1.04Microbial Polysaccharide Structures
O.Holst and S.Mu¨ller-Loennies,Research Center Borstel–Leibniz-Center for Medicine and Biosciences, Borstel,Germany
ß2007Elsevier Ltd.All rights reserved.
1.04.1Introduction:Bacterial Polysaccharides123 1.04.2Lipopolysaccharides(Endotoxins)124 1.04.
2.1Introduction124 1.04.2.2Functional Aspects of LPS125 1.04.2.3The Chemical Structure of Smooth(S)-Type LPS126
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2.
3.1The lipid A128
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党内法规清理的
处理方式包括
2.
3.2The core oligosaccharide135
2.
3.3The O-polysaccharide142 1.0
4.2.4Bacteria Expressing Only Rough(R)-Type LPS145
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2.4.1Yersinia pestis145
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2.4.2Chlamydia146
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2.4.3Neisseria,Haemophilus149 1.04.2.5LPS of the Gram-Positive:Pectinatus151 1.04.2.6Minimal L
PS Structure Supporting Survival of Gram-Negative Bacteria151 1.04.2.7Structural Analysis of LPS154 1.04.2.8Concluding Remarks155 1.04.3The Rigid Layer of Bacteria:The Peptidoglycan156 1.04.4Lipoteichoic Acids156 1.04.5Capsular and Exo-Polysaccharides157 1.04.5.1Capsular Polysaccharides157 1.04.5.2Exopolysaccharides159 1.04.6Gram-Positive Bacterial Cell Wall-Associated Polyols and Polysaccharides162 1.04.7Mycobacterial Cell Wall Polysaccharides164 1.04.7.1Mycoloyl-Arabinogalactan165 1.04.7.2Lipoarabinomannan165 1.04.8Mycobacterial Capsule Polysaccharides166 1.04.9Polysaccharides of Pathogenic Fungi and Yeasts167 1.04.10Final Remarks168
1.04.1Introduction:Bacterial Polysaccharides
Bacteria possess a cell envelope which is a highly complex structure with a number of functions which may be ,separation from the environment,protection from harmful influences)or ,transport of substances inside-out/outside-in,communication with the environment).1Bacterial cell envelopes provide the bacteria with sufficient rigidity and enable metabolism,growth,and multiplication.In general,bacterial cell envelopes allow for all of these functions;however,different bacteria involve different cell envelope molecules and architectures to be optimally operable.Thus,bacterial cell envelopes are complex and vary in a number of details.Based
on overall architecture,the general classification distinguishes between Gram-negative,Gram-positive,mycobacterial and archaebacterial cell envelopes which does not reflect a variety of structural details.
常熟市淼泉中学123
124Microbial Polysaccharide Structures
In addition to protein and lipid components,bacterial cell envelopes contain a variety of glycans,which are classified as polysaccharides,lipoglycans,and peptidoglycans.This overview summarizes general features and recently analyzed chemical structures of the latter three groups.A number of reviews have been published which summarize earlier findings,some of which are mentioned here.2–13
1.04.2Lipopolysaccharides(Endotoxins)
1.04.
2.1Introduction
According to the behavior in the Gram-stain,the domain of bacteria is divided into Gram-positive and Gram-negative bacteria and the outcome of this staining procedure is based on the cell wall architecture.14,15The cell walls of Gram-negative and Gram-positive bacteria differ fundamentally(Figure1),and only the former contain an additional membrane,the outer membrane(OM),thereby creating an additional compartment,the periplasmic space.The outer membrane of Gram-negative bacteria is asymmetric with respect to the distribution of lipids whereby the outer leaflet is made from a phosphoglycolipid called lipopolysaccharide(LPS,endotoxin),while the inner leaflet is made from phospholipids.16The number of LPS molecules per cell in Gram-negative organisms has been estimated to$2Â106molecules.17
The genera Escherichia,Salmonella,Klebsiella,Proteus,Yersinia,and Shigella,among others,make up the family of Enterobacteriaceae and thus are members of the harmless or even vitally important commensal flora of mammals. However,the same genera also comprise important pathogens which are able to cause infections.Infection may occur by invasive bacteria,,Escherichia coli,Salmonella enterica,Shigella)and nonenterobacterial (e.g.,Neisseria),which are able to penetrate the mucosa and the endothelium and may reach subepithelial tissues and the bloodstream.Also following traumatic stress,surgery,and,for example,severe burns,the protective barriers are broken a
nd leakage of bacteria may occur,whereby LPS and other microbial products reach the blood circulation. Primary immune recognition by dendritic cells,neutrophils,and macrophages lead to the activation of innate immune responses which have developed to identify and,if possible,eliminate the potentially life-threatening microbes from the infected site.Recognized target molecules comprise LPS and other microbial products which are commonly referred to as pathogen-associated molecular patterns(PAMPs).Various receptors of immune cells are involved in the innate immune recognition(pattern recognition receptors,PRRs),among which T oll-like receptors(TLRs)have been shown to play a prominent role and specifically recognize molecules belonging to PAMP.18,19Apart from the innate defense system,the adaptive immune response is also activated in mammals.By orders of magnitude the most potent
Bacterial cell envelope
Gram-negative Gram-positive
Cytoplasm
Figure1Schematic representation of the membrane organization of Gram-positive and Gram-negative bacteria.Only Gram-negative bacteria contain an outer membrane(OM)creating an addition
al compartment,the periplasmic space.The outer layer of the OM is composed of lipopolysaccharides(LPSs)whereas the inner layer is composed of phospholipids.
Microbial Polysaccharide Structures125 stimulus of all PAMPs is LPS of a characteristic chemical structure exemplified by LPS ica li.20 Biophysical studies have revealed that LPSs adopt certain physical aggregate structures and a conical shape of the individual molecules is associated with biological activity.21
The activation of the immune system may lead to the eradication of the infectious bacteria at the infected site; however,dissemination of bacteria may occur accompanied by an initial overwhelming hyper inflammatory response (systemic inflammatory response syndrome,SIRS),which is counter-regulated leading to suppression of the immune system and the failure to accurately manage the infection.22This scenario may lead to organ failure and death,a complication known as septic shock.Septic episodes,with an incidence of estimated>750000cases each year in the United States of America,are associated with a high mortality rate among patients who develop septic shock,ranging in severe cases from30%to70%.23Apart from Gram-negative bacteria,sepsis can be elicited also by other pathogens such as Gram-positive bacteria,viruses,and fungi.
石志高
Already in1892,Richard Pfeiffer attributed toxic effects to the action of heat-stable components of the Gram-negative bacterium Vibrio cholerae.The term endotoxin was thus introduced to distinguish this class of toxins from actively secreted heat-labile exotoxins,and subsequently LPSs were recognized as endotoxins(see below).Reviews on the history of endotoxin have been published recently.24,25Despite the fact that many of the molecular events during the activation of the immune system by LPS have been elucidated in in vitro test systems18,23,25and animal models of septic shock,26and despite the availability of improved antibiotic treatment even today,the treatment of septic shock is still difficult.22,25
The amphiphilic nature of LPS leading to larger aggregates in solution and difficulties in obtaining pure prepara-tions precluded a detailed structural analysis for decades and therefore an establishment of structure–activity relation-ships.Only after the development of extraction procedures for the preparation of homogeneous LPS,27,28chemical and biological meaningful experiments could be performed.For the structural analysis,the application of degradation by chemical means was necessary and it was realized that upon treatment with mild acid in water,a precipitate could be obtained which was termed lipid A and which represented the lipid anchor of the LPS molecule.29Its exact chemical structure(see below)remained elusive for several decades due to
the difficult chemistry of this complex molecule.In1983the structure was elucidated and unequivocally proved to be correct after its chemical synthesis.30 The available material also paved the way for the identification of lipid A as the endotoxic principle of LPS31–33and the acquired knowledge allowed then the detailed investigation of structure–activity relationships,34–36including a biophysical characterization of biologically active and inactive lipid A.37
Although biological activities of the isolated LPS have been well established in vitro and in vivo,there was some uncertainty with regard to the role as virulence factor during natural infections.The improved knowledge of the biosynthesis of lipid A38together with the established structure–activity relationships,allowed the construction of mutant bacteria which express functional but nontoxic lipid A39which showed that upon infection of mice despite an in vivo growth rate comparable to the wild-type strain,the mutant was unable to cause disease.LPS can thus be regarded an important virulence factor also during infections with Gram-negative bacteria.It is therefore expected that a detailed chemical characterization of bioactive LPS and the establishment of structure–function relationships in terms of immune cell activation will allow an antiendotoxic treatment during infections with Gram-negative bacteria and possibly help to prevent the development of septic shock.Since LPS is essential for the viability of most Gram-negative bacteria(discussed below),critical enzymatic
steps for its biosynthesis represent attractive targets for the development of novel antibiotics40and inhibitors of lipid A and Kdo biosynthesis have been developed.41,42 A detailed knowledge of structure–function relationships of LPS in bacterial membranes may also lead to novel antiinfectious agents.Since LPS are surface molecules which are frequently accessible to antibodies,they represent potential targets for vaccination.
