The Klebsiella pneumoniae wabG gene: role in biosynthesis of the core lipopolysaccharide and virulence

To determine the function of the wabG gene in the biosynthesis of the core lipopolysaccharide (LPS) of Klebsiella pneumoniae, we constructed wabG nonpolar mutants. Data obtained from the comparative chemical and structural analysis of LPS samples obtained from the wild type, the mutant strain, and the complemented mutant demonstrated that the wabG gene is involved in attachment to alpha-L-glycero-D-manno-heptopyranose II (L,D-HeppII) at the O-3 position of an alpha-D-galactopyranosyluronic acid (alpha-D-GalAp) residue. K. pneumoniae nonpolar wabG mutants were devoid of the cell-attached capsular polysaccharide but were still able to produce capsular polysaccharide. Similar results were obtained with K. pneumoniae nonpolar waaC and waaF mutants, which produce shorter LPS core molecules than do wabG mutants. Other outer core K. pneumoniae nonpolar mutants in the waa gene cluster were encapsulated. K. pneumoniae waaC, waaF, and wabG mutants were avirulent when tested in different animal models. Furthermore, these mutants were more sensitive to some hydrophobic compounds than the wild-type strains. All these characteristics were rescued by reintroduction of the waaC, waaF, and wabG genes from K. pneumoniae.     

Genetic characterization of the Klebsiella pneumoniae waa gene cluster, involved in core lipopolysaccharide biosynthesis

A recombinant cosmid containing genes involved in Klebsiella pneumoniae C3 core lipopolysaccharide biosynthesis was identified by its ability to confer bacteriocin 28b resistance to Escherichia coli K-12. The recombinant cosmid contains 12 genes, the whole waa gene cluster, flanked by kbl and coaD genes, as was found in E. coli K-12. PCR amplification analysis showed that this cluster is conserved in representative K. pneumoniae strains. Partial nucleotide sequence determination showed that the same genes and gene order are found in K. pneumoniae subsp. ozaenae, for which the core chemical structure is known. Complementation analysis of known waa mutants from E. coli K-12 and/or Salmonella enterica led to the identification of genes involved in biosynthesis of the inner core backbone that are shared by these three members of the Enterobacteriaceae. K. pneumoniae orf10 mutants showed a two-log-fold reduction in a mice virulence assay and a strong decrease in capsule amount. Analysis of a constructed K. pneumoniae waaE deletion mutant suggests that the WaaE protein is involved in the transfer of the branch beta-D-Glc to the O-4 position of L-glycero-D-manno-heptose I, a feature shared by K. pneumoniae, Proteus mirabilis, and Yersinia enterocolitica.     

Full structural characterization of Shigella flexneri M90T serotype 5 wild-type R-LPS and its delta galU mutant: glycine residue location in the inner core of the lipopolysaccharide

Shigella flexneri is a gram-negative bacterium responsible for serious enteric infections that occur mainly in the terminal ileum and colon. High interest in Shigella, as a human pathogen, is driven by its antibiotic resistance and the necessity to develop a vaccine against its infections. Vaccines of the last generation use carbohydrate moieties of the lipopolysaccharide as probable candidates. For this reason, the primary structure of the core oligosaccharide from the R-LPS produced by S. flexneri M90T serotype 5 using chemical analysis, nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MALDI), is herein reported. This is the first time that the core oligosaccharide primary structure by S. flexneri M90T is established in an unambiguous multidisciplinary approach. Chemical and spectroscopical investigation of the de-acetylated LPS showed that the inner core structure is characterized by a L,D-Hep-(1 -->7)-L,D-Hep-(1 -->3)-L,D-Hep-(1 -->5)-[Kdo-(2 -->4)]-Kdo sequence that is the common structural theme identified in Enterobacteriaceae. In particular, in S. flexneri M90T serotype 5 LPS, a glucosamine residue is additionally sitting at O-7 of the last heptose whereas the outer core is characterized by glucose and galactose residues. Also, in order to exactly define the position of glycine that is an integral constituent of the core region of the LPS, we created a S. flexneri M90T delta galU mutant and studied its LOS. In this way it was possible to establish that glycine is sitting at O-6 of the second heptose in the inner core.     

