Mechanism of O-antigen distribution in lipopolysaccharide.

O-antigen units are nonuniformly distributed among lipid A-core molecules in lipopolysaccharide (LPS) from gram-negative bacteria, as revealed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; the actual distribution patterns are complex, multimodal, and strain specific. Although the basic biochemical steps involved in synthesis and polymerization of O-antigen monomers and their subsequent attachment to lipid A-core are known, the mechanism by which specific multimodal distribution patterns are attained in mature LPS has not been previously considered theoretically or experimentally. We have developed probability equations which completely describe O-antigen distribution among lipid A-core molecules in terms of the probability of finding a nascent polymer (O antigen linked to carrier lipid) of length k (Tk) and the probability that a nascent polymer of length k will be extended to k + 1 by polymerase (pk) or transferred to lipid A-core by ligase (qk). These equations were used to show that multimodal distribution patterns in mature LPS cannot be produced if all pk are equal to p and all qk are equal to q, conditions which indicate a lack of selectivity of polymerase and ligase, respectively, for nascent O-antigen chain lengths. A completely stochastic model (pk = p, qk = q) of O-antigen polymerization and transfer to lipid A-core was also inconsistent with observed effects of mutations which resulted in partial inhibition of O-antigen monomer synthesis, lipid A-core synthesis, or ligase activity. The simplest explanation compatible with experimental observations is that polymerase or ligase, or perhaps both, have specificity for certain O-antigen chain lengths during biosynthesis of LPS. Our mathematical model indicates selectively probably was associated with the polymerase reaction. Although one may argue for a multimodal distribution pattern based on a kinetic mechanism i.e., varying reaction parameters in space or in time during cell growth, such a model requires complex sensory and regulatory mechanisms to explain the mutant data and mechanisms for sequestering specific components of LPS biosynthesis to explain the distribution pattern in normal cells. We favor the simple alternative of enzyme specificity and present generalized equations which should be useful in analysis of other analogous biochemical systems.     

Defining function of lipopolysaccharide O-antigen ligase WaaL using chemoenzymatically synthesized substrates

The WaaL-mediated ligation of O-antigen onto the core region of the lipid A-core block is an important step in the lipopolysaccharide (LPS) biosynthetic pathway. Although the LPS biosynthesis has been largely characterized, only a limited amount of in vitro biochemical evidence has been established for the ligation reaction. Such limitations have primarily resulted from the barriers in purifying WaaL homologues and obtaining chemically defined substrates. Accordingly, we describe herein a chemical biology approach that enabled the reconstitution of this ligation reaction. The O-antigen repeating unit (O-unit) of Escherichia coli O86 was first enzymatically assembled via sequential enzymatic glycosylation of a chemically synthesized GalNAc-pyrophosphate-undecaprenyl precursor. Subsequent expression of WaaL through use of a chaperone co-expression system then enabled the demonstration of the in vitro ligation between the synthesized donor (O-unit-pyrophosphate-undecaprenyl) and the isolated lipid A-core acceptor. The previously reported ATP and divalent metal cation dependence were not observed using this system. Further analyses of other donor substrates revealed that WaaL possesses a highly relaxed specificity toward both the lipid moiety and the glycan moiety of the donor. Lastly, three conserved amino acid residues identified by sequence alignment were found essential for the WaaL activity. Taken together, the present work represents an in vitro systematic investigation of the WaaL function using a chemical biology approach, providing a system that could facilitate the elucidation of the mechanism of WaaL-catalyzed ligation reaction.     

Occurence of antibodies against chlamydial lipopolysaccharide in human sera as measured by ELISA using an artificial glycoconjugate antigen

Abstract An artificial glycoconjugate containing, as a ligand, the deacylated carbohydrate backbone of a recombinant Chlamydia -specific lipopolysaccharide was used as a solid-phase antigen in ELISA to measure antibodies against chlamydial LPS. The specificity and reproducibility of the assay was shown by using a panel of prototype monoclonal antibodies representing the spectrum of antibodies also occuring in patient sera. These mAbs recognized Chlamydia -specific epitopes [ α 2→8-linked disaccharide of 3-deoxy- d - manno -octulosonic acid (Kdo) or the trisaccharide α Kdo-(2→8)-→Kdo] or those shared between chlamydial and Re-type LPS ( α Kdo, α →4-linked Kdo disacccharide). The assay was used to measure IgG, IgA and IgM antibodies against chlamydial LPS in patients with genital or respiratory tract infections. In comparison to the results obtained with sera from blood donors, it became evident that both types of infection result in significant changes in the profile of LPS antibodies.     

