Inner Core Of Lipopolysaccharide Research Articles

Characterization of the ADP-β-d-manno-heptose biosynthetic enzymes from two pathogenic Vibrio strains

ADP-activated β-d-manno-heptoses (ADP-β-d-manno-heptoses) are precursors for the biosynthesis of the inner core of lipopolysaccharide in Gram-negative bacteria. Recently, ADP-d-glycero-β-d-manno-heptose (ADP-d,d-manno-heptose) and its C-6′′ epimer, ADP-l-glycero-β-d-manno-heptose (ADP-l,d-manno-heptose), were identified as potent pathogen-associated molecular patterns (PAMPs) that can trigger robust innate immune responses. Although the production of ADP-d,d-manno-heptose has been studied in several different pathogenic Gram-negative bacteria, current knowledge of ADP-β-d-manno-heptose biosynthesis in Vibrio strains remains limited. Here, we characterized the biosynthetic enzymes of ADP-d,d-manno-heptose and the epimerase that converts it to ADP-l,d-manno-heptose from Vibrio cholerae (the causative agent of pandemic cholera) and Vibrio parahaemolyticus (non-cholera pathogen causing vibriosis with clinical manifestations of gastroenteritis and wound infections) in comparison with their isozymes from Escherichia coli. Moreover, we discovered that β-d-mannose 1-phosphate, but not α-d-mannose 1-phosphate, could be activated to its ADP form by the nucleotidyltransferase domains of bifunctional kinase/nucleotidyltransferases HldEVC (from V. cholerae) and HldEVP (from V. parahaemolyticus). Kinetic analyses of the nucleotidyltransferase domains of HldEVC and HldEVP together with the E. coli–derived HldEEC were thus carried out using β-d-mannose 1-phosphate as a mimic sugar substrate. Overall, our works suggest that V. cholerae and V. parahaemolyticus are capable of synthesizing ADP-β-d-manno-heptoses and lay a foundation for further physiological function explorations on manno-heptose metabolism in Vibrio strains.Key points• Vibrio strains adopt the same biosynthetic pathway as E. coli in synthesizing ADP-β-d-manno-heptoses.• HldEs from two Vibrio strains and E. coli could activate β-d-mannose 1-phosphate to ADP-β-d-mannose.• Comparable nucleotidyltransfer efficiencies were observed in the kinetic studies of HldEs.

Characterization of a gene cluster containing four genes relevant to biosynthesis of inner core of lipopolysaccharide in Cronobacter sakazakii.

Many genes in the biosynthetic pathway of lipopolysaccharide in Cronobacter sakazakii have not been identified. In this study, we demonstrate that an operon containing four genes ESA_RS18945, ESA_RS18950, ESA_RS18955, and ESA_RS18960 is responsible for L-glycero-D-mannoheptose addition on the inner core of lipopolysaccharide in C. sakazakii. The proteins encoded by these four genes are homologous to E. coli WaaQ, WaaC, WaaF, and WaaD. Lipopolysaccharide from the deletion mutants of ESA_RS18945, ESA_RS18950, ESA_RS18955, and ESA_RS18960 (named as △RS18945, △RS18950, △RS18955 and △RS18960, respectively) were analyzed by SDS-PAGE. △RS18945 synthesized lipopolysaccharide with similar length to the wildtype BAA-894, whereas △RS18950, △RS18955, and △RS18960 synthesized much shorter lipopolysaccharide. This suggests that the enzyme encoded by ESA_RS18945 might function as E. coli WaaQ on the sidechain of lipopolysaccharide. When E. coli WaaC, WaaF, and WaaD were overexpressed in △RS18950, △RS18955, and △RS18960, respectively, the full length of lipopolysaccharide was recovered. Mass spectrometry analysis indicates that △RS18950 and △RS18960 only synthesized Kdo2 -lipid A, confirming that enzymes encoded by ESA_RS18950 and ESA_RS18960 have similar functions to E. coli WaaC and WaaD, respectively. Hep-Kdo2 -lipid A with a phosphoethanolamine was produced in △RS18955, suggesting that the enzyme encoded by ESA_RS18955 has similar function to E. coli WaaF.