Structural analysis of the carbohydrate backbone of Vibrio parahaemolyticus O2 lipopolysaccharides

A structural investigation has been carried out on the carbohydrate backbone of Vibrio parahaemolyticus O2 lipopolysaccharides (LPS) isolated by dephosphorylation, O-deacylation and N-deacylation. The carbohydrate backbone is a short-chain saccharide consisting of nine monosaccharide units i.e., 1 mol each of D-galactose (Gal), D-glucose (Glc), D-glucuronic acid (GlcA), L-glycero-D-manno-heptose (L,D-Hep), D-glycero-D-manno-heptose (D,D-Hep), 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (NonlA), and 2 mol of 2-amino-2-deoxy-D-glucose (D-glucosamine, GlcN). Based on the data obtained by NMR spectroscopy, fast-atom bombardment mass spectrometry (FABMS) and methylation analysis, a structure was elucidated for the carbohydrate backbone of O2 LPS. In the native O2 LPS, the 2-amino-2-deoxy-D-glucitol (GlcN-ol) at the reducing end of the nonasaccharide is present as GlcN. The lipid A backbone is a beta-D-GlcN-(1-->6)-D-GlcN disaccharide as is the case for many Gram-negative bacterial LPS. The lipid A proximal Kdo is substituted by the distal part of the carbohydrate chain at position-5. In the native O2 LPS, D-galacturonic acid, which is liberated from LPS by mild acid treatment or by dephosphorylation in hydrofluoric acid, is present although its binding position is unknown at present.     

Structural diversity in lipopolysaccharide expression in nontypeable Haemophilus influenzae. Identification of L-glycerol-D-manno-heptose in the outer-core region in three clinical isolates.

Structural elucidation of the lipopolysaccharide (LPS) from three nontypeable Haemophilus influenzae clinical isolates, 1209, 1207 and 1233 was achieved using NMR spectroscopy and ESI-MS on O-deacylated LPS and core oligosaccharide (OS) material as well as ESI-MS(n) on permethylated dephosphorylated OS. It was found that the organisms expressed a tremendous heterogeneous glycoform mixture resulting from the variable length of the OS chains attached to the common structural element of H. influenzae, L-alpha-D-Hepp-(1-->2)-[PEtn-->6]-L-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)]-L-alpha-D-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdop-(2-->6)-Lipid A. Notably, the O-6 position of the beta-D-Glcp residue could either be occupied by PCho or L-glycero-D-manno-heptose (L,D-Hep), which is a location for L,D-Hep that has not been seen previously in H. influenzae LPS. The outer-core L,D-Hep residue was further chain elongated at the O-6 position by the structural element beta-D-GalpNAc-(1-->3)-alpha-D-Galp-(1-->4)-beta-D-Galp, or sequentially truncated versions thereof. The distal heptose residue in the inner-core was found to be chain elongated at O-2 by the globotetraose unit, beta-D-GalpNAc-(1-->3)-alpha-D-Galp-(1-->4)-beta-D-Galp-(1-->4)-beta-D-Glcp, or sequentially truncated versions thereof. Investigation of LPS from an lpsA mutant of isolate 1233 and a lic1 mutant of isolate 1209 was also performed, which aside from confirming the functions of the gene products, simplified elucidation of the OS extending from the proximal heptose (the lpsA mutant), and showed that the organism exclusively expresses LPS glycoforms comprising the outer-core l,d-Hep residue when PCho is not expressed (the lic1 mutant).     

Overexpression of the waaZ gene leads to modification of the structure of the inner core region of Escherichia coli lipopolysaccharide,truncation of the outer core,and reduction of the amount of O polysaccharide on the cell surface

The waa gene cluster is responsible for the biosynthesis of the lipopolysaccharide (LPS) core region in Escherichia coli and Salmonella: Homologs of the waaZ gene product are encoded by the waa gene clusters of Salmonella enterica and E. coli strains with the K-12 and R2 core types. Overexpression of WaaZ in E. coli and S. enterica led to a modified LPS structure showing core truncations and (where relevant) to a reduction in the amount of O-polysaccharide side chains. Mass spectrometry and nuclear magnetic resonance spectroscopy were used to determine the predominant LPS structures in an E. coli isolate with an R1 core (waaZ is lacking from the type R1 waa gene cluster) with a copy of the waaZ gene added on a plasmid. Novel truncated LPS structures, lacking up to 3 hexoses from the outer core, resulted from WaaZ overexpression. The truncated molecules also contained a KdoIII residue not normally found in the R1 core.     

Isolation and structural analysis of phosphorylated oligosaccharides obtained from Escherichia coli J-5 lipopolysaccharide.

The chemical structure of the phosphorylated lipopolysaccharide (LPS) of Escherichia coli J-5 was investigated because it is of biomedical interest in the context of septic shock, a syndrome often encountered in nosocomial infections with gram-negative pathogens. The successive de-O-acylation and de-N-acylation of J-5 LPS yielded phosphorylated oligosaccharides which represent the complete carbohydrate backbone. Five compounds were separated by high-performance anion-exchange chromatography and analysed by one-dimensional and two-dimensional homonuclear and heteronuclear 1H-NMR, 13C-NMR and 31P-NMR spectroscopy. The main product was a nonasaccharide of the structure alpha-D-Glcp-(1-->3)-[alpha-D-GlcpN- (1-->7)-alpha-L,D-Hepp-(1-->7)]-alpha-L,D-Hepp-(1-->3)-alpha -L, D-Hepp-4P-(1-->5)-[alpha-Kdop-(2-->4)]-alpha-Kdop-(2-- >6)-beta-D-GlcpN-4p- (1-->6)-alpha-D-GlcN-1P wherein all sugars are present as D-pyranoses. Hep and Kdo represent L-glycero-D-manno-heptose and 3-deoxy-D-manno-oct-2-ulosonic acid, respectively. In addition, two octasaccharides and two heptasaccharides were isolated that were partial structures of the nonasaccharide. In one octasaccharide the terminal alpha-D-GlcpN was missing and an additional phosphate group linked to O4 of the branched heptose was present, whereas in the other octasaccharide the side-chain Kdo was missing. In both heptasaccharides the side-chain alpha-D-GlcpN-(1-->7)-L-alpha-D-Hepp-disaccharide was absent; they differed in their phosphate substitution. Whereas both heptasaccharides contained two phosphates in the lipid-A backbone (beta-1,6-linked GlcpN-disaccharide at the reducing end) and one phosphate group at O4 of the first heptose, only one of them was additionally substituted with phosphate at O4 of the second heptose.     

A second galacturonic acid transferase is required for core lipopolysaccharide biosynthesis and complete capsule association with the cell surface in Klebsiella pneumoniae

The core lipopolysaccharide (LPS) of Klebsiella pneumoniae contains two galacturonic acid (GalA) residues, but only one GalA transferase (WabG) has been identified. Data from chemical and structural analysis of LPS isolated from a wabO mutant show the absence of the inner core beta-GalA residue linked to L-glycero-D-manno-heptose III (L,D-Hep III). An in vitro assay demonstrates that the purified WabO is able to catalyze the transfer of GalA from UDP-GalA to the acceptor LPS isolated from the wabO mutant, but not to LPS isolated from waaQ mutant (deficient in l,d-Hep III). The absence of this inner core beta-GalA residue results in a decrease in virulence in a capsule-dependent experimental mouse pneumonia model. In addition, this mutation leads to a strong reduction in cell-bound capsule. Interestingly, a K66 Klebsiella strain (natural isolate) without a functional wabO gene shows reduced levels of cell-bound capsule in comparison to those of other K66 strains. Thus, the WabO enzyme plays an important role in core LPS biosynthesis and determines the level of cell-bound capsule in Klebsiella pneumoniae.     

