Investigation of bacterial resistance to the immune system response: cepacian depolymerisation by reactive oxygen species

Reactive oxygen species (ROS) are part of the weapons used by the immune system to kill and degrade infecting microorganisms. Bacteria can produce macromolecules, such as polysaccharides, that are able to scavenge ROS. Species belonging to the Burkholderia cepacia complex are involved in serious lung infection in cystic fibrosis patients and produce a characteristic polysaccharide, cepacian. The interaction between ROS and bacterial polysaccharides was first investigated by killing experiments, where bacteria cells were incubated with sodium hypochlorite (NaClO) with and without prior incubation with cepacian. The results showed that the polysaccharide had a protective effect towards bacterial cells. Cepacian was then treated with different concentrations of NaClO and the course of reactions was followed by means of capillary viscometry. The degradation products were characterised by size-exclusion chromatography, NMR and mass spectrometry. The results showed that hypochlorite depolymerised cepacian, removed side chains and O-acetyl groups, but did not cleave the glycosidic bond between glucuronic acid and rhamnose. The structure of some oligomers produced by NaClO oxidation is reported.     

细菌多糖的生物合成机制

近些年来,细菌多糖因其重要的医学价值和工业用途引起了人们的广泛关注。随着细菌基因组的不断测序,众多与细菌多糖合成相关的基因簇被发现。不同多糖合成基因簇的比对分析结果表明,尽管自然界中多糖的结构复杂多变,但是它们的合成机制却是相对单一的。本文就当前国际国内不同细菌多糖的合成机制,以及多糖合成途径中相关糖基转移酶和聚合酶的研究进展进行了论述。     

Recognition of bacterial capsular polysaccharides and lipopolysaccharides by the macrophage mannose receptor

The in vitro binding of the macrophage mannose receptor to a range of different bacterial polysaccharides was investigated. The receptor was shown to bind to purified capsular polysaccharides from Streptococcus pneumoniae and to the lipopolysaccharides, but not capsular polysaccharides, from Klebsiella pneumoniae. Binding was Ca(2+)-dependent and inhibitable with d-mannose. A fusion protein of the mannose receptor containing carbohydrate recognition domains 4-7 and a full-length soluble form of the mannose receptor containing all domains external to the transmembrane region both displayed very similar binding specificities toward bacterial polysaccharides, suggesting that domains 4-7 are sufficient for recognition of these structures. Surprisingly, no direct correlation could be made between polysaccharide structure and binding to the mannose receptor, suggesting that polysaccharide conformation may play an important role in recognition. The full-length soluble form of the mannose receptor was able to bind simultaneously both polysaccharide via the carbohydrate recognition domains and sulfated oligosaccharide via the cysteine-rich domain. The possible involvement of the mannose receptor, either cell surface or soluble, in the innate and adaptive immune responses to bacterial polysaccharides is discussed.     

Novel bacterial exopolysaccharides from deep-sea hydrothermal vents

Five bacterial strains recovered from deep-sea hydrothermal vents were studied for their ability to secrete extracellular polymers. A preliminary characterization displayed four different polysaccharides in terms of both chemical composition and rheological properties. One of them was secreted by Alteromonas macleodii subsp. fijiensis and exhibited similarities with xanthan, a commercial polysaccharide. Two of the three Pseudoalteromonas species were shown to produce the same polymer. The last polymer was secreted by a bacterium belonging to the Vibrio genus. They all contained glucose, galactose, mannose, glucuronic and galacturonic acids as the main sugars with the exception of the last one which was only constituted by uronic acids and hexosamines, in that similar to the structure of heparin, a glycosaminoglycan useful in pharmaceutical area. Applications for these polysaccharides could be expected in various biotechnological fields including the food industry, the wastewater treatment and pharmaceutical areas.     

Low-molecular-weight polysaccharide antigens isolated from Rhodopseudomonas gelatinosa.

Strain-specific low-molecular-weight polysaccharides of different chemical compositions were obtained from cells of nine different wild-type strains of the phototrophic bacterium Rhodopseudomonas gelatinosa. The polysaccharides are free of typical capsule components like hexuronic or aminohexuronic acids but contain (except that of strain 39/2) substantial amounts of phosphorus. A number of unusual o-methyl sugars (2-o-methyl-D-galactose, 2,3-di-o-methyl-D-galactose, 2-o-methyl-L-fucose) as well as 3,6-dideoxy-D-xylo-hexose (abequose) were identified in the R. gelatinosa polysaccharides. o-Methyl and dideoxy sugars however, are typical constituents of O-specific chains of the lipopolysaccharides of gram-negative bacteria (Rhodospirillaceae and Enterobacteriaceae, respectively). Considering both the R-type character of the R. gelatinosa lipopolysaccharides and the occurrence of these strain-specific ETEROPOLYSACCHARIDES, THE ASSUMPTION SEEMS TO BE JUSTIFIED THAT THE LOW-MOLECULAR-WEIGHT POLYSACCHARIDES ARE RELATED TO O-specific chains of lipopolysaccharides (haptens) rather than to capsular or slime antigens. In serological terms the polysaccharides of R. gelatinosa have to be classified as K-antigens. They are able to cover the O-specificity of the respective different strains and confer on them additional specificity which is demonstrable by bacterial agglutination.     

Biological chemistry of immunomodulation by zwitterionic polysaccharides

Capsular polysaccharides isolated from pathogenic bacteria are comprised typically of many repeating units from one to eight or more monosaccharides in length. These polysaccharides stimulate the murine humoral immune system to elicit primarily IgM antibody responses. Studies conducted primarily in the mouse have characterized these polymers as T cell-independent antigens. These mouse studies and the relatively poor immunogenicity of polysaccharides in human hosts have led to the design of vaccines by coupling these polysaccharides to protein carriers to stimulate a T cell-dependent response. However, a newly described class of bacterial polysaccharides has been characterized that have the ability to modulate the cellular immune system. They are structurally diverse, but all share a zwitterionic charge motif that allows them to directly interact with T cells and antigen-presenting cells to initiate an immunomodulatory T cell response. These polymers, termed zwitterionic polysaccharides (ZPSs), elicit T cell-derived chemokines and cytokines that influence the immune response governing at least one classic host response to bacterial infection: abscess formation. This review will describe the biological and structural aspects of ZPSs that convey these activities.     

Ruthenium red adsorption method for measurement of extracellular polysaccharides in sludge flocs

A nondestructive method of measuring extracellular polysaccharides (ECP) in activated sludge floes using Ruthenium Red dye adsorption was developed at the Environmental Engineering Laboratory at the University of Colorado at Boulder. The effects of pH, buffer solution, dye concentration, sludge mass, temperature, and incubation time on dye adsorption was determined. Ruthenium Red dye adsorption to bacterial floes was found to fit a Brunauer-Emmett-Teller (BET) isotherm model. Of the other environmental conditions in the system, pH was found to have the strongest effect on dye adsorption to bacterial flocs. The amount of extra cellular polysaccharides (ECP) measured by Ruthenium Red adsorption was compared with extracellular polysaccharides measured by two chemical extraction methods. Of all methods considered Ruthenium Red dye adsorption measured the highest amount of extracellular polysaccharide with the lowest amount of bacterial cell disruption. Thus, Ruthenium Red dye adsorption was more effective than extraction procedures for measurement of extracellular polysaccharides in activated sludge flocs.     

Deacylated lipopolysaccharides inhibit biofilm formation by Gram-negative bacteria

The extracellular polysaccharides of Vibrio vulnificus play different roles during biofilm development. Among them, the effect of lipopolysaccharide (LPS), which is crucial for bacterial adherence to surfaces during the initial stage of biofilm formation, on the formation process was examined using various types of LPS extracts. Exogenously added LPS strongly inhibited biofilm formation in a dose-dependent manner. In addition, the exogenous addition of a deacylated form of LPS (dLPS) also inhibited biofilm formation. However, an LPS fraction extracted from a mutant not able to produce O-antigen polysaccharides (O-Ag) did not have an inhibitory effect. Furthermore, biofilm formation by several Gram-negative bacteria was inhibited by dLPS addition. In contrast, biofilm formation by Gram-positive bacteria was not influenced by dLPS but was affected by lipoteichoic acid. Therefore, this study demonstrates that O-Ag in LPS is important for inhibiting biofilm formation and may serve an efficient anti-biofilm agent specific for Gram-negative bacteria.     

Distinct roles of extracellular polymeric substances in Pseudomonas aeruginosa biofilm development

Bacteria form surface attached biofilm communities as one of the most important survival strategies in nature. Biofilms consist of water, bacterial cells and a wide range of self-generated extracellular polymeric substances (EPS). Biofilm formation is a dynamic self-assembly process and several distinguishable stages are observed during bacterial biofilm development. Biofilm formation is shown to be coordinated by EPS production, cell migration, subpopulation differentiation and interactions. However, the ways these different factors affect each other and contribute to community structural differentiation remain largely unknown. The distinct roles of different EPS have been addressed in the present report. Both Pel and Psl polysaccharides are required for type IV pilus-independent microcolony formation in the initial stages of biofilm formation by Pseudomonas aeruginosa PAO1. Both Pel and Psl polysaccharides are also essential for subpopulation interactions and macrocolony formation in the later stages of P. aeruginosa PAO1 biofilm formation. Pel and Psl polysaccharides have different impacts on Pseudomonas quinolone signal-mediated extracellular DNA release in P. aeruginosa PAO1 biofilms. Psl polysaccharide is more important than Pel polysaccharide in P. aeruginosa PAO1 biofilm formation and antibiotic resistance. Our study thus suggests that different EPS materials play distinct roles during bacterial biofilm formation.     

Strong seasonal differences of bacterial polysaccharide utilization in the North Sea over an annual cycle

Marine heterotrophic bacteria contribute considerably to global carbon cycling, in part by utilizing phytoplankton-derived polysaccharides. The patterns and rates of two different polysaccharide utilization modes – extracellular hydrolysis and selfish uptake – have previously been found to change during spring phytoplankton bloom events. Here we investigated seasonal changes in bacterial utilization of three polysaccharides, laminarin, xylan and chondroitin sulfate. Strong seasonal differences were apparent in mode and speed of polysaccharide utilization, as well as in bacterial community compositions. Compared to the winter month of February, during the spring bloom in May, polysaccharide utilization was detected earlier in the incubations and a higher portion of all bacteria took up laminarin selfishly. Highest polysaccharide utilization was measured in June and September, mediated by bacterial communities that were significantly different from spring assemblages. Extensive selfish laminarin uptake, for example, was detectible within a few hours in June, while extracellular hydrolysis of chondroitin was dominant in September. In addition to the well-known Bacteroidota and Gammaproteobacteria clades, the numerically minor verrucomicrobial clade Pedosphaeraceae could be identified as a rapid laminarin utilizer. In summary, polysaccharide utilization proved highly variable over the seasons, both in mode and speed, and also by the bacterial clades involved.     

哈氏噬纤维菌吸附纤维素的影响因素

[目的]探索哈氏噬纤维菌(Cytophaga hutchinsonii)吸附纤维素的作用机制.[方法]通过比较不同因素对哈氏噬纤维菌吸附纤维素的影响,包括:菌龄、pH、温度、表面电荷、细胞活力、细胞表面蛋白、细胞表面多糖以及纤维素类似物等,寻找在吸附过程中起重要作用的细胞成分.[结果]菌体经蛋白酶及热处理,对纤维素的吸附能力完全丧失;叠氮化钠、甲醛和戊二醛处理对菌体吸附能力影响不明显;菌体经刚果红和高碘酸钠处理,吸附能力变化不大;菌体对纤维素底物的吸附具有特异性,吸附作用不受纤维二糖和羧甲基纤维素的抑制.[结论]实验表明,哈氏噬纤维菌吸附纤维素的能力与菌体表面蛋白密切相关,而受细胞的代谢活性和胞外多糖影响较小,推测细胞表面可能存在特异性的纤维素结合蛋白.     

Effect of natural modifications on the functional properties of extracellular bacterial polysaccharides

Extracellular bacterial polysacharides comprise the capsules and slimes secreted by many bacteria. Little is known about the features of the chemical structure which are of importance in determining the helical conformation and inter- or intramolecular associations of these polysaccharides. An understanding of such structure-function relationships is hampered by the often complex chemical repeat units of these bacterial polysaccharides. One approach is to investigate and compare the properties of families of polysaccharides in which individual members of the group show small naturally arising modifications to the chemical structure. This approach is illustrated by studies which show the effects of changes in the polymer backbone, polymer side chains and non-carbohydrate substituents on polymer functionality. It is shown how such studies form a basis for explaining and optimizing the industrial applications of bacterial polysaccharides and for understanding the natural roles of extracellular polysaccharides.     

赤灵芝GL-2发酵液中多糖组分及活性研究

对赤灵芝ＧＬ－２菌株发酵液中胞外多糖的提取进行了研究,通过乙醇分级沉淀对发酵液中多糖进行了分离,并采用凝胶柱层析法检测了各级份中多糖的分子量分布范围,动物实验证明,分级沉淀各级份均能促进小鼠体重增长,增强小鼠白细胞吞噬细胞的能力,提高红细胞中ＳＯＤ酶的活力。其中６０％级份的效果最为明显。     

Mannose-containing polysaccharides of yeasts

The known species of yeasts produce at lest 180 different polysaccharides having mannose as a major constituent. The type of mannan formed is characteristic of the yeast species, and can be used as an aid in identification and classification. The immunological characteristics of yeasts depend largely upon the nature of the mannans. Some of the yeast mannans can be produced in relatively high yields, and have been suggested as replacements for plant and bacterial gums as thickeners, dispersing agents, and similar applications. However, more significant uses, based on the finer differences in their chemical structures and the specific reactions they undergo, may result from a closer study of these polymers.     

Two species of culturable bacteria associated with degradation of brown algae Fucus evanescens

The heterotrophic microbial enrichment community established during degradation of brown algae Fucus evanescens was characterized. A two-species bacterial community of marine culturable gamma-proteobacteria consisted of Pseudoalteromonas and Halomonas. The first member of the community, Pseudoalteromonas sp., was highly metabolically active, had bacteriolytic and hemolytic activities, produced proteinases (gelatinase and caseinase), lipases, DNases, and fucoidanhydrolases, laminaranases, alginases, pustulanases, beta-glucosidases, beta-galactosidases, beta-N-acetylglucosaminidases, and beta-xylosidases. The second member of the community, Halomonas marina, produced only caseinase and DNase, and it did not hydrolyze algal polysaccharides. Both members of the studied bacterial community utilized a range of easily assimilable monosaccharides and other low molecular weight organic substances. The results provide an evidence of the complex metabolic interrelations between two members of this culturable community. One of them Pseudoalteromonas sp., most likely plays the major role in the initial stages of algal degradation; the other one, H. marina, resistant to the bacteriolytic activity of the former, is able to utilize the products of degradation of polysaccharides.     

细菌荚膜多糖的化学结构

荚膜是一些细菌所具有的表层结构,与多种疾病有着密切联系。细菌荚膜多糖不仅结构复杂,而且在免疫活性方面发挥着重要的作用。同一种细菌根据其荚膜多糖的抗原性不同可分为不同的血清型,不同血清型细菌荚膜多糖的化学结构也存在差异。以荚膜多糖为基础的疫苗正在积极研究开发当中,对不同致病细菌荚膜多糖具体化学结构的掌握是疫苗得到许可的必备条件之一。本文对致病细菌荚膜多糖的化学结构进行了归纳和总结,以期为荚膜多糖的化学结构研究和疫苗开发提供参考。     

Use of methylotrophic bacteria Methylobacillus flagellatum KT for isolation of deuterated exogenous carbohydrates

The cultivation conditions optimal for biosynthesis of exogenous polysaccharides by methylotrophic bacteria Methylobacillus flagellatum were evaluated. The mutant strain most active in accumulating exogenous polysaccharides was selected. Gradual adaptation of this strain to the deuterated medium containing 1 vol % CD3OD in deuterium oxide intensified biosynthesis of exogenous polysaccharides and inhibited bacterial growth (compared to the standard medium). The monomeric composition of exogenous polysaccharides obtained during cultivation on standard and deuterated media was estimated by high-performance liquid chromatography and nuclear magnetic resonance spectroscopy. Nondeuterated exogenous polysaccharides contained only fructose, while deuterated exogenous polysaccharides included 98% fructose and 2% ribose. Paramagnetic resonance spectroscopy showed that the degree of deuterium incorporation into molecules of biosynthetic carbohydrates was 89%.     

Use of Methylotrophic Bacteria Methylobacillus flagellatumKT for Isolation of Deuterated Exogenous Carbohydrates

Cultivation conditions optimal for biosynthesis of exogenous polysaccharides by methylotrophic bacteria Methylobacillus flagellatum KT were evaluated. The mutant strain most active in accumulating exogenous polysaccharides was selected. Gradual adaptation of this strain to the deuterated medium containing 1 vol % CD₃OD in deuterium oxide intensified biosynthesis of exogenous polysaccharides and inhibited bacterial growth (compared to the standard medium). The monomeric composition of exogenous polysaccharides obtained during cultivation on standard and deuterated media was estimated by HPLC and NMR spectroscopy. Nondeuterated exogenous polysaccharides contained only fructose, whereas deuterated exogenous polysaccharides contained 98% fructose and 2% ribose. Paramagnetic resonance spectroscopy showed that the degree of deuterium incorporation into molecules of biosynthetic carbohydrates was 89%.     

Structural studies of the capsular polysaccharides from Klebsiella types 8 and 82, a reinvestigation

The structures of the capsular polysaccharides elaborated by Klebsiella types 8 (K8) and 82 (K82) have been reinvestigated. N.m.r. spectroscopy of the original and chemically modified polysaccharides was the principal method used. It is concluded that the polysaccharides are composed of repeating units having the following structures. (Formula: see text). The presence of L-glutamic acid, linked as an amide to the carboxyl group of a uronic acid, has not been observed hitherto in bacterial polysaccharides.     

Role of protein phosphorylation on serine/threonine and tyrosine in the virulence of bacterial pathogens

Bacterial pathogens have developed a diversity of strategies to interact with host cells, manipulate their behaviors, and thus to survive and propagate. During the process of pathogenesis, phosphorylation of proteins on hydroxyl amino acids (serine, threonine, tyrosine) occurs at different stages, including cell-cell interaction and adherence, translocation of bacterial effectors into host cells, and changes in host cellular structure and function induced by infection. The phosphorylation reactions are catalyzed in a reversible fashion by specific protein kinases and phosphatases that belong to either the invading bacterial cells or the infected eukaryotic host cells. Among the various virulence factors involved in bacterial pathogenesis, special attention has been paid recently to the cell wall components, exopolysaccharides. A major breakthrough has been made by showing the existence of a biological link between the activity of certain protein-tyrosine kinases/phosphatases and the production and/or transport of surface polysaccharides. In addition, genetic studies have revealed a key role played by some serine/threonine kinases in pathogenesis. Considering the structural organization and membrane topology of these different kinases, it can be envisaged that they operate as one-component systems in signal transduction pathways, in the form of single proteins containing input and output domains on the same polypeptide chain. From a general standpoint, the demonstration of a direct relationship between protein phosphorylation on serine/threonine/tyrosine and bacterial virulence represents a novel concept of great importance in deciphering the molecular and cellular mechanisms that underlie pathogenesis.