Variable expression of O-antigen and the role of lipopolysaccharide as an adhesin in Aeromonas sobria

Abstract On initial isolation of Aeromonas sobria 3767 from a diarrhoeal stool specimen, two colony types were obtained: opaque (3767O) and translucent (3767T). Strain 3767O consistently produced lipopolysaccharide (LPS) core and O-antigen side chain, detectable by SDS-PAGE and by Western blotting with an O-antigen-specific monoclonal antibody. Strain 3767T produced LPS core but the amount of O-antigen was dependent on factors including growth medium and bacterial growth phase. Strain 3767T exhibited significantly lower levels of adhesion to HEp-2 cells than 3767O and this correlated with the level of LPS expression, with the greatest reduction (61%) at stationary phase when no LPS was detectable. The results implicate LPS as an adhesin for A. sobria 3767.     

Common themes and variations in the rhodanese superfamily

The rhodanese homology domain is a ubiquitous fold found in several phylogenetically related proteins encoded by eubacterial, archeal, and eukaryotic genomes. Although rhodanese-like proteins share evolutionary relationships, analysis of their sequences highlights that they are so heterogeneous to form the rhodanese superfamily. The variability occurs at different levels including sequence, active site loop length, presence of a critical catalytic Cys residue, and domain arrangement. Even within the same genome, multiple genes encode rhodanese-like proteins presenting with variably arranged rhodanese domain(s): as single or tandem domain(s), or combined with other protein domain(s). Given the highly variable organization of the rhodanese domain(s) and the context where it is found, here we review the structural organization and function of the rhodanese-like proteins. The overview of the most recent findings about rhodanese allow us to depict a superfamily of versatile proteins relying on persulfide chemistry to accomplish cellular functions spanning from resistance to environmental threats, such as cyanide, and key cellular reactions related to sulfur metabolism and progression of cell cycle.     

Common themes in mechanisms of gene silencing

The assembly of DNA into regions of inaccessible chromatin, called silent chromatin, is involved in the regulation of gene expression and maintenance of chromosome stability in eukaryotes. Recent studies on Sir2-containing silencing complexes in budding yeast and HP1- and Swi6-containing silencing complexes in metazoans and fission yeast suggest a common mechanism for the assembly of these domains, which involves the physical coupling of histone modifying enzymes to histone binding proteins.     

Structure and heterogeneity of the O-antigen chain of Salmonella Agona lipopolysaccharide

Lipopolysaccharide of Salmonella Agona smooth-type cells was obtained from bacteria by a hot phenol-water extraction procedure. Mild acid hydrolysis of lipopolysaccharide, followed by gel filtration, yielded the pure O-polysaccharide. Abequose, rhamnose, mannose, galactose and glucose in the molar ratio 0.8 : 1.0 : 1.0 : 1.1 : 0.5 were detected, and their linkages were established. Sugar configurations were determined by gas chromatography. Two repeating units, namely -->2)-[alpha-Abep-(1-->3)-]-alpha-d-Manp-(1-->4)-alpha-l-Rhap-(1-->3)-alpha-d-Galp-(1-->and -->2)-[alpha-Abep-(1-->3)-]-alpha-d-Manp-(1-->4)-alpha-l-Rhap-(1-->3)-[alpha-d-Glcp-(1-->4)-]-alpha-d-Galp-(1-->, were deduced from nuclear magnetic resonance studies. The effort to separate them was unsuccessful. An immunochemical test performed by means of Western blotting with anti O12 serum demonstrated that glucose was present in the longer lipopolysaccharide chains, at some distance from the core region.     