Synthesis of a Klebsiella pneumoniae O-antigen heteropolysaccharide (O12) requires an ABC 2 transporter

A recombinant clone encoding enzymes for Klebsiella pneumoniae O12-antigen lipopolysaccharide (LPS) was found when we screened for serum resistance of a cosmid-based genomic library of K. pneumoniae KT776 (O12:K80) introduced into Escherichia coli DH5alpha. A total of eight open reading frames (ORFs) (wb(O12) gene cluster) were necessary to produce K. pneumoniae O12-antigen LPS in E. coli K-12. A complete analysis of the K. pneumoniae wb(O12) cluster revealed an interesting coincidence with the wb(O4) cluster of Serratia marcescens from ORF5 to ORF8 (or WbbL to WbbA). This prompted us to generate mutants of K. pneumoniae strain KT776 (O12) and to study complementation between the two enterobacterial wb clusters using mutants of S. marcescens N28b (O4) obtained previously. Both wb gene clusters are examples of ABC 2 transporter-dependent pathways for O-antigen heteropolysaccharides. The wzm-wzt genes and the wbbA or wbbB genes were not interchangeable between the two gene clusters despite their high level of similarity. However, introduction of three cognate genes (wzm-wzt-wbbA or wzm-wzt-wbbB) into mutants unable to produce O antigen allowed production of the specific O antigen. The K. pneumoniae O12 WbbL protein performs the same function as WbbL from S. marcescens O4 in either the S. marcescens O4 or E. coli K-12 genetic background.     

Effects of lipopolysaccharide biosynthesis mutations on K1 polysaccharide association with the Escherichia coli cell surface

The presence of cell-bound K1 capsule and K1 polysaccharide in culture supernatants was determined in a series of in-frame nonpolar core biosynthetic mutants from Escherichia coli KT1094 (K1, R1 core lipopolysaccharide [LPS] type) for which the major core oligosaccharide structures were determined. Cell-bound K1 capsule was absent from mutants devoid of phosphoryl modifications on L-glycero-D-manno-heptose residues (HepI and HepII) of the inner-core LPS and reduced in mutants devoid of phosphoryl modification on HepII or devoid of HepIII. In contrast, in all of the mutants, K1 polysaccharide was found in culture supernatants. These results were confirmed by using a mutant with a deletion spanning from the hldD to waaQ genes of the waa gene cluster to which individual genes were reintroduced. A nuclear magnetic resonance (NMR) analysis of core LPS from HepIII-deficient mutants showed an alteration in the pattern of phosphoryl modifications. A cell extract containing both K1 capsule polysaccharide and LPS obtained from an O-antigen-deficient mutant could be resolved into K1 polysaccharide and core LPS by column chromatography only when EDTA and deoxycholate (DOC) buffer were used. These results suggest that the K1 polysaccharide remains cell associated by ionically interacting with the phosphate-negative charges of the core LPS.     

Construction of a deep-rough mutant of Burkholderia cepacia ATCC 25416 and characterization of its chemical and biological properties

Burkholderia cepacia is a bacterium with increasing importance as a pathogen in patients with cystic fibrosis. The deep-rough mutant Ko2b was generated from B. cepacia type strain ATCC 25416 by insertion of a kanamycin resistance cassette into the gene waaC encoding heptosyltransferase I. Mass spectrometric analysis of the de-O-acylated lipopolysaccharide (LPS) of the mutant showed that it consisted of a bisphosphorylated glucosamine backbone with two 3-hydroxyhexadecanoic acids in amide-linkage, 4-amino-4-deoxyarabinose (Ara4N) residues on both phosphates, and a core oligosaccharide of the sequence Ara4N-(1 --> 8) D-glycero-D-talo-oct-2-ulosonic acid (Ko)-(2 --> 4)3-deoxy-D-manno-oct-2-ulosonic acid (Kdo). The mutant allowed investigations on the biosynthesis of the LPS as well as on its role in human infection. Mutant Ko2b showed no difference in its ability to invade human macrophages as compared with the wild type. Furthermore, isolated LPS of both strains induced the production of tumor necrosis factor alpha from macrophages to the same extent. Thus, the truncation of the LPS did not decrease the biological activity of the mutant or its LPS in these aspects.     

The incorporation of glucosamine into enterobacterial core lipopolysaccharide: two enzymatic steps are required

The core lipopolysaccharide (LPS) of Klebsiella pneumoniae is characterized by the presence of disaccharide alphaGlcN-(1,4)-alphaGalA attached by an alpha1,3 linkage to l-glycero-d-manno-heptopyranose II (ld-HeppII). Previously it has been shown that the WabH enzyme catalyzes the incorporation of GlcNAc from UDP-GlcNAc to outer core LPS. The presence of GlcNAc instead of GlcN and the lack of UDP-GlcN in bacteria indicate that an additional enzymatic step is required. In this work we identified a new gene (wabN) in the K. pneumoniae core LPS biosynthetic cluster. Chemical and structural analysis of K. pneumoniae non-polar wabN mutants showed truncated core LPS with GlcNAc instead of GlcN. In vitro assays using LPS truncated at the level of d-galacturonic acid (GalA) and cell-free extract containing WabH and WabN together led to the incorporation of GlcN, whereas none of them alone were able to do it. This result suggests that the later enzyme (WabN) catalyzes the deacetylation of the core LPS containing the GlcNAc residue. Thus, the incorporation of the GlcN residue to core LPS in K. pneumoniae requires two distinct enzymatic steps. WabN homologues are found in Serratia marcescens and some Proteus strains that show the same disaccharide alphaGlcN-(1,4)-alphaGalA attached by an alpha1,3 linkage to ld-HeppII.     

A Salmonella typhimurium rfaE mutant recovers invasiveness for human epithelial cells when complemented by wild type rfaE (controlling biosynthesis of ADP-L-glycero-D-mannoheptose-containing lipopolysaccharide)

Invasion of host cells is essential for the pathogenicity of Salmonella. The author's group has recently reported the cloning of the rfaE gene of Salmonella typhimurium, previously implicated in biosynthesis of the lipopolysaccharide (LPS)-inner core [Jin et al. (2001); Kim (2002)]. The product of the rfaE gene is involved in ADP-L-glycero-D-manno-heptose biosynthesis. rfaE mutants synthesize heptose-deficient LPS (Re-LPS) consisting only of lipid A and 3-deoxy-D-manno-octulosonic acid (KDO). Mutants that make incomplete LPS are rough mutants and "deep-rough" mutants affected in the heptose region of the inner core have reduced growth rate and increased sensitivity to high temperature. Complementation of S. typhimurium rfaE mutant strain SL1102 (rfaE543) with rfaE demonstrated conclusively that this gene restored the smooth phenotype, and the LPS produced by the complemented strain was indistinguishable from that of wild type smooth strains. In vitro infection experiments showed that complementation with rfaE permitted invasion of human Chang epithelial cells, larynx epidermal carcinoma HEp-2 cells and intestinal epithelial Henle-407 cells. These data imply that the structure of the LPS that is synthesized is critical for Salmonella invasiveness.     

Three enzymatic steps required for the galactosamine incorporation into core lipopolysaccharide

The core lipopolysaccharides (LPS) of Proteus mirabilis as well as those of Klebsiella pneumoniae and Serratia marcescens are characterized by the presence of a hexosamine-galacturonic acid disaccharide (αHexN-(1,4)-αGalA) attached by an α1,3 linkage to L-glycero-D-manno-heptopyranose II (L-glycero-α-D-manno-heptosepyranose II). In K. pneumoniae, S. marcescens, and some P. mirabilis strains, HexN is D-glucosamine, whereas in other P. mirabilis strains, it corresponds to D-galactosamine. Previously, we have shown that two enzymes are required for the incorporation of D-glucosamine into the core LPS of K. pneumoniae; the WabH enzyme catalyzes the incorporation of GlcNAc from UDP-GlcNAc to outer core LPS, and WabN catalyzes the deacetylation of the incorporated GlcNAc. Here we report the presence of two different HexNAc transferases depending on the nature of the HexN in P. mirabilis core LPS. In vivo and in vitro assays using LPS truncated at the level of galacturonic acid as acceptor show that these two enzymes differ in their specificity for the transfer of GlcNAc or GalNAc. By contrast, only one WabN homologue was found in the studied P. mirabilis strains. Similar assays suggest that the P. mirabilis WabN homologue is able to deacetylate both GlcNAc and GalNAc. We conclude that incorporation of d-galactosamine requires three enzymes: Gne epimerase for the generation of UDP-GalNAc from UDP-GlcNAc, N-acetylgalactosaminyltransferase (WabP), and LPS:HexNAc deacetylase.     

