Studies on the reductive amination of 3-deoxy-D-manno-octulosonic acid (Kdo)

Reductive amination of 3-deoxy-D-manno-octulosonic acid (Kdo) with allylamine (AllN) or 2-(4-aminophenyl)ethylamine (APEA) yields epimer pairs of 2-N-allylamino and 2-N-[2-(4-aminophenyl)ethylamino]-2,3-dideoxy-D-glycero-D-galacto- and-2,3-dideoxy-D-glycero-D-talo-octonic acid. The yields were 50–60% due to reduction of Kdo to the respective polyols as side reaction products. Mass spectrometric analyses proved the amination derivatives to be the expected glycamines. Nuclear magnetic resonance (NMR) studies were performed on 2-N-allylamino-2,3-dideoxyoctonic acid which represents the chain terminus of allylaminated oligosaccharides derived from bacterial lipopolysaccharides (LPS) after acid hydrolysis and reductive allylamination.     

A novel 3-deoxy-D-manno-octulosonic acid (Kdo) hydrolase that removes the outer Kdo sugar of Helicobacter pylori lipopolysaccharide

The lipid A domain anchors lipopolysaccharide (LPS) to the outer membrane and is typically a disaccharide of glucosamine that is both acylated and phosphorylated. The core and O-antigen carbohydrate domains are linked to the lipid A moiety through the eight-carbon sugar 3-deoxy-D-manno-octulosonic acid known as Kdo. Helicobacter pylori LPS has been characterized as having a single Kdo residue attached to lipid A, predicting in vivo a monofunctional Kdo transferase (WaaA). However, using an in vitro assay system we demonstrate that H. pylori WaaA is a bifunctional enzyme transferring two Kdo sugars to the tetra-acylated lipid A precursor lipid IV(A). In the present work we report the discovery of a Kdo hydrolase in membranes of H. pylori capable of removing the outer Kdo sugar from Kdo2-lipid A. Enzymatic removal of the Kdo group was dependent upon prior removal of the 1-phosphate group from the lipid A domain, and mass spectrometric analysis of the reaction product confirmed the enzymatic removal of a single Kdo residue by the Kdo-trimming enzyme. This is the first characterization of a Kdo hydrolase involved in the modification of gram-negative bacterial LPS.     

A Haemophilus influenzae gene that encodes a membrane bound 3-deoxy-D-manno-octulosonic acid (Kdo) kinase. Possible involvement of kdo phosphorylation in bacterial virulence.

The lipopolysaccharide of Haemophilus influenzae contains a single 3-deoxy-D-manno-octulosonic acid (Kdo) residue derivatized with either a phosphate or an ethanolamine pyrophosphate moiety at the 4-OH position. In previous studies, we identified a kinase unique to H. influenzae extracts that phosphorylates Kdo-lipid IV(A), a key precursor of lