Biosynthesis Of Bacterial Polysaccharides Research Articles

Connection between protein-tyrosine kinase inhibition and coping with oxidative stress in Bacillus subtilis

In bacteria, attenuation of protein-tyrosine phosphorylation occurs during oxidative stress. The main described mechanism behind this effect is the H2O2-triggered conversion of bacterial phospho-tyrosines to protein-bound 3,4-dihydroxyphenylalanine. This disrupts the bacterial tyrosine phosphorylation-based signaling network, which alters the bacterial polysaccharide biosynthesis. Herein, we report an alternative mechanism, in which oxidative stress leads to a direct inhibition of bacterial protein-tyrosine kinases (BY-kinases). We show that DefA, a minor peptide deformylase, inhibits the activity of BY-kinase PtkA when Bacillus subtilis is exposed to oxidative stress. High levels of PtkA activity are known to destabilize B. subtilis pellicle formation, which leads to higher sensitivity to oxidative stress. Interaction with DefA inhibits both PtkA autophosphorylation and phosphorylation of its substrate Ugd, which is involved in exopolysaccharide formation. Inactivation of defA drastically reduces the capacity of B. subtilis to cope with oxidative stress, but it does not affect the major oxidative stress regulons PerR, OhrR, and Spx, indicating that PtkA inhibition is the main pathway for DefA involvement in this stress response. Structural analysis identified DefA residues Asn95, Tyr150, and Glu152 as essential for interaction with PtkA. Inhibition of PtkA depends also on the presence of a C-terminal α-helix of DefA, which resembles PtkA-interacting motifs from known PtkA activators, TkmA, SalA, and MinD. Loss of either the key interacting residues or the inhibitory helix of DefA abolishes inhibition of PtkA in vitro and impairs postoxidative stress recovery in vivo, confirming the involvement of these structural features in the proposed mechanism.

Glycosyltransferase-Coupled Assays for 4-Epimerase WbpP from Pseudomonas aeruginosa.

The donor substrates for the biosynthesis of bacterial polysaccharides include UDP-Glc/Gal and UDP-GlcNAc/GalNAc. The conversion of these nucleotide sugars is catalyzed by 4-epimerases. The wbpP gene of Pseudomonas aeruginosa encodes a 4-epimerase that has a preference for UDP-GlcNAc/GalNAc as substrates. Other 4-epimerases have broad specificities or preference for UDP-Glc/Gal. We have developed coupled assays where the 4-epimerase product is used as a donor substrate for glycosyltransferases that are highly specific for the nucleotide sugar structure. We describe here a method for the study of substrate specificity of WbpP, using coupled assays employing four different glycosyltransferases. These protocols can be applied to the identification and characterization of novel 4-epimerases and to determine their substrate specificities.

Advancing Homogeneous Antimicrobial Glycoconjugate Vaccines.

Since 2004, when the first synthetic glycoconjugate vaccine against the pneumonia and meningitis causing bacterium Haemophilus influenza type b (Hib) approved for human use in Cuba was reported, 34 million doses of the synthetic vaccine have been already distributed in several countries under the commercial name of Quimi-Hib. However, despite the success of this product, no other synthetic glycoconjugate vaccine has been licensed in the following 13 years. As well as avoiding the need to handle pathogens, synthetic glycoconjugates offer clear advantages in terms of product characterization and the possibility to understand the parameters influencing immunogenicity. Nevertheless, large scale application of synthetic sugars has been perceived as challenging because of manufacturing costs and process complexity compared to natural polysaccharides. Chemoenzymatic approaches, one-pot protocols, and automated solid-phase synthesis are rendering carbohydrate production considerably more attractive for industrialization. Here we identify three areas where chemical approaches can advance this progress: (i) chemical or enzymatic methods enabling the delivery of the minimal polysaccharide portion responsible for an effective immune response; (ii) site-selective chemical or enzymatic conjugation strategies for the exploration of the conjugation point in immune responses against carbohydrate-based vaccines, and the consistent preparation of more homogeneous products; (iii) multicomponent constructs targeting receptors responsible for immune response modulation in order to control its quality and magnitude. We discuss how synthesis of bacterial oligosaccharides is useful toward understanding the polysaccharide portion responsible for immunogenicity, and for developing robust and consistent alternatives to natural heterogeneous polysaccharides. The synthesis of sugar analogues can lead to the identification of hydrolytically more stable versions of oligosaccharide antigens. The study of bacterial polysaccharide biosynthesis aids the development of in vitro hazard-free oligosaccharide production. Novel site-selective conjugation methods contribute toward deciphering the role of conjugation sites in the immunogenicity of glycoconjugates and prove to be particularly useful when glycans are conjugated to protein serving as carrier and antigen. The orthogonal incorporation of two different carbohydrate haptens enables the reduction of vaccine components. Finally, coordinated conjugation of glycans and small molecule immunopotentiators supports simplification of vaccine formulation and localization of adjuvant. Synergistic advancement of these areas, combined with competitive manufacturing processes, will contribute to a better understanding of the features guiding the immunological activity of glycoconjugates and, ultimately, to the design of improved, safer vaccines.

Structural determination of Streptococcus pneumoniae repeat units in serotype 41A and 41F capsular polysaccharides to probe gene functions in the corresponding capsular biosynthetic loci

We report the repeating unit structures of the native capsular polysaccharides of Streptococcus pneumoniae serotypes 41A and 41F. Structural determinations yielded six carbohydrate units in the doubly branched repeating unit to give the following structure for serotype 41A: [Display omitted] The structure determinations were motivated (1) by an ambition to help close the remaining gaps in S. pneumoniae capsular polysaccharide structures, and (2) by the attempt to derive functional annotations of carbohydrate active enzymes in the biosynthesis of bacterial polysaccharides from the determined structures. An activity present in 41F but not 41A is identified as an acetyltransferase acting on the rhamnopyranosyl sidechain E. The genes encoding the formation of the six glycosidic bonds in serogroup 41 were determined from the capsular polysaccharide structures of serotype 41A, 41F, and genetically related serotypes, in conjunction with corresponding genomic information and computational homology searches. In combination with complementary information, NMR spectroscopy considerably simplifies the functional annotation of carbohydrate active enzymes in the biosynthesis of bacterial polysaccharides.

Functional identification of a galactosyltransferase critical to Bacteroides fragilis Capsular Polysaccharide A biosynthesis

Capsular Polysaccharide A (CPSA), a polymer of a four-sugar repeating unit that coats the surface of the mammalian symbiont Bacteroides fragilis, has therapeutic potential in animal models of Multiple Sclerosis and other autoinflammatory diseases. Genetic studies have demonstrated that CPSA biosynthesis is dependent primarily on a single gene cluster within the B. fragilis genome. However, the precise functions of the individual glycosyltransferases encoded by this cluster have not been identified. In this report each of these glycosyltransferases (WcfQ, WcfP, and WcfN) have been expressed and tested for their function in vitro. Using a reverse phase high performance liquid chromatography (HPLC) assay, WcfQ and WcfP were found to transfer galactose from uridine diphosphate (UDP)-linked galactose (Gal) to N-acetyl-4-amino-6-deoxy-galactosamine (AADGal) linked to a fluorescent mimic of bactoprenyl diphosphate, the native isoprenoid anchor for bacterial polysaccharide biosynthesis. The incorporation of galactose to form a bactoprenyl-linked disaccharide was confirmed by radiolabel incorporation and mass spectrometry (MS) of purified product. Using varying concentrations of UDP-Gal and enzyme, WcfQ was found to be the most effective protein at transferring galactose, and is the most likely candidate for in vivo incorporation of the sugar. WcfQ also cooperated in the presence of three preceding biosynthetic enzymes to form an isoprenoid-linked disaccharide in a single-pot reaction. This work represents a critical step in understanding the biosynthetic pathway responsible for the formation of CPSA, an unusual and potentially therapeutic biopolymer.

Domain organization of the polymerizing mannosyltransferases involved in synthesis of the Escherichia coli O8 and O9a lipopolysaccharide O-antigens.

The Escherichia coli O9a and O8 polymannose O-polysaccharides (O-PSs) serve as model systems for the biosynthesis of bacterial polysaccharides by ATP-binding cassette transporter-dependent pathways. Both O-PSs contain a conserved primer-adaptor domain at the reducing terminus and a serotype-specific repeat unit domain. The repeat unit domain is polymerized by the serotype-specific WbdA mannosyltransferase. In serotype O9a, WbdA is a bifunctional α-(1→2)-, α-(1→3)-mannosyltransferase, and its counterpart in serotype O8 is trifunctional (α-(1→2), α-(1→3), and β-(1→2)). Little is known about the detailed structures or mechanisms of action of the WbdA polymerases, and here we establish that they are multidomain enzymes. WbdA(O9a) contains two separable and functionally active domains, whereas WbdA(O8) possesses three. In WbdC(O9a) and WbdB(O9a), substitution of the first Glu of the EX(7)E motif had detrimental effects on the enzyme activity, whereas substitution of the second had no significant effect on activity in vivo. Mutation of the Glu residues in the EX(7)E motif of the N-terminal WbdA(O9a) domain resulted in WbdA variants unable to synthesize O-PS. In contrast, mutation of the Glu residues in the motif of the C-terminal WbdA(O9a) domain generated an enzyme capable of synthesizing an altered O-PS repeat unit consisting of only α-(1→2) linkages. In vitro assays with synthetic acceptors unequivocally confirmed that the N-terminal domain of WbdA(O9a) possesses α-(1→2)-mannosyltransferase activity. Together, these studies form a framework for detailed structure-function studies on individual domains and a strategy applicable for dissection and analysis of other multidomain glycosyltransferases.

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.

In vitro bacterial polysaccharide biosynthesis: defining the functions of Wzy and Wzz

Polysaccharides constitute a major component of bacterial cell surfaces and play critical roles in bacteria/host interactions. The biosynthesis of such molecules, however, has mainly been characterized through in vivo genetic studies, thus precluding discernment of the details of this pathway. Accordingly, we present a chemical approach which enabled reconstitution of the E. coli O-polysaccharide biosynthetic pathway in vitro. Starting with chemically prepared N-Acetyl-D-galactosamine-diphospho-undecaprenyl, the E. coli O86 oligosaccharide repeating unit was assembled via sequential enzymatic glycosylation. Successful expression of the putative polymerase Wzy via a chaperone co-expression system then allowed demonstration of polymerization in vitro using this substrate. Analysis of additional substrates revealed a defined mode of recognition for Wzy towards the lipid moiety. Specific polysaccharide chain length modality was furthermore demonstrated to result from the action of Wzz. Collectively, polysaccharide biosynthesis was chemically reconstituted in vitro, providing a well-defined system for further underpinning molecular details of this biosynthetic pathway.

Microbial exopolysaccharides

This web entry provides general definitions and a list of exopolysaccahrides. It provides a starting point for searches on specific structural classes. Exopolysaccharides are important for bacterial survival, particularly for some pathogenic bacteria. This page focuses on this aspect of exopolysaccharides. This database represents a beginning effort to catalogue genes and metabolic pathways involved in the biosynthesis of bacterial polysaccharides. Gellan gum is a polysaccharide that is sometimes used as a gelling agent in place of agar. It is known commercially as Gelrite. Gellan gum is prepared commercially from Sphingomonas elodea. This web page describes the chemical structure and properties of gellan. This web page describes the specifications of gellan that are important in its commercial uses. This commercial page contains excellent information on the microbial polysaccahride levan. Levan is unusual in that it packs into spherical structures. This web page describes the specifications of alginate, a commercially important polysaccharide used in enzyme immobilization. Pullulan is a neutral exopolysaccharide made by different strains of Aureobasidium pullulans. The web page contains basic information about the polymer. Kefir is a popular fermented food made by lactobacillus fermentation. The Lactobacillus strain making kefir generates an exopolysaccharide known as kefiran. This web page contains interesting scanning probe microscopic images of various microbial exopolysaccahrides. Curdlan gum is an exopolymer prepared from cultures of Alcaligenes faecalis var. myxogenes. This page describes the chemical properties of the polymer. This commercial web page describes commercially relevant properties of the exopolysaccharide known as welan gum. This web abstract describes the properties of succinoglycan as a useful polymer. This web page describes an interesting exopolysaccharide produced by Schizophyllan commune. This website focuses on advanced oil recovery methods. Surprisingly, some microbial exopolysaccharides are used in advanced oil recovery where they plug rock fractures and prevent oil seepage.

Stop and go: regulation of chain length in the biosynthesis of bacterial polysaccharides

An immense range of polysaccharide structures is expressed on the bacterial surface. The length of the polymer can be a crucial attribute for virulence. Members of a family of 'polysaccharide copolymerase' proteins are essential for the regulation of polymer chain length, participating in one widespread biosynthesis scheme.

Genetic Analysis of the Capsular Biosynthetic Locus from All 90 Pneumococcal Serotypes

Several major invasive bacterial pathogens are encapsulated. Expression of a polysaccharide capsule is essential for survival in the blood, and thus for virulence, but also is a target for host antibodies and the basis for effective vaccines. Encapsulated species typically exhibit antigenic variation and express one of a number of immunochemically distinct capsular polysaccharides that define serotypes. We provide the sequences of the capsular biosynthetic genes of all 90 serotypes of Streptococcus pneumoniae and relate these to the known polysaccharide structures and patterns of immunological reactivity of typing sera, thereby providing the most complete understanding of the genetics and origins of bacterial polysaccharide diversity, laying the foundations for molecular serotyping. This is the first time, to our knowledge, that a complete repertoire of capsular biosynthetic genes has been available, enabling a holistic analysis of a bacterial polysaccharide biosynthesis system. Remarkably, the total size of alternative coding DNA at this one locus exceeds 1.8 Mbp, almost equivalent to the entire S. pneumoniae chromosomal complement.

Structures of the biological repeating units in the O-chain polysaccharides of Hafnia alvei strains having a typical lipopolysaccharide outer core region

Earlier, the structures of the O-chain polysaccharides of the lipopolysaccharides (LPS) of a number of Hafnia alvei strains have been established. However, it remained unknown, which is the first and the last monosaccharide of the O-chain. This is defined by the structure of the so-called biological repeating unit (O-unit), which is pre-assembled and then polymerised in the course of biosynthesis of bacterial polysaccharides by the Wzy-dependent pathway. Now we report on the structures of the O-units in 10 H. alvei strains. The LPS were cleaved by mild acid hydrolysis and oligosaccharide fractions IIIa and IIIb were isolated by gel chromatography subsequently on Sephadex G-50 and BioGel P-2 and studied by methylation analysis and NMR spectroscopy. Fraction IIIb was found to represent the core oligosaccharide containing a terminal upstream α- d-Glc-(1→3)-α- d-Glc or α- d-Gal-(1→3)-α- d-Glc disaccharide in the outer region that is typical of H. alvei. Fraction IIIa consists of the LPS core with one O-unit linked by a 3-substituted β- d-GalNAc residue (in strains PCM 1189 and PCM 1546) or a 3-substituted β- d-GlcNAc residue (in the other strains studied). In most strains examined the β-configuration of the d-GlcNAc linkage in the first O-unit attached to the core is the same and in some strains is opposite to that found in the interior O-units of the O-chain polysaccharide. Various monosaccharides, including d-Glc, d-Gal, d-GlcA and acyl derivatives of 3-amino-3,6-dideoxy- d-glucose or 4-amino-4,6-dideoxy- d-glucose, occupy the non-reducing end of the O-unit.

Analysis of a capsular polysaccharide biosynthesis locus of Bacteroides fragilis.

A major clinical manifestation of infection with Bacteroides fragilis is the formation of intra-abdominal abscesses, which are induced by the capsular polysaccharides of this organism. Transposon mutagenesis was used to locate genes involved in the synthesis of capsular polysaccharides. A 24,454-bp region was sequenced and found to contain a 15,379-bp locus (designated wcf) with 16 open reading frames (ORFs) encoding products similar to those encoded by genes of other bacterial polysaccharide biosynthesis loci. Four genes encode products that are similar to enzymes involved in nucleotide sugar biosynthesis. Seven genes encode products that are similar to sugar transferases. Two gene products are similar to O-acetyltransferases, and two products are probably involved in polysaccharide transport and polymerization. The product of one ORF, WcfH, is similar to a set of deacetylases of the NodB family. Deletion mutants demonstrated that the wcf locus is necessary for the synthesis of polysaccharide B, one of the two capsular polysaccharides of B. fragilis 9343. The virulence of the polysaccharide B-deficient mutant was comparable to that of the wild type in terms of its ability to induce abscesses in a rat model of intra-abdominal infection.

The type II O-antigenic polysaccharide moiety of Burkholderia pseudomallei lipopolysaccharide is required for serum resistance and virulence.

Melioidosis, an infection caused by the gram-negative bacterial pathogen Burkholderia pseudomallei, is endemic in south-east Asia and northern Australia. Acute septicaemic melioidosis is a major cause of morbidity and mortality, especially in north-east Thailand. B. pseudomallei is highly resistant to the bactericidal activity of normal human serum (NHS), and we have found that B. pseudomallei 1026b multiplies in 10-30% NHS. We developed a simple screen for the identification of serum-sensitive mutants based on this novel phenotype. Approximately 1200 Tn5-OT182 mutants were screened, and three serum-sensitive mutants were identified. The type II O-antigenic polysaccharide (O-PS) moiety of lipopolysaccharide was not present in the serum-sensitive mutants. A representative serum-sensitive mutant, SRM117, was killed by the alternative pathway of complement and was less virulent than 1026b in three animal models of melioidosis. The Tn5-OT182 integrations in the serum-sensitive mutants were physically linked on the B. pseudomallei chromosome, and further genetic analysis of this locus revealed a cluster of 15 genes required for type II O-PS production. The proteins encoded by these genes were similar to proteins involved in bacterial polysaccharide biosynthesis. The results presented here demonstrate that type II O-PS is essential for B. pseudomallei serum resistance and virulence.

Assignment of biochemical functions to glycosyl transferase genes which are essential for biosynthesis of exopolysaccharides in Sphingomonas strain S88 and Rhizobium leguminosarum.

Glycosyl transferases which recognize identical substrates (nucleotide-sugars and lipid-linked carbohydrates) can substitute for one another in bacterial polysaccharide biosynthesis, even if the enzymes originate in different genera of bacteria. This substitution can be used to identify the substrate specificities of uncharacterized transferase genes. The spsK gene of Sphingomonas strain S88 and the pssDE genes of Rhizobium leguminosarum were identified as encoding glucuronosyl-(B1-->4)-glucosyl transferases based on reciprocal genetic complementation of mutations in the spsK gene and the pssDE genes by segments of cloned DNA and by the SpsK-dependent incorporation of radioactive glucose (Glc) and glucuronic acid (GlcA) into lipid-linked disaccharides in EDTA-permeabilized cells. By contrast, glycosyl transferases which form alternative sugar linkages to the same substrate caused inhibition of polysaccharide synthesis or were deleterious or lethal in a foreign host. The negative effects also suggested specific substrate requirements: we propose that spsL codes for a glucosyl-(beta1-->4)-glucuronosyl transferase in Sphingomonas and that pssC codes for a glucuronosyl-(beta1-->4)-glucuronosyl transferase in R. leguminosarum. Finally, the complementation results indicate the order of attachment of sphingan main-chain sugars to the C55-isoprenylphosphate carrier as -Glc-GlcA-Glc-isoprenylpyrophosphate.

Functional Analysis of Glycosyltransferases Encoded by the Capsular Polysaccharide Biosynthesis Locus of Streptococcus pneumoniae Serotype 14

Bacteria belonging to the species Streptococcus pneumoniae vary in their capsule. Presently, 90 capsular serotypes are known, all possessing their own specific polysaccharide structure. Little is known about the biosynthesis of these capsular polysaccharides. The cps locus of S. pneumoniae serotype 14 was cloned. So far, 7 open reading frames have been sequenced, cps14B to cps14H. The gene products are similar to proteins involved in bacterial polysaccharide biosynthesis, both of Gram-negative and -positive micro-organisms. Gene-specific mutants were created for cps14D to cps14H by insertional mutagenesis. All mutants no longer agglutinated with a monoclonal antibody against type 14 capsule polysaccharides. The biosynthetic function of cps14E and cps14G was determined by analysis of the intermediates in the synthesis of the oligosaccharide subunit, formed in membrane preparations of the wild-type and mutant strains and in membrane preparations of Escherichia coli expressing the pneumococcal glycosyltransferases. The enzyme encoded by cps14E is a glucosyl-1-phosphate transferase that links glucose to a lipid carrier, the first step in the biosynthesis of the type 14 repeating unit. The gene product of cps14G encodes a beta-1,4-galactosyltransferase, the enzyme responsible for the second step in the subunit synthesis, the transfer of galactose to lipid-linked glucose.

The biosynthesis of bacterial polysaccharides.

The biosynthesis of bacterial polysaccharides.