1.04.
2.2Functional Aspects of LPS
In bacteria which are not encapsulated,LPSs are exposed on the surface of the bacterial cell and thus represent the first-line defense against various chemical and physical stresses associated with the natural habitats of bacteria.In particular,in situations where an infection in mammals is established,some pathogenic bacteria are able to resist the mounted counterattack consisting of a whole range of antimicrobials in activated serum,for example,antibacterial peptides,antibodies,the deposition of complement and formation of the membrane attack complex,in addition to ingestion and destruction by cellular phagocytosis.Some Gram-negative bacteria are able to evade the serum attack by their ability to grow intracellulary within mammalian ,Chlamydia,Yersinia,Salmonella,Brucella abortus).
A similar ecological niche is occupied by nitrogen-fixing symbiotic bacteria such as Rhizobium in plants which has been
126Microbial Polysaccharide Structures
suggested to involve similar protective mechanisms,43and the chronic intracellular infection of alfalfa nodules by Sinorhizobium meliloti has been shown to depend on the structure of its LPS.44
LPSs have evolved to support bacterial growth in these very different environments.The environmental condi-tions may change accidentally or such changes are regular events as part of the biology of the bacteria,for example, the change of host species from rodent to insect and mammal by Yersinia pestis.45–48Bacteria have thus developed the ability to sense environmental changes in pH,salt concentration,and temperature by two-component regulatory systems such as PhoP/PhoQ and PmrA/PmrB.Such systems are also involved in the structural modification of LPS.49–55Random phase variation may also occur in a bacterial ,in Neisseria and Haemophilus)which is based on regular genetic events56and leads to a preadaptation of a certain percentage of the culture to likely encountered environmental conditions.
The fact that LPSs have been preserved during evolution indicates their biological importance for the
survival of Gram-negative bacteria.The outer membrane of these bacteria represents a permeation barrier which very effectively prevents the lateral diffusion of hydrophobic molecules such as detergents,bile salts,antibiotics,and large glycopep-tides.57–59This is attributed to tight lateral interactions between a large number of anionic groups present in LPS molecules which are bridged by divalent cations such as Mg2þand Ca2þ.These charged groups are mainly located close to the surface of the membrane.The fatty acids in LPS are highly ordered in a gel-like state and in a nearly crystalline arrangement with transition temperatures up to about60 C.59The unsubstituted hydroxyl groups of b-hydroxylated fatty acids have been suggested to participate in intermolecular hydrogen bonds,further strengthening the ordered structure of LPS in membranes.59
Due to the barrier properties of the outer membrane,Gram-negative bacteria have developed transport mechanisms to allow uptake of nutrients and export of waste products.They are able to use fatty acids as energy source,and their transport through the outer membrane is mediated by outer membrane proteins.60A recently solved crystal structure of the FadL protein li,which is involved in long-chain fatty acid uptake,gives an indication of how this transport is achieved.61T ransport of hydrophilic substrates across the outer membrane is primarily mediated by passive diffusion through nonspecific or substrate-specific porins.59An exception is the energy-driven active
transport of siderophores across the outer membrane.59LPSs form the matrix for those proteins which are embedded in the outer membrane and have been shown to be important for their correct folding,62,63oligomerization,64,65and functioning.66,67Therefore,mutations which lead to severely truncated LPS are known to affect the formation of a functional outer membrane referred to as the deep-rough phenotype which is characterized by a higher permeability toward hydrophobic agents.57,59,68,69Mutations which affect the available amount of functional LPS molecules in a cell are accompanied by the loss of the barrier function and lead to a hypersensitivity against hydrophobic substances59 which has been explained by the introduction of patches of phospholipids into the outer leaflet of the outer membrane in order to compensate the reduced amount of available LPS.
Mutations in genes which are involved in early LPS biosynthesis are known to interfere also with the assembly of flagella and pili.70–75Structural analysis of the outer membrane protein FhuA which belongs to a family of proteins that mediates the active transport of siderophores has revealed by accidental co-crystallization a crystal structure li K-12LPS and details of the interaction with this outer membrane protein.76In this complex most of the important hydrogen-bonding or electrostatic interactions with LPS were provided by eight positively charged residues of FhuA.A data
base search based on this complex has identified a similar structural motif of a subset of four amino acids in various proteins which are known to bind to lipid A and of which some are involved in innate immune responses.77
Cationic antimicrobial peptides(CAMPs)and proteins,like defensins and polymyxins,among others,effectively disintegrate the LPS assembly by targeting the negatively charged groups,and compromise the barrier function.57,59,78 When grown in the presence of such ica bacteria were isolated which showed a resistant phenotype which is correlated with the expression of structurally modified LPS(see below).79Some bacteria,like Pseudomonas aeruginosa and Burkholderia cepacia are known to be intrinsically resistant toward CAMP and have been shown to contain similarly modified LPS80(and references cited therein).
1.04.
2.3The Chemical Structure of Smooth(S)-Type LPS芥川龙之介河童
LPSs are phosphorylated glycolipids which possess a complex chemical structure and many reviews on LPS structures and their biosynthesis has been published,some of which appeared more recently.38,40,80–85The lipid anchor of LPS, called lipid A,in most bacteria studied consists of an N-
and O-acylated(b1!6)-linked D-glucosamine(GlcN)
disaccharide which is phosphorylated in positions 1and 40(lipid A backbone).b -Hydroxylated fatty acids are characteristic components of lipid A which,for biosynthetic reasons,38always quantitatively substitute the 2-and 20-positions of the backbone in an amide linkage.They may be further located at the 3-and 30-positions in the ester-linkage and esterified at the b -hydroxyl group (secondary fatty acids).80Attached to the 60-position of the lipid A backbone is a heteropolysaccharide of varying length via a ketosidic linkage involving either D -glycero -D -talo -oct-2-ulosonic acid (Ko)in Acinetobacter or 3-deoxy-D -manno -oct-2-ulosonic acid (Kdo)in all other bacteria (for structural differences,see Figure 2).pacia,Ko replaces the terminal Kdo residue 86and in Y .pestis either a single Kdo or an a -Ko-(2!4)-a -Kdo-disaccharide is expressed.87,88The ketosidic linkage between the core and the lipid A is generally labile toward mild acid;however,replacement of Kdo by Ko leads to an increased acid stability of this linkage.Thus,mild acid treatment is often applied to cleave the lipid A from the remaining saccharides.Alternatively,strong alkaline hydrolysis may be applied for complete deacylation (see below).
In many bacteria,a core-oligosaccharide (core-OS),which,based on genetic and structural differences,may be further subdivided into an inner and outer core,connects a long carbohydrate cha
in,the O-polysaccharide (O-PS),to the lipid A.Within enterobacteria,a characteristic component of the inner core region is heptose which li and Salmonella mostly possesses the L -glycero -D -manno configuration (L ,D -Hep,Figure 2);D ,D -Hep has been found in LPS li 89and bacteria other li and Salmonella .82,83However,after many structures have been elucidated,it becomes evident that a clear distinction between an inner and an outer core based solely on composition cannot be applied easily to LPS from many other bacterial species.Although Kdo or Ko have so far always been found in LPS,connecting the core with the lipid A,some LPSs do not contain heptoses.83In addition,heptose has been identified as component of the outer core (e.g.,li K-12and Klebsiella )and even the O-PS and,as we know now,the same holds true for Kdo.T ypical examples of LPSs in which the core region can be divided into an inner and an outer core are LPSs from Enterobacteriaceae and Pseudomonadaceae .
In general,LPSs which contain an O-PS are referred to as smooth (S)-type LPSs due to a smooth colony appearance of these bacteria.Such bacteria express a mixture of LPS molecules which differ in the length of the O-PS leading to a characteristic banding pattern in polyacrylamide electrophoresis.Additional heterogeneity results from molecules devoid of the O-PS,called rough (R)-type LPS,and from nonstoichiometrical structural variations in all parts of the LPS molecule including
the lipid A.R-type LPS can be isolated in large amounts from mutant bacteria which have a defect in LPS biosynthesis.Due to the low proportion of R-type LPS in wild-type bacteria,the first chemical analysis of core-OS was only possible from LPS of such mutant bacteria and phage typing was often used as a helpful tool.90–97Apart from these mutants,in certain other nonenteric pathogenic bacteria such as Neisseria ,Haemophilus ,Chlamydia ,and Y .pestis only R-type LPSs are present which are naturally devoid of O-PS and contain short oligosac-charide chains instead.82The term lipooligosaccharides (LOSs)was introduced by some authors to distinguish
Kdo L,D -Hep
D,D -Hep Ko Figure 2Chemical structures of 3-deoxy-D -manno -oct-2-ulosonic acid (Kdo),D -glycero -D -talo -oct-2-ulosonic acid (Ko),L -glycero -D -manno -heptose (L ,D -Hep),and D -glycero -D -manno -heptose (D ,D -Hep).Microbial Polysaccharide Structures 127
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