Peripheral nervous system defects in a mouse model for peroxisomal biogenesis disorders

Peroxisome biogenesis disorders (PBD) are autosomal recessive disorders in humans characterized by skeletal, eye and brain abnormalities. Despite the fact that neurological deficits, including peripheral nervous system (PNS) defects, can be observed at birth in some PBD patients including those with PEX10 mutations, the embryological basis of the PNS defects is unclear. Using a forward genetic screen, we identified a mouse model for Pex10 deficiency that exhibits neurological abnormalities during fetal development. Homozygous Pex10 mutant mouse embryos display biochemical abnormalities related to a PBD deficiency. During late embryogenesis, Pex10 homozygous mutant mice experience progressive loss of movement and at birth they become cyanotic and die shortly thereafter. Homozygous Pex10 mutant fetuses display decreased integrity of axons and synapses, over-extension of axons in the diaphragm and decreased Schwann cell numbers. Our neuropathological, molecular and electrophysiological studies provide new insights into the embryological basis of the PNS deficits in a PBD model. Our findings identify PEX10 function, and likely other PEX proteins, as an essential component of the spinal locomotor circuit.     

Characterization of the lipopolysaccharide O-antigen of Francisella novicida (U112)

Francisella novicida (U112), a close relative of the highly virulent bacterium F. tularensis, was shown to produce a lipopolysaccharide in which the antigenic O-polysaccharide component was found by chemical, 1H and 13C NMR and MS analyses to be an unbranched neutral linear polymer of a repeating tetrasaccharide unit composed of 2-acetamido-2-deoxy-D-galacturonamide (D-GalNAcAN) and 2,4-diacetamido-2,4,6-trideoxy-D-glucose (D-Qui2NAc4NAc, di-N-acetylbacillosamine) residues (3:1) and had the structure: -->4)-alpha-D-GalNAcAN-(1-->4)-alpha-D-GalNAcAN-(1-->4)-alpha-D-GalNAcAN-(1-->3)-alpha-D-QuiNAc4NAc-(1-->. With polyclonal murine antibody, the F. novicida O-antigen did not show serological cross-reactivity with the O-antigen of F. tularensis despite the occurrence of a common -->4)-D-GalpNAcAN-(1-->4)-alpha-D-GalpNAcAN-(1--> disaccharide unit in their respective O-antigens. Thus, O-PS serology offers a practical way to distinguish between the two Francisella species.     

Evolution of metabolic diversity in polyketide-derived pyrones: Using the non-colinear aureothin assembly line as a model system

Polyketide-derived pyrones are structurally diverse secondary metabolites that are represented in all three kingdoms of life and are endowed with various biological functions. The aureothin family of Streptomyces metabolites was chosen as a model to study the factors governing structural diversity and the evolutionary processes involved. This review highlights recent insights into the non-colinear aureothin and neoaureothin modular type I polyketide synthase (PKS), aromatic starter unit biosynthesis, polyketide tailoring reactions, and a non-enzymatic polyene splicing cascade. Pyrone biosynthesis in bacteria, fungi, and plants is compared. Finally, various strategies to increase metabolic diversity of aureothin derivatives through mutasynthesis, pathway engineering, and biotransformation are presented. The unusual aureothin and neoaureothin assembly lines thus not only represent a model for PKS evolution, but provided important insights into non-canonical enzymatic processes that could be employed for the production of antitumor and antifungal agents.     

Investigating heavy-metal hyperaccumulation using Thlaspi caerulescens as a model system

Background

Metal-hyperaccumulating plant species are plants that are endemic to metalliferous soils and are able to tolerate and accumulate metals in their above-ground tissues to very high concentrations. One such hyperaccumulator, Thlaspi caerulescens, has been widely studied for its remarkable properties to tolerate toxic levels of zinc (Zn), cadmium (Cd) and sometimes nickel (Ni) in the soil, and accumulate these metals to very high levels in the shoot. The increased awareness regarding metal-hyperaccumulating plants by the plant biology community has helped spur interest in the possible use of plants to remove heavy metals from contaminated soils, a process known as phytoremediation. Hence, there has been a focus on understanding the mechanisms that metal-hyperaccumulator plant species such as Thlaspi caerulescens employ to absorb, detoxify and store metals in order to use this information to develop plants better suited for the phytoremediation of metal-contaminated soils.

Scope

In this review, an overview of the findings from recent research aimed at better understanding the physiological mechanisms of Thlaspi caerulescens heavy-metal hyperaccumulation as well as the underlying molecular and genetic determinants for this trait will be discussed. Progress has been made in understanding some of the fundamental Zn and Cd transport physiology in T. caerulescens. Furthermore, some interesting metal-related genes have been identified and characterized in this plant species, and regulation of the expression of some of these genes may be important for hyperaccumulation.