Thin pilus PilV adhesins of plasmid R64 recognize specific structures of the lipopolysaccharide molecules of recipient cells

IncI1 plasmid R64 encodes a type IV pilus called a thin pilus, which includes PilV adhesins. Seven different sequences for the C-terminal segments of PilV adhesins can be produced by shufflon DNA rearrangement. The expression of the seven PilV adhesins determines the recipient specificity in liquid matings of plasmid R64. Salmonella enterica serovar Typhimurium LT2 was recognized by the PilVA' and PilVB' adhesins, while Escherichia coli K-12 was recognized by the PilVA', PilVC, and PilVC' adhesins. Lipopolysaccharide (LPS) on the surfaces of recipient cells was previously shown to be the specific receptor for the seven PilV adhesins. To identify the specific receptor structures of LPS for various PilV adhesins, R64 liquid matings were carried out with recipient cells consisting of various S. enterica serovar Typhimurium LT2 and E. coli K-12 waa mutants and their derivatives carrying various waa genes of different origins. From the mating experiments, including inhibition experiments, we propose that the GlcNAc(alpha1-2)Glc and Glc(alpha1-2)Gal structures of the LPS core of S. enterica serovar Typhimurium LT2 function as receptors for the PilVB' and PilVC' adhesins, respectively, while the PilVC' receptor in the wild-type LT2 LPS core may be masked. We further propose that the GlcNAc(beta1-7)Hep and Glc(alpha1-2)Glc structures of the LPS core of E. coli K-12 function as receptors for the PilVC and PilVC' adhesins, respectively.     

Cloning and characterization of two Serratia marcescens genes involved in core lipopolysaccharide biosynthesis.

Bacteriocin 28b from Serratia marcescens binds to Escherichia coli outer membrane proteins OmpA and OmpF and to lipopolysaccharide (LPS) core (J. Enfedaque, S. Ferrer, J. F. Guasch, J. Tomás, and M. Requé, Can. J. Microbiol. 42:19-26, 1996). A cosmid-based genomic library of S. marcescens was introduced into E. coli NM554, and clones were screened for bacteriocin 28b resistance phenotype. One clone conferring resistance to bacteriocin 28b and showing an altered LPS core mobility in polyacrylamide gel electrophoresis was found. Southern blot experiments using DNA fragments containing E. coli rfa genes as probes suggested that the recombinant cosmid contained S. marcescens genes involved in LPS core biosynthesis. Subcloning, isolation of subclones and Tn5tac1 insertion mutants, and sequencing allowed identification of two apparently cotranscribed genes. The deduced amino acid sequence from the upstream gene showed 80% amino acid identity to the KdtA protein from E. coli, suggesting that this gene codes for the 3-deoxy-manno-octulosonic acid transferase of S. marcescens. The downstream gene (kdtX) codes for a protein showing 20% amino acid identity to the Haemophilus influenzae kdtB gene product. The S. marcescens KdtX protein is unrelated to the KdtB protein of E. coli K-12. Expression of the kdtX gene from S. marcescens in E. coli confers resistance to bacteriocin 28b.     

Comparative analyses of secondary gene products of 3-deoxy-D-manno-oct-2-ulosonic acid transferases from Chlamydiaceae in Escherichia coli K-12.

The waaA gene encoding the essential, lipopolysaccharide (LPS)-specific 3-deoxy-Dmanno-oct-2-ulosonic acid (Kdo) transferase was inactivated in the chromosome of a heptosyltransferase I and II deficient Escherichia coli K-12 strain by insertion of gene expression cassettes encoding the waaA genes of Chlamydia trachomatis, Chlamydophila pneumoniae or Chlamydophila psittaci. The three chlamydial Kdo transferases were able to complement the knockout mutation without changing the growth or multiplication behaviour. The LPS of the mutants were serologically and structurally characterized in comparison to the LPS of the parent strain using compositional analyses, high performance anion exchange chromatography, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and specific monoclonal antibodies. The data show that chlamydial Kdo transferases can replace in E. coli K-12 the host's Kdo transferase and retain the product specificities described in their natural background. In addition, we unequivocally proved that WaaA from C. psittaci transfers predominantly four Kdo residues to lipid A, forming a branched tetrasaccharide with the structure alpha-Kdo-(2-->8)-[alpha-Kdo-(2-->4)]-alpha-Kdo-(2-->4)-alpha-Kdo.     