A triple‐targeting inhibitory activity of Rose Bengal on polysaccharide biosynthesis of Burkholderia pseudomallei

Sugar nucleotidyltransferases (SNTs) participate in various biosynthesis pathways constructing polysaccharides in Gram-negative bacteria. In this study, a triple-targeting inhibitory activity of Rose Bengal against SNTs such as d-glycero-α-d-manno-heptose-1-phosphate guanylyltransferase (HddC), d-glycero-β-d-manno-heptose-1-phosphate adenylyltransferase (HldC), and 3-deoxy-d-manno-oct-2-ulosonic acid cytidylyltransferase (KdsB) from Burkholderia pseudomallei is provided. Rose Bengal effectively suppresses the nucleotidyltransferase activity of the three SNTs, and its IC50 values are 10.42, 0.76, and 5.31 µM, respectively. Interestingly, Rose Bengal inhibits the three enzymes regardless of their primary, secondary, tertiary, and quaternary structural differences. The experimental results indicate that Rose Bengal possesses the plasticity to shape its conformation suitable to interact with the three SNTs. As HddC functions in the formation of capsular polysaccharides and HldC and KdsB produce building blocks to constitute the inner core of lipopolysaccharide, Rose Bengal is a potential candidate to design antibiotics in a new category. In particular, it can be developed as a specific antimelioidosis agent. As the mortality rate of the infected people caused by B. pseudomallei is quite high, there is an urgent need for specific antimelioidosis agents. Therefore, a further study is being carried out with derivatives of Rose Bengal.

Expression and crystallographic studies of D-glycero-β-D-manno-heptose-1-phosphate adenylyltransferase from Burkholderia pseudomallei.

The Gram-negative bacterium Burkholderia pseudomallei is the causative agent of melioidosis. D-glycero-β-D-manno-Heptose-1-phosphate adenylyltransferase (HldC) is the fourth enzyme of the ADP-L-glycero-β-D-manno-heptose biosynthesis pathway, which produces an essential carbohydrate comprising the inner core of lipopolysaccharide. Therefore, HldC is a potential target of antibiotics against melioidosis. In this study, HldC from B. pseudomallei has been cloned, expressed, purified and crystallized. Synchrotron X-ray data from a selenomethionine-substituted HldC crystal were also collected to 2.8 Å resolution. The crystal belonged to the primitive triclinic space group P1, with unit-cell parameters a = 74.0, b = 74.0, c = 74.9 Å, α = 108.4, β = 108.4, γ = 108.0°. Eight protomers are present in the unit cell and three out of five selenomethionines were found in each protomer using the PHENIX software suite. A full structural determination is in progress to elucidate the structure-function relationship of the protein.

Characterization of a biofilm-forming Shigella flexneri phenotype due to deficiency in Hep biosynthesis.

Deficiency in biosynthesis of inner core of lipopolysaccharide (LPS) rendered a characteristic biofilm-forming phenotype in E. coli. The pathological implications of this new phenotype in Shigella flexneri, a highly contagious enteric Gram-negative bacteria that is closely related to E. coli, were investigated in this study. The ΔrfaC (also referred as waaC) mutant, with incomplete inner core of LPS due to deficiency in Hep biosynthesis, was characteristic of strong biofilm formation ability and exhibited much more pronounced adhesiveness and invasiveness to human epithelial cells than the parental strain and other LPS mutants, which also showed distinct pattern of F-actin recruitment. Failure to cause keratoconjunctivitis and colonize in the intestine in guinea pigs revealed that the fitness gain on host adhesion resulted from biofilm formation is not sufficient to offset the loss of fitness on survivability caused by LPS deletion. Our study suggests a clear positive relationship between increased surface hydrophobicity and adhesiveness of Shigella flexneri, which should be put into consideration of virulence of Shigella, especially when therapeutic strategy targeting the core oligosaccharide (OS) is considered an alternative to deal with bacterial antibiotics-resistance.

The antibacterial toxin colicin N binds to the inner core of lipopolysaccharide and close to its translocator protein

Colicins are a diverse family of large antibacterial protein toxins, secreted by and active against Escherichia coli and must cross their target cell's outer membrane barrier to kill. To achieve this, most colicins require an abundant porin (e.g. OmpF) plus a low-copy-number, high-affinity, outer membrane protein receptor (e.g. BtuB). Recently, genetic screens have suggested that colicin N (ColN), which has no high-affinity receptor, targets highly abundant lipopolysaccharide (LPS) instead. Here we reveal the details of this interaction and demonstrate that the ColN receptor-binding domain (ColN-R) binds to a specific region of LPS close to the membrane surface. Data from in vitro studies using calorimetry and both liquid- and solid-state NMR reveal the interactions behind the in vivo requirement for a defined oligosaccharide region of LPS. Delipidated LPS (LPS(Δ) (LIPID) ) shows weaker binding; and thus full affinity requires the lipid component. The site of LPS binding means that ColN will preferably bind at the interface and thus position itself close to the surface of its translocon component, OmpF. ColN is, currently, unique among colicins in requiring LPS and, combined with previous data, this implies that the ColN translocon is distinct from those of other known colicins.