Conclusions

Thlaspi caerulescens is a fascinating and useful model system not only for studying metal hyperaccumulation, but also for better understanding micronutrient homeostasis and nutrition. Considerable future research is still needed to elucidate the molecular, genetic and physiological bases for the extreme metal tolerance and hyperaccumulation exhibited by plant species such as T. caerulescens.Key words: Zn, Cd, Ni, Thlaspi caerulescens, hyperacumulator, phytoremediation, heavy metal     

Elucidation of the structure of the lipopolysaccharide core and the linkage between the core and the O-antigen in Pseudomonas aeruginosa immunotype 5 using strong alkaline degradation of the lipopolysaccharide

The products of the strong alkaline degradation of the lipopolysaccharide (LPS) of Pseudomonas aeruginosa immunotype 5 were separated by anion-exchange HPLC and studied by electrospray ionization mass spectrometry and NMR spectroscopy.It was found that two major products have the same inner core region and lipid A carbohydrate backbone but different outer core regions.The difference is in the position of a rhamnose residue,which is substituted with either an additional glucose residue or a disaccharide remainder of the degraded O-polysaccharide.The site and the configuration of the linkage between the O-polysaccharide and the core were determined and,together with published data,the structure of the so-called biological repeating unit of the O-antigen was defined.The glycosidic linkage of the 2-acetamido-2,6-dideoxy D-glucose (N-acetyl-D-quinovosamine) residue is when it links the O-polysaccharide to the core and when it connects the interior repeating units of the O-polysaccharide to each other.     

Glucosylated N-acetyllactosamine O-antigen chain in the lipopolysaccharide from Helicobacter pylori strain UA861

The O-antigen chain from the lipopolysaccharide of Helicobacter pylori strain UA861 was determined to be composed of an elongated type 2 N - acetyllactosamine backbone, -[-->3)-beta-D-Gal-(1-->4)-beta-D-GlcNAc-(1- ]n-->, with approximately half of the GlcNAc units carrying a terminal alpha-d-Glc residue at the O -6 position. The O-chain of H.pylori UA861 was terminated by a N -acetyllactosamine [beta-D-Gal-(1-->4)-beta-D- GlcNAc] (LacNAc) epitope and did not express terminal Lewis X or Lewis Y blood-group determinants as previously found in other H.pylori strains. The absence of terminal Lewis X and Lewis Y blood-group epitopes and the replacement of Fuc by Glc as a side chain in the O- chain of H.pylori UA861 represents yet another type of lipopolysaccharide structure from H.pylori species. These structural differences in H.pylori lipopolysaccharide molecules carry implications with regard to possible different pathogenic events between strains and respective hosts.      

Functional genomic approaches using the nematode Caenorhabditis elegans as a model system

Since the completion of the genome project of the nematode C. elegans in 1998, functional genomic approaches have been applied to elucidate the gene and protein networks in this model organism. The recent completion of the whole genome of C. briggsae, a close sister species of C. elegans, now makes it possible to employ the comparative genomic approaches for identifying regulatory mechanisms that are conserved in these species and to make more precise annotation of the predicted genes. RNA interference (RNAi) screenings in C. elegans have been performed to screen the whole genome for the genes whose mutations give rise to specific phenotypes of interest. RNAi screens can also be used to identify genes that act genetically together with a gene of interest. Microarray experiments have been very useful in identifying genes that exhibit co-regulated expression profiles in given genetic or environmental conditions. Proteomic approaches also can be applied to the nematode, just as in other species whose genomes are known. With all these functional genomic tools, genetics will still remain an important tool for gene function studies in the post genome era. New breakthroughs in C. elegans biology, such as establishing a feasible gene knockout method, immortalized cell lines, or identifying viruses that can be used as vectors for introducing exogenous gene constructs into the worms, will augment the usage of this small organism for genome-wide biology.     

