯源,在传染病预防控制中具有重要意义。     

志贺菌属致病的研究进展

致病岛是指细菌染色体上具有典型结构特征的基因片段,主要编码细菌毒力及代谢相关产物。志贺菌属基因组中已发现了多个致病岛,并广泛存在于该菌属的各群细菌中,与致病性和耐药性等密切相关。对志贺菌属致病岛的研究为进一步深入理解其致病机制、开发防治细菌性痢疾的新策略提供了理论依据。     

幽门螺杆菌细胞毒素相关基因致病岛及Ⅳ型分泌系统的研究进展

致病岛是指细菌染色体上一段具有典型结构特征的基因簇,主要编码与细菌毒力及代谢等功能相关的产物。Ⅳ型分泌系统指革兰阴性菌中由多种蛋白分子构成的、通过菌毛样结构向宿主细胞注入毒力因子的分泌系统。幽门螺杆菌细胞毒素相关基因致病岛及其编码的Ⅳ型分泌系统是幽门螺杆菌关键性致病因子,有可能成为药物作用的新靶标,是近年相关研究的热点。     

大肠杆菌P4 FliC蛋白结构分析及同源建模

为了进一步认识大肠杆菌鞭毛蛋白(Escherichia coliFliC)的结构及功能,深入研究细菌鞭毛的生物学特性,采用生物信息学方法,对组成Escherichia coliP4 FliC蛋白质的理化性质、二级结构及三级结构等进行生物信息学分析,并在三级结构的基础上进行同源建模。理化性质分析结果表明,E.coliP4 FliC蛋白为酸性结构蛋白,与沙门氏菌鞭毛蛋白性质较为接近,二级结构以α-螺旋和随机卷曲为主要构件,其空间结构与Salmonella typhimurium的3a5xA蛋白相似性较高,以此为模板成功构建了可靠的三维结构分子模型。FliC蛋白为多种细菌的鞭毛蛋白,与细菌的运动功能关系密切,本研究为深入研究细菌的运动机制及致病机理奠定基础。     

革兰氏阴性细菌脂多糖分子Kdo2-lipid A基团的结构修饰

在大多数革兰氏阴性细菌中,脂多糖分子的Kdo2-lipid A基团是构成其外膜外层的主要成分.一些细菌通过修饰其脂多糖分子的Kdo2-lipid A基团来适应新的生存环境.Kdo2-lipid A在细胞内膜内层合成,被连上核心糖并翻转到内膜外层,再连上O-抗原重复单元,形成脂多糖分子.Kdo2-lipid A可以通过TLR4受体激活先天性免疫系统,所以其结构修饰机制的研究有助于开发新的细菌疫苗和疫苗佐剂.     

差异表达分析法在细菌致病相关基因研究中的应用

差异表达分析是比较相关细胞特殊表型基因背景的研究方法,应用于致病菌可以研究细菌的致病性、抗药性、遗传特性等。基因差异表达分析主要分四类,其代表性的方法分别为差异显示、消减杂交、基于数据库的基因表达连续分析和DNA微阵列。本文综述此四类方法及其衍生技术的基本策略、应用特点及近年来在细菌致病相关基因研究中的成功应用。     

The structure and proinflammatory activity of the lipopolysaccharide from Burkholderia multivorans and the differences between clonal strains colonizing pre and posttransplanted lungs

The Burkholderia cepacia complex is a group of Gram-negative bacteria that are opportunistic pathogens for humans especially in cystic fibrosis patients. Lipopolysaccharide (LPS) molecules are potent virulence factors of Gram-negative bacteria organisms essential for bacterial survival. A complete analysis of the bacterial lipopolysaccharide structure to function relationship is required to understand the chemical basis of the inflammatory process. We have therefore investigated the structures of lipopolysaccharides from clonally identical Burkholderia multivorans strains (genomovar II) isolated pre- and post-lung transplantation through compositional analysis, mass spectrometry, and 2D NMR spectroscopy. We tested the LPS proinflammatory activity as a stimulant of human myelomonocytic U937 cell cytokine induction and assessed TLR4/MD2 signaling. Marked changes between the paired strains were found in the lipid A-inner core region. Such structural variations can contribute to the bacterial survival and persistence of infections despite the loss of a CF milieu following lung transplantation.     

Unusual outer membrane lipid composition of the gram-negative, lipopolysaccharide-lacking myxobacterium Sorangium cellulosum So ce56

The gram-negative myxobacterium Sorangium cellulosum So ce56 bears the largest bacterial genome published so far, coding for nearly 10,000 genes. Careful analysis of this genome data revealed that part of the genes coding for the very well conserved biosynthesis of lipopolysaccharides (LPS) are missing in this microbe. Biochemical analysis gave no evidence for the presence of LPS in the membranes of So ce56. By analyzing the lipid composition of its outer membrane sphingolipids were identified as the major lipid class, together with ornithine-containing lipids (OL) and ether lipids. A detailed analysis of these lipids resulted in the identification of more than 50 structural variants within these three classes, which possessed several interesting properties regarding to LPS replacement, mediators in myxobacterial differentiation, as well as potential bioactive properties. The sphingolipids with the basic structure C9-methyl-C(20)-sphingosine possessed as an unusual trait C9-methylation, which is common to fungi but highly uncommon to bacteria. Such sphingolipids have not been found in bacteria before, and they may have a function in myxobacterial development. The OL, also identified in myxobacteria for the first time, contained acyloxyacyl groups, which are also characteristic for LPS and might replace those in certain functions. Finally, the ether lipids may serve as biomarkers in myxobacterial development.     

Thermodynamic analysis of the lipopolysaccharide-dependent resistance of gram-negative bacteria against polymyxin B

Cationic antimicrobial cationic peptides (CAMP) have been found in recent years to play a decisive role in hosts' defense against microbial infection. They have also been investigated as a new therapeutic tool, necessary in particular due to the increasing resistance of microbiological populations to antibiotics. The structural basis of the activity of CAMPs has only partly been elucidated and may comprise quite different mechanism at the site of the bacterial cell membranes or in their cytoplasm. Polymyxin B (PMB) is a CAMP which is effective in particular against Gram-negative bacteria and has been well studied with the aim to understand its interaction with the outer membrane or isolated membrane components such as lipopolysaccharide (LPS) and to define the mechanism by which the peptides kill bacteria or neutralize LPS. Since PMB resistance of bacteria is a long-known phenomenon and is attributed to structural changes in the LPS moiety of the respective bacteria, we have performed a thermodynamic and biophysical analysis to get insights into the mechanisms of various LPS/PMB interactions in comparison to LPS from sensitive strains. In isothermal titration calorimetric (ITC) experiments considerable differences of PMB binding to sensitive and resistant LPS were found. For sensitive LPS the endothermic enthalpy change in the gel phase of the hydrocarbon chains converts into an exothermic reaction in the liquid crystalline phase. In contrast, for resistant LPS the binding enthalpy change remains endothermic in both phases. As infrared data show, these differences can be explained by steric changes in the headgroup region of the respective LPS.     

O-antigen structural variation: mechanisms and possible roles in animal/plant-microbe interactions

Current data from bacterial pathogens of animals and from bacterial symbionts of plants support some of the more general proposed functions for lipopolysaccharides (LPS) and underline the importance of LPS structural versatility and adaptability. Most of the structural heterogeneity of LPS molecules is found in the O-antigen polysaccharide. In this review, the role and mechanisms of this striking flexibility in molecular structure of the O-antigen in bacterial pathogens and symbionts are illustrated by some recent findings. The variation in O-antigen that gives rise to an enormous structural diversity of O-antigens lies in the sugar composition and the linkages between monosaccharides. The chemical composition and structure of the O-antigen is strain-specific (interstrain LPS heterogeneity) but can also vary within one bacterial strain (intrastrain LPS heterogeneity). Both LPS heterogeneities can be achieved through variations at different levels. First of all, O-polysaccharides can be modified non-stoichiometrically with sugar moieties, such as glucosyl and fucosyl residues. The addition of non-carbohydrate substituents, i.e. acetyl or methyl groups, to the O-antigen can also occur with regularity, but in most cases these modifications are again non-stoichiometric. Understanding LPS structural variation in bacterial pathogens is important because several studies have indicated that the composition or size of the O-antigen might be a reliable indicator of virulence potential and that these important features often differ within the same bacterial strain. In general, O-antigen modifications seem to play an important role at several (at least two) stages of the infection process, including the colonization (adherence) step and the ability to bypass or overcome host defense mechanisms. There are many reports of modifications of O-antigen in bacterial pathogens, resulting either from altered gene expression, from lysogenic conversion or from lateral gene transfer followed by recombination. In most cases, the mechanisms underlying these changes have not been resolved. However, in recent studies some progress in understanding has been made. Changes in O-antigen structure mediated by lateral gene transfer, O-antigen conversion and phase variation, including fucosylation, glucosylation, acetylation and changes in O-antigen size, will be discussed. In addition to the observed LPS heterogeneity in bacterial pathogens, the structure of LPS is also altered in bacterial symbionts in response to signals from the plant during symbiosis. It appears to be part of a molecular communication between bacterium and host plant. Experiments ex planta suggest that the bacterium in the rhizosphere prepares its LPS for its roles in symbiosis by refining the LPS structure in response to seed and root compounds and the lower pH at the root surface. Moreover, modifications in LPS induced by conditions associated with infection are another indication that specific structures are important. Also during the differentiation from bacterium to bacteroid, the LPS of Rhizobium undergoes changes in the composition of the O-antigen, presumably in response to the change of environment. Recent findings suggest that, during symbiotic bacteroid development, reduced oxygen tension induces structural modifications in LPS that cause a switch from predominantly hydrophilic to predominantly hydrophobic molecular forms. However, the genetic mechanisms by which the LPS epitope changes are regulated remain unclear. Finally, the possible roles of O-antigen variations in symbiosis will be discussed.     

Mass Spectrometry-based Structural Analysis and Systems Immunoproteomics Strategies for Deciphering the Host Response to Endotoxin

One cause of sepsis is systemic maladaptive immune response of the host to bacteria and specifically, to Gram-negative bacterial outer-membrane glycolipid lipopolysaccharide (LPS). On the host myeloid cell surface, proinflammatory LPS activates the innate immune system via Toll-like receptor-4/myeloid differentiation factor-2 complex. Intracellularly, LPS is also sensed by the noncanonical inflammasome through caspase-11 in mice and 4/5 in humans. The minimal functional determinant for innate immune activation is the membrane anchor of LPS called lipid A. Even subtle modifications to the lipid A scaffold can enable, diminish, or abolish immune activation. Bacteria are known to modify their LPS structure during environmental stress and infection of hosts to alter cellular immune phenotypes. In this review, we describe how mass spectrometry-based structural analysis of endotoxin helped uncover major determinations of molecular pathogenesis. Through characterization of LPS modifications, we now better understand resistance to antibiotics and cationic antimicrobial peptides, as well as how the environment impacts overall endotoxin structure. In addition, mass spectrometry-based systems immunoproteomics approaches can assist in elucidating the immune response against LPS. Many regulatory proteins have been characterized through proteomics and global/targeted analysis of protein modifications, enabling the discovery and characterization of novel endotoxin-mediated protein translational modifications.     

The dual role of lipopolysaccharide as effector and target molecule.

Lipopolysaccharides (LPS) are major integral components of the outer membrane of Gram-negative bacteria being exclusively located in its outer leaflet facing the bacterial environment. Chemically they consist in different bacterial strains of a highly variable O-specific chain, a less variable core oligosaccharide, and a lipid component, termed lipid A, with low structural variability. LPS participate in the physiological membrane functions and are, therefore, essential for bacterial growth and viability. They contribute to the low membrane permeability and increase the resistance towards hydrophobic agents. They are also the primary target for the attack of antibacterial drugs and proteins such as components of the host's immune response. When set free LPS elicit, in higher organisms, a broad spectrum of biological activities. They play an important role in the manifestation of Gram-negative infection and are therefore termed endotoxins. Physico-chemical parameters such as the molecular conformation and the charges of the lipid A portion, which is responsible for endotoxin-typical biological activities and is therefore termed the 'endotoxic principle' of LPS, are correlated with the biological activity of chemically different LPS.     

Alterations in Helicobacter pylori outer membrane and outer membrane vesicle-associated lipopolysaccharides under iron-limiting growth conditions

Outer membrane vesicles (OMVs) shed from the gastroduodenal pathogen Helicobacter pylori have measurable effects on epithelial cell responses. The aim of this study was to determine the effect of iron availability, and its basis, on the extent and nature of lipopolysaccharide (LPS) produced on H. pylori OMVs and their parental bacterial cells. Electrophoretic, immunoblotting and structural analyses revealed that LPSs of bacterial cells grown under iron-limited conditions were notably shorter than those of bacteria and OMVs obtained from iron-replete conditions. Structural analysis and serological probing showed that LPSs of iron-replete cells and OMVs expressed O-chains of Lewis(x) with a terminal Lewis(y) unit, whereas Lewis(y) expression was notably reduced on bacteria and OMVs from iron-limiting conditions. Unlike the O-chain, the core oligosaccharide and lipid A moieties of iron-replete and iron-limited bacteria and their OMVs were similar. Quantitatively, shed OMVs from iron-replete bacteria were found to be LPSenriched, whereas shed OMVs from iron-limited bacteria had a significantly reduced content of LPS. These differences were linked to bacterial ATP levels. Since iron availability affects the extent and nature of LPS expressed by H. pylori, host iron status may contribute to H. pylori pathogenesis.     

Modification of lipopolysaccharides as one of the ways of adaptation of pathogenic bacteria and their populations to biotic and abiotic environmental factors

The review is devoted to a problem of adaptive role of lipopolysaccharide (LPS) modification of pathogenic bacteria cells. The biological activity of LPS depends on their molecular conformation, which is determined by primary structure and charges of their molecules and can change through regulation of enzyme activity of LPS separate component synthesis. Regulation of gene expression, which determines LPS synthesis, can occur at a level of DNA primary structure (through mutations and recombinations); on highest levels of DNA organization through alteration of its conformation; can be mediated by signal transduction systems of bacterial cells. The changes of LPS structure can have spontaneous character due both to the instability of the appropriate genes and to the induction under the influence of various factors. Spontaneous variation of LPS structure is one of the reasons of heterogeneity of bacterial cell populations and creates a basis of their preadaptation. The influence of the environment factors on structure and functions of LPS can be both direct and mediated through regulation of expression of chromosome and plasmid genes by direct influence of the factors on DNA and through signal transduction systems of bacterial cells. Pathogenic bacteria can use the LPS variations at adaptation to biotic and abiotic factors.     

Computer simulation of the rough lipopolysaccharide membrane of Pseudomonas aeruginosa.

Lipopolysaccharides (LPSs) form the major constituent of the outer membrane of Gram-negative bacteria, and are believed to play a key role in processes that govern microbial metal binding, microbial adsorption to mineral surfaces, and microbe-mediated oxidation/reduction reactions at the bacterial exterior surface. A computational modeling capability is being developed for the study of geochemical reactions at the outer bacterial envelope of Gram-negative bacteria. A molecular model for the rough LPS of Pseudomonas aeruginosa has been designed based on experimentally determined structural information. An electrostatic model was developed based on Hartree-Fock SCF calculations of the complete LPS molecule to obtain partial atomic charges. The exterior of the bacterial membrane was assembled by replication of a single LPS molecule and a single phospholipid molecule. Molecular dynamics simulations of the rough LPS membrane of P. aeruginosa were carried out and trajectories were analyzed for the energetic and structural factors that determine the role of LPS in processes at the cell surface.     

Biosynthesis, transport, and modification of lipid A.

Lipopolysaccharide (LPS) is the major surface molecule of Gram-negative bacteria and consists of three distinct structural domains: O-antigen, core, and lipid A. The lipid A (endotoxin) domain of LPS is a unique, glucosamine-based phospholipid that serves as the hydrophobic anchor of LPS and is the bioactive component of the molecule that is associated with Gram-negative septic shock. The structural genes encoding the enzymes required for the biosynthesis of Escherchia coli lipid A have been identified and characterized. Lipid A is often viewed as a constitutively synthesized structural molecule. However, determination of the exact chemical structures of lipid A from diverse Gram-negative bacteria shows that the molecule can be further modified in response to environmental stimuli. These modifications have been implicated in virulence of pathogenic Gram-negative bacteria and represent one of the molecular mechanisms of microbial surface remodeling used by bacteria to help evade the innate immune response. The intent of this review is to discuss the enzymatic machinery involved in the biosynthesis of lipid A, transport of the molecule, and finally, those enzymes involved in the modification of its structure in response to environmental stimuli.