161 Discovery of potent KdsA inhibitors of Leptospira interrogans through homology modeling, docking, and molecular dynamics simulations

Leptospira interrogans is the foremost cause of human leptospirosis. Discovery of novel lead molecules for common drug targets of more than 250 Leptospira serovars is of significant research interest. Lipopolysaccharide (LPS) layer prevent entry of hydrophobic agents into the cell and protect structural integrity of the bacterium. KDO-8-phosphate synthase (KdsA) catalyzes the first step of KDO biosynthesis that leads to formation of inner core of LPS. KdsA was identified as a potential drug target against Leptospira interrogans through subtractive genomic approach, metabolic pathway analysis, and comparative analysis (Amineni et al., 2010). The present study rationalizes a systematic implementation of homology modeling, docking, and molecular dynamics simulations to discover potent KdsA inhibitors (Pradhan et al., 2013; Umamaheswari et al., 2010). A reliable tertiary structure of KdsA in complex with substrate PEP was constructed based on co-crystal structure of Aquifex aeolicus KdsA synthase with PEP using Modeller9v10. Geometry-based analog search for PEP was performed from LigandInfo database to generate an in house library of 352 ligands. The ligand data-set was docked into KdsA active site through three-stage docking technique (HTVS, SP, and XP) using Glidev5.7. Thirteen lead molecules were found to have better binding affinity compared to PEP (XP Gscore = −7.38 kcal/mol; Figure 1). The best lead molecule (KdsA- lead1 docking complex) showed XP Gscore of −10.26 kcal/mol and the binding interactions (Figure 2) were correlated favorably with PEP–KdsA interactions (Figure 1). Molecular dynamics simulations of KdsA– lead1 docking complex for 10 ns had revealed that the complex (Figure 3) remained stable in closer to physiological environmental condition. The predicted pharmacological properties of lead1 were well within the range of a drug molecule with good ADME profile, hence, would be intriguing towards development of potent inhibitor molecule against KdsA of Leptospira.

Glycosyltransferases involved in the biosynthesis of the inner core region of different lipopolysaccharides

The inner core of lipopolysaccharide (LPS) structures in Gram-negative bacteria is considered a highly conserved region. The sugar connecting the membrane-associated lipid A moiety with the hydrophilic saccharide moiety, 3-deoxy-α- d- manno-oct-2-ulosonic acid (Kdo) is present in every LPS molecule investigated but it may be partially replaced by d- glycero-α- d- talo-oct-2-ulosonic acid (Ko). l- Glycero-α- d- manno-heptose (Hep) and phosphate residues are part of most but not all LPS structures and additionally, modifications with 4-amino-4-deoxy-β- l-arabinose (Ara4N) residues occur in some. A number of different glycosyltransferases is involved in the biosynthesis of the inner core region of different lipopolysaccharides. Here, we report the characterization of Kdo transferases, heptosyltransferases and Ara4N transferases from a variety of bacteria.

A female‐specific pentraxin, CrOctin, bridges pattern recognition receptors to bacterial phosphoethanolamine

Pathogen recognition and binding are crucial functions of innate immunity. It has been observed that the short pentraxin superfamily including C-reactive protein (CRP) and serum amyloid P component are pathogen pattern recognition receptors (PRR) in the plasma. We isolated and characterized a novel and distinctive pentraxin from the plasma of horseshoe crab, Carcinoscorpius rotundicauda, henceforth named CrOctin, which binds to bacteria via phosphoethanolamine (PE), a chemical component present on lipid A and core polysaccharide moieties of bacterial lipopolysaccharide (LPS). Infection enhances the formation of the PRR interactome constituting CrOctin, CRP and galactose-binding protein. In particular, infection increases the affinity of CRP to CrOctin by 1000-fold. Furthermore, we observed that by binding to PE, CrOctin acts as a linker that bridges the PRR interactome to the inner core of LPS. On the other hand, under normal physiological conditions, binding of CrOctin to PE appears to obscure other PRR from interacting directly with PE. Interestingly, the cluster of "CrOctin-interactive PRR" is sex specific. We report, for the first time, the change in PRR protein profiles with a distinctive gender difference during Pseudomonas infection.

Llama Single-Chain Antibody That Blocks Lipopolysaccharide Binding and Signaling: Prospects for Therapeutic Applications

Sepsis is a considerable health problem and a burden on the health care system. Endotoxin, or lipopolysaccharide (LPS), present in the outer membrane of gram-negative bacteria, is responsible for more than 50% of the sepsis cases and is, therefore, a legitimate target for therapeutic approaches against sepsis. In this study, we selected and characterized a llama single-chain antibody fragment (VHH) directed to Neisseria meningitidis LPS. The VHH, designated VHH 5G, showed affinity to purified LPS as well as to LPS on the surfaces of the bacteria. Epitope mapping using a panel of N. meningitidis mutants revealed that VHH 5G recognizes an epitope in the inner core of LPS, and as expected, the VHH proved to have broad specificity for LPS from different bacteria. Furthermore, this VHH blocked binding of LPS to target cells of the immune system, resulting in the inhibition of LPS signaling in whole blood. Moreover, it was found to remove LPS efficiently from aqueous solutions, including serum. The selected anti-LPS VHH is a leading candidate for therapies against LPS-mediated sepsis.

Cross-reactive polyclonal antibodies to the inner core of lipopolysaccharide from Neisseria meningitidis.

Sera from mice immunized with native or detergent-extracted outer membrane vesicles derived from lipopolysaccharide (LPS) mutant 44/76(Mu-4) of Neisseria meningitidis were analyzed for antibodies to LPS. The carbohydrate portion of 44/76(Mu-4) LPS consists of the complete inner core, Glc beta 1-->4[GlcNAc alpha 1-->2Hep alpha 1-->3]Hep alpha 1-->5KDO[4-->2 alpha KDO]. Immunoblot analysis revealed that some sera contained antibodies to wild-type LPS which has a fully extended carbohydrate chain of immunotype L3,7, as well as to the homologous LPS. Sera reacted only weakly to LPS from 44/76(Mu-3), which lacks the terminal glucose of the inner core. No binding to more truncated LPS was observed. Consequently, the cross-reactive epitopes are expressed mainly by the complete inner core. Dephosphorylation of wild-type LPS abolished antibody binding to LPS in all but one serum. Thus, at least two specificities of cross-reactive antibodies exist: one is dependent on phosphoethanolamine groups in LPS, and one is not. Detection of these cross-reactive antibodies strongly supports the notion that epitopes expressed by meningococcal LPS inner core are also accessible to antibodies when the carbohydrate chain is fully extended. Also, these inner core epitopes are sufficiently immunogenic to induce antibody levels detectable in polyclonal antibody responses. Meningococci can escape being killed by antibodies to LPS that bind only to a specific LPS variant, by altering the carbohydrate chain length. Cross-reactive antibodies may prevent such escape. Therefore, inner core LPS structures may be important antigens in future vaccines against meningococcal disease.

Molecular characterization of a gene region involved in lipopolysaccharide biosynthesis in Bradyrhizobium japonicum: cloning, sequencing and expression of rfaf gene.

A 3.0-kb region involved in lipopolysaccharide biosynthesis in Bradyrhizobium japonicum was sequenced. One complete open reading frame was identified which encodes a polypeptide of 354 amino acid residues with a predicted molecular mass of 38 209 Da. Expression of the protein using a T7 gene expression system revealed a band of similar molecular mass after sodium dodecyl sulfate polyacrylamide gel electrophoresis. A database search against known gene sequences revealed a significant sequence similarity to the rfaF gene cloned from several Gram-negative bacteria. The rfaF gene is known to encode heptosyltransferase II that transfers a second heptose to the inner core of lipopolysaccharide. The cloned B. japonicum open reading frame was able to functionally complement a rfaF mutant of Salmonella typhimurium SL3789. Transformation of this mutant with the B. japonicum gene restored production of an intact lipopolysaccharide and resistance to the hydrophobic antibiotic, novobiocin. An additional open reading frame having a significant sequence similarity to the rfaD gene was found to be divergently oriented to the rfaF gene.

Molecular characterization of a gene region involved in lipopolysaccharide biosynthesis in Bradyrhizobium japonicum: cloning, sequencing and expression of rfaf gene

A 3.0-kb region involved in lipopolysaccharide biosynthesis in Bradyrhizobium japonicum was sequenced. One complete open reading frame was identified which encodes a polypeptide of 354 amino acid residues with a predicted molecular mass of 38 209 Da. Expression of the protein using a T7 gene expression system revealed a band of similar molecular mass after sodium dodecyl sulfate–polyacrylamide gel electrophoresis. A database search against known gene sequences revealed a significant sequence similarity to the rfaF gene cloned from several Gram-negative bacteria. The rfaF gene is known to encode heptosyltransferase II that transfers a second heptose to the inner core of lipopolysaccharide. The cloned B. japonicum open reading frame was able to functionally complement a rfaF mutant of Salmonella typhimurium SL3789. Transformation of this mutant with the B. japonicum gene restored production of an intact lipopolysaccharide and resistance to the hydrophobic antibiotic, novobiocin. An additional open reading frame having a significant sequence similarity to the rfaD gene was found to be divergently oriented to the rfaF gene.