Structure of the O-antigen of the main lipopolysaccharide isolated from Sinorhizobium fredii SMH12

The lipopolysaccharide of Sinorhizobium fredii SMH12, a wide-range host bacterium isolated from nodulated soybean plants growing in Vietnam, has been studied. Isolation of lipopolysaccharide by the phenol-water method leads to a mixture of two polysaccharides; polyacrylamide gel electrophoresis indicates that both are possibly lipopolysaccharides. The structures of the O-antigen of the main lipopolysaccharide and its deacetylated form are determined by sugar and methylation analysis, partial hydrolysis, lithium degradation, ESI-MS/MS, and NMR studies. Here we show that the fast-growing S. fredii SMH12 produces a lipopolysaccharide whose O-antigen has a repeating unit consisting of the trisaccharide -->4)-alpha-D-Gal pA-(1-->3)-2-O-Ac-alpha-L-Rha p-(1-->3)-2-O-Ac-alpha-D-Man p-(1-->. The position O-6 of the mannose residue in the repeating unit is unsubstituted, acetylated, or methylated in an approximate ratio 1:1:2. The tandem mass spectrometry studies rule out both an alternating and a random distribution of methyl groups and suggest the existence of zones in the polysaccharide rich in methyl groups interspersed with zones without methyl groups.     

Chemical and biological studies on the lipopolysaccharide (O-antigen) of Anacystis nidulans

The O-antigen (lipopolysaccharide) of Anacystis nidulans, strain KM, has been isolated from whole cells and from cell wall preparations by phenolwater extraction. The polysaccharide moiety consists of a D-mannose polymer accompanied by smaller amounts of 3- and 4-O-methyl-D-mannoses, D-galactose, D-glucose, L-fucose, D-glucosamine, mannosamine and 2-keto-3-deoxyoctonate. Aldoheptoses are lacking. The degraded polysaccharide is split from lipid A by acid hydrolysis (10% acetic acid, 100°C, 3 h) whereby 2-keto-3-deoxyoctonate is released in small amounts. Degraded polysaccharide forms only one major fraction by Sephadex G-50 gel-filtration. This fraction includes all the sugars mentioned above except L-fucose, which is released during the acetic acid degradation. Periodate studies and methylation analysis revealed that the poly-mannose chain consists of about 75% 13 linked and of 25% 14 linked D-mannose units.Lipid A of A. nidulans is phosphate-free. The main fatty acid, -hydroxypalmitic acid, is exclusively amide-bound, presumably to the amino group of D-glucosamine. Other fatty acids, found as minor constituents, are -hydroxymyristic, palmitic and stearic acids. Lipopolysaccharide of A. nidulans KM exhibits high anticomplementary activity in guineapig serum. It is about 800 times less toxic for adrenalectomized mice than endotoxin from Salmonella typhimurium.The isolated lipopolysaccharide reacts with rabbit antisera against living or heat-killed cells of A. nidulans in passive hemagglutination, when untreated or heated, but not when alkali-treated lipopolysaccharide is used for red blood cell sensibilization. It is concluded that lipopolysaccharide of A. nidulans KM is exposed on the surface of the cell.     

Characterization of functional oligosaccharide mimics of the Shigella flexneri serotype 2a O-antigen: implications for the development of a chemically defined glycoconjugate vaccine

Protection against reinfection with noncapsulated Gram-negative bacteria, such as Shigella, an enteroinvasive bacterium responsible for bacillary dysentery, is mainly achieved by Abs specific for the O-Ag, the polysaccharide part of the LPS, the major bacterial surface Ag. The use of chemically defined glycoconjugates encompassing oligosaccharides mimicking the protective determinants carried by the O-Ag, thus expected to induce an efficient anti-LPS Ab response, has been considered an alternative to detoxified LPS-protein conjugate vaccines. The aim of this study was to identify such functional oligosaccharide mimics of the S. flexneri serotype 2a O-Ag. Using protective murine mAbs specific for S. flexneri serotype 2a and synthetic oligosaccharides designed to analyze the contribution of each sugar residue of the branched pentasaccharide repeating unit of the O-Ag, we demonstrated that the O-Ag exhibited an immunodominant serotype-specific determinant. We also showed that elongating the oligosaccharide sequence improved Ab recognition. From these antigenicity data, selected synthetic oligosaccharides were assessed for their potential to mimic the O-Ag by analyzing their immunogenicity in mice when coupled to tetanus toxoid via single point attachment. Our results demonstrated that induction of an efficient serotype 2a-specific anti-O-Ag Ab response was dependent on the length of the oligosaccharide sequence. A pentadecasaccharide representing three biological repeating units was identified as a potential candidate for further development of a chemically defined glycoconjugate vaccine against S. flexneri 2a infection.