Structural analysis of the core region of the lipopolysaccharides from eight serotypes of Klebsiella pneumoniae

The core regions of the lipopolysaccharides (LPS) from Klebsiella pneumoniae serotypes O1, O2a, O2a,c, O3, O4, O5, O8, and O12 were analysed using NMR spectroscopy, ESI-MS spectroscopy, and chemical methods. All the LPSs had similar core structures, as shown below, differing only in the number and position of beta-D-galacturonic acid substituents: [carbohydrate structure: see text] where P is H or alpha-Hep, J, K is H or beta-GalA. LPS from all serotypes contained varying proportions of structures having additional or missing phosphate substituents. The core from serotype O1 contained a minor amount of a previously described variant with alpha-DD-Hep-(1-->2)-alpha-DD-Hep-(1-->6)-alpha-GlcN-(1--> replacing the alpha-Hep-(1-->4)-alpha-Kdo-(2-->6)-alpha-GlcN-(1--> component.     

Synthesis and biological evaluation of novel azole derivatives as selective potent inhibitors of brassinosteroid biosynthesis

Brassinosteroids (BRs) are phytohormones that control several important agronomic traits, such as flowering, plant architecture, seed yield, and stress tolerance. To manipulate the BR levels in plant tissues using specific inhibitors of BR biosynthesis, a series of novel azole derivatives were synthesized and their inhibitory activity on BR biosynthesis was investigated. Structure–activity relationship studies revealed that 2RS, 4RS-1-[4-(2-allyloxyphenoxymethyl)-2-(4-chlorophenyl)-[1,3]dioxolan-2-ylmethyl]-1H-[1,2,4]triazole (G₂) is a highly selective inhibitor of BR biosynthesis, with an IC₅₀ value of approximately 46 ± 2 nM, which is the most potent BR biosynthesis inhibitor observed to date. Use of gibberellin (GA) biosynthesis mutants and BR signaling mutants to analyze the mechanism of action of this synthetic series indicated that the primary site of action is BR biosynthesis. Experiments feeding BR biosynthesis intermediates to chemically treated Arabidopsis seedlings suggested that the target sites of this synthetic series are CYP90s, which are responsible for the C-22 and/or C-23 hydroxylation of campesterol.     

A new complex triterpenoid saponin from Calliandra pulcherrima with haemolytic activity and adjuvant effect

A new complex triterpenoid saponin was isolated from the leaves of Calliandra pulcherrima by using chromatographic methods. On the basis of chemical evidence, spectroscopic analyses and comparison of known compounds its structure was established as 3-[(O-α-l-arabinopyranosyl-(1 → 2)-O-α-l-arabinopyranosyl-(1 → 6)-2-(acetylamino)-2-deoxy-β-d-glucopyranosyl)oxy]-(3β)-olean-12-en-28-oic acid O-β-d-xylopyranosyl-(1 → 3)-O-β-d-xylopyranosyl-(1 → 4)-O-[(β-d-glucopyranosyl-(1 → 3)]-O-6-deoxy-α-l-mannopyranosyl-(1 → 2)-6-O-[(2E,6S)-6-[[2-O-[(2E,6S)-6-[[6-deoxy-2-O-[(2E,6S)-2,6-dimethyl-1-oxo-6-(β-d-xylopyranosyloxy)-2,7-octadienyl]-β-d-glucopyranosyl]oxy]-2,6-dimethyl-1-oxo-2,7-octadienyl]-β-d-xylopyranosyl]oxy]-2,6-dimethyl-1-oxo-2,7-octadienyl]-β-d-glucopyranosyl ester (1). The haemolytic activity of the saponin was evaluated using in vitro assays, and its adjuvant potential on the cellular immune response against ovalbumin antigen was investigated using in vivo models