细菌中D-氨基酸生物合成及调控作用研究进展

细菌中普遍存在L/D型氨基酸,与L-氨基酸(L-AAs)不同,D-氨基酸(D-AAs)不参与蛋白质合成,而与细胞壁肽聚糖的合成有关,直接影响细菌细胞壁的形状、数量和强度。D-AAs在细菌表征、药物抑菌性、靶标确定等方面具有重要的作用。目前,外源添加D-AAs参与肽聚糖合成的机制已有一些研究进展,其荧光衍生物已应用于细菌可视化,特异性探测细胞壁形成/重塑、细菌生长和细胞形态。但D-AAs如何影响细菌生长及其抗逆性的机制尚未研究清楚。对D-AAs的研究现状进行综述,重点介绍D-AAs在细菌中的生物合成机制和参与细胞壁合成的机制、非典型DAAs对细菌的调控以及在细菌可视化中的应用,并对D-AAs未来研究方向进行了展望。     

乳酸菌肽聚糖的研究进展

肽聚糖是乳酸菌细胞壁的必需成分,它的化学结构较为保守固定,而其合成是一个涉及多步反应的复杂过程。乳酸菌肽聚糖具有多种生物学活性,比如免疫增强功能、抗感染、抗肿瘤及抗过敏等。本文对乳酸菌肽聚糖的组成结构和生物学活性进行了简要的介绍,重点综述了近年来乳酸菌肽聚糖代谢及其调控过程的研究进展,并指出了乳酸菌肽聚糖未来研究的方向。     

细菌细胞壁结构物质的生物合成

肽聚糖、磷壁酸、脂多糖是细菌细胞壁的主要结构物质,了解这些物质的合成过程,对于阐明细胞壁与细胞膜之间的关系,阐明某些抗生素的作用机制等具有重要意义。本文简要介绍上述几种物质生物合成过程以及某些抗生素对肽聚糖合成的影响。     

大肠杆菌细胞壁肽聚糖的化学修饰及荧光标记

【目的】发展一种活细菌细胞壁荧光标记方法,为后期研究细菌肽聚糖的生物合成和代谢规律以及其与细菌感染致病的关系提供新的工具。【方法】对细菌肽聚糖的生物合成前体N-乙酰葡萄糖胺-1-磷酸(GlcNAc-1-P)进行化学修饰,设计并合成含有叠氮基的GlcNAc-1-P类似物(化合物5:Ac3GlcNAz-1-P)。将该类似物与细菌共同孵育,使其作为探针进入细菌肽聚糖天然合成途径。之后提取并酶解肽聚糖组分,用红外光谱(FTIR)和液质联用(LC/MS)检测探针是否通过代谢进入细菌肽聚糖结构中。同时用外源荧光素对代谢掺入细菌肽聚糖中的探针进行染色。在激光共聚焦显微镜下观察对活细菌的荧光标记效果。【结果】通过四步有机合成反应,以79%的总收率成功获得了化合物5。将大肠杆菌(Escherichia coli BL21)作为模式菌株与化合物5共孵育后,其肽聚糖组分的LC/MS和FTIR分析结果均显示探针可以被细菌利用并被代谢掺入到肽聚糖结构中。激光共聚焦显微镜观察结果显示,荧光素可以高效标记表面携带有生物正交探针的大肠杆菌。【结论】设计合成了一种新型探针,可用于活细菌成像,为深入研究细菌肽聚糖的生物学功能及其与细菌感染致病的关系提供了一种简便的方法。     

抗细菌药物默诺霉素的化学生物学研究进展

默诺霉素(moenomycins)家族类化合物主要是由链霉菌产生,属于磷酸糖脂类抗生素。该类化合物通过与细菌细胞壁肽聚糖糖基转移酶(peptidoglycan transferase,PGT)的活性位点结合,可以抑制众多革兰氏阳性细菌细胞壁的合成,具有很强的生物活性和重要的应用开发潜力。本文针对默诺霉素的化学结构、生物活性机制、抗性机制、生物合成研究途径、外排机制和新结构创制等化学生物学方面进行了系统综述,并对默诺霉素化学生物学研究现状以及可能存在的问题进行了总结,旨在为高活性磷酸糖脂类临床药物的研究与开发提供借鉴。     

双歧杆菌及其表面分子对小鼠肠内致癌相关性细菌酶活性的影响

本文研究了分叉双歧杆菌及其脂磷壁酸、细胞壁肽聚糖对小鼠粪便中肠内细菌酶－β－葡萄糖醛酸酶、偶氮还原酶和硝酸盐还原酶活性的影响。结果表明,小鼠摄入双歧杆菌活菌、死菌、脂磷壁酸、细胞壁肽聚糖３周期间,肠内细菌偶氮还原酶、β－葡萄糖醛酸酶、硝酸盐还原酶活性降低,以活菌、死菌作用更为显著,二者无显著差异。     

乳酸杆菌肽聚糖免疫作用的研究进展

乳酸杆菌是一群生活在机体内益于宿主健康的微生物,它维护人体健康和调节免疫功能的作用已被广泛认可.而对于乳酸杆菌里起免疫调节作用的具体成分目前还不十分清楚.大量资料显示乳酸杆菌细胞壁成分肽聚糖可能在这方面起到了重要作用,乳酸杆菌的细胞壁肽聚糖(whole peptidoglycan,WPG)能够增强吞噬细胞功能、诱导细胞因子和一氧化氮等免疫介质的释放,在抗感染和抗肿瘤免疫中发挥重要作用.本研究综述了乳酸杆菌肽聚糖在免疫方面作用的研究进展.     

昆虫肽聚糖识别蛋白研究进展

在脊椎动物和非脊椎动物中,识别非己是天生免疫反应中的第一步。肽聚糖是细菌细胞壁的必需成分,属于进化上保守的微生物表面病原相关分子模式(pathogen-associated molecular pattern, PAMP),可以被模式识别蛋白(pattern recognition proteins, PRRs)如肽聚糖识别蛋白(peptidoglycan recognition proteins, PGRPs)识别。 在昆虫的天生免疫系统中,有些PGRPs能够利用细菌独有的肽聚糖识别入侵细菌,并将细菌入侵信号传递给下游的抗菌肽(antimicrobial peptide, AMP)合成途径,启动抗菌肽基因的转录及合成;PGRPs对肽聚糖的识别也会启动酚氧化酶原途径的激活,引起黑化反应。有些具有酰胺酶活性的PGRPs可以促进吞噬作用;有些可以抑制抗菌肽合成以减弱过度免疫反应带来的损伤。还有一些PGRPs作为效应因子直接作用于细菌将细菌杀死。本文主要从昆虫PGRPs作为识别受体(recognition receptor)、调节子(regulator)和效应因子(effector) 3个方面进行了综述,并分析了目前PGRPs研究中仍不清楚的问题和未来研究的方向。     

细菌细胞壁生长调控机制研究进展

在细菌生长过程中,细胞壁起到维持细胞形状和完整性,抵抗内部膨胀压的作用。细胞壁的合成、分裂、再生、循环再利用等与细菌自身生长繁殖和应对环境压力息息相关。目前,细胞壁生长机理,细菌如何调控细胞壁生长及如何与其他细胞过程相协调的机制尚未研究清楚。细胞壁调控机制的解析对了解细菌细胞壁功能、确定药物的作用方式和发展新一代的治疗方法至关重要。对细菌调控细胞壁生长机制的国外研究进展进行了概述,重点阐述了支架蛋白、转录因子、非编码小RNA及蛋白相互作用调控细胞壁的合成、细胞分裂、压力响应的机制,总结了细胞壁调控机制在抗菌药物研发中的应用,并对未来的研究方向进行了展望。     

半胱氨酸合成酶复合体形成机制的研究进展

在细菌和植物中,O-乙酰丝氨酸硫解酶(OASTL)和丝氨酸乙酰转移酶(SAT)的结合形成了半胱氨酸合成酶复合物(CSC),这个酶复合体在硫同化和半胱氨酸生物合成中起调节作用。综述植物和微生物中CSC的结构、组装形成过程及其调控机制的研究进展。     

The different shapes of cocci

The shape of bacteria is determined by their cell wall and can be very diverse. Even among genera with the suffix 'cocci', which are the focus of this review, different shapes exist. While staphylococci or Neisseria cells, for example, are truly round-shaped, streptococci, lactococci or enterococci have an ovoid shape. Interestingly, there seems to be a correlation between the shape of an organism and its set of penicillin-binding proteins--the enzymes that assemble the peptidoglycan, the main constituent of the cell wall. While only one peptidoglycan biosynthesis machinery seems to exist in staphylococci, two of these machineries are proposed to function in ovoid-shaped bacteria, reinforcing the intrinsic differences regarding the morphogenesis of different classes of cocci. The present review aims to integrate older ultra-structural data with recent localization studies, in order to clarify the relation between the mechanisms of cell wall synthesis and the determination of cell shape in various cocci.     

Differential methicillin susceptibilities of peptidoglycan syntheses in methicillin-resistant Staphylococcus aureus.

The mechanism of staphylococcal resistance to methicillin is unknown. Peptidoglycan synthesis was studied in a methicillin-resistant and a derived methicillin-sensitive Staphylococcus aureus strain. Although the methicillin minimum inhibitory concentration for growth of the methicillin-resistant strain was 1,600 micrograms/ml, peptidoglycan synthesis by the organism incubated in a wall synthesis solution was inhibited about 90% by 5 micrograms of methicillin per ml. In contrast, high concentrations of methicillin added to actively growing cultures of the methicillin-resistant strain had little effect on growth or peptidoglycan synthesis. Peptidoglycan synthesis in chloramphenicol-treated cultures was more susceptible to methicillin than it was in actively growing cultures of the methicillin-resistant strain. It is proposed that in this strain cell wall thickening peptidoglycan synthesis which predominates in cell wall synthesis solution and chloramphenicol-treated cultures is methicillin sensitive, whereas peptidoglycan synthesis involved in cell division, primarily in the region of the septum, which predominates in actively growing cultures is methicillin resistant. Both cell wall thickening and septal peptidoglycan syntheses are methicillin sensitive in the methicillin-sensitive strain.     

Peptidoglycan fragments elicit antibacterial protein synthesis in larvae of Manduca sexta

In several insect species, serum lysozyme and antibacterial peptide concentration increases after injection of bacteria and other foreign substances. The purpose of this study was to characterize the specificity of this induction in the tobacco hornworm, Manduca sexta. By 48 h after injection of killed bacteria, lysozyme activity was approximately tenfold greater than in untreated insects. This maximal response was observed after injection of every bacterial species tested and after injection of purified cell walls of Micrococcus luteus. A variety of acellular particles, soluble molecules, and bacterial cell wall components were either poor lysozyme inducers or elicited no change in lysozyme concentration. The polysaccharide zymosan from yeast cell walls was a moderate lysozyme inducer. Peptidoglycan from M. luteus cell walls was found to induce lysozyme to a level as great or greater than whole cell walls. Small fragments of peptidoglycan generated by hen egg white lysozyme digestion were isolated, partially characterized, and shown to be good inducers of lysozyme as well as other antibacterial peptides. It appears that peptidoglycan provides a signal that initiates antibacterial responses in the insect.     

High-throughput,Highly Sensitive Analyses of Bacterial Morphogenesis Using Ultra Performance Liquid Chromatography

The bacterial cell wall is a network of glycan strands cross-linked by short peptides (peptidoglycan); it is responsible for the mechanical integrity of the cell and shape determination. Liquid chromatography can be used to measure the abundance of the muropeptide subunits composing the cell wall. Characteristics such as the degree of cross-linking and average glycan strand length are known to vary across species. However, a systematic comparison among strains of a given species has yet to be undertaken, making it difficult to assess the origins of variability in peptidoglycan composition. We present a protocol for muropeptide analysis using ultra performance liquid chromatography (UPLC) and demonstrate that UPLC achieves resolution comparable with that of HPLC while requiring orders of magnitude less injection volume and a fraction of the elution time. We also developed a software platform to automate the identification and quantification of chromatographic peaks, which we demonstrate has improved accuracy relative to other software. This combined experimental and computational methodology revealed that peptidoglycan composition was approximately maintained across strains from three Gram-negative species despite taxonomical and morphological differences. Peptidoglycan composition and density were maintained after we systematically altered cell size in Escherichia coli using the antibiotic A22, indicating that cell shape is largely decoupled from the biochemistry of peptidoglycan synthesis. High-throughput, sensitive UPLC combined with our automated software for chromatographic analysis will accelerate the discovery of peptidoglycan composition and the molecular mechanisms of cell wall structure determination.     

Cytoplasmic steps of peptidoglycan biosynthesis

The biosynthesis of bacterial cell wall peptidoglycan is a complex process that involves enzyme reactions that take place in the cytoplasm (synthesis of the nucleotide precursors) and on the inner side (synthesis of lipid-linked intermediates) and outer side (polymerization reactions) of the cytoplasmic membrane. This review deals with the cytoplasmic steps of peptidoglycan biosynthesis, which can be divided into four sets of reactions that lead to the syntheses of (1) UDP-N-acetylglucosamine from fructose 6-phosphate, (2) UDP-N-acetylmuramic acid from UDP-N-acetylglucosamine, (3) UDP-N-acetylmuramyl-pentapeptide from UDP-N-acetylmuramic acid and (4) D-glutamic acid and dipeptide D-alanyl-D-alanine. Recent data concerning the different enzymes involved are presented. Moreover, special attention is given to (1) the chemical and enzymatic synthesis of the nucleotide precursor substrates that are not commercially available and (2) the search for specific inhibitors that could act as antibacterial compounds.     

Functional and Morphological Adaptation to Peptidoglycan Precursor Alteration in Lactococcus lactis

Cell wall peptidoglycan assembly is a tightly regulated process requiring the combined action of multienzyme complexes. In this study we provide direct evidence showing that substrate transformations occurring at the different stages of this process play a crucial role in the spatial and temporal coordination of the cell wall synthesis machinery. Peptidoglycan substrate alteration was investigated in the Gram-positive bacterium Lactococcus lactis by substituting the peptidoglycan precursor biosynthesis genes of this bacterium for those of the vancomycin-resistant bacterium Lactobacillus plantarum. A set of L. lactis mutant strains in which the normal d-Ala-ended precursors were partially or totally replaced by d-Lac-ended precursors was generated. Incorporation of the altered precursor into the cell wall induced morphological changes arising from a defect in cell elongation and cell separation. Structural analysis of the muropeptides confirmed that the activity of multiple enzymes involved in peptidoglycan synthesis was altered. Optimization of this altered pathway was necessary to increase the level of vancomycin resistance conferred by the utilization of d-Lac-ended peptidoglycan precursors in the mutant strains. The implications of these findings on the control of bacterial cell morphogenesis and the mechanisms of vancomycin resistance are discussed.     

The biosynthesis of peptidoglycan lipid-linked intermediates

The biosynthesis of bacterial cell wall peptidoglycan is a complex process involving many different steps taking place in the cytoplasm (synthesis of the nucleotide precursors) and on the inner and outer sides of the cytoplasmic membrane (assembly and polymerization of the disaccharide-peptide monomer unit, respectively). This review summarizes the current knowledge on the membrane steps leading to the formation of the lipid II intermediate, i.e. the substrate of the polymerization reactions. It makes the point on past and recent data that have significantly contributed to the understanding of the biosynthesis of undecaprenyl phosphate, the carrier lipid required for the anchoring of the peptidoglycan hydrophilic units in the membrane, and to the characterization of the MraY and MurG enzymes which catalyze the successive transfers of the N-acetylmuramoyl-peptide and N-acetylglucosamine moieties onto the carrier lipid, respectively. Enzyme inhibitors and antibacterial compounds interfering with these essential metabolic steps and interesting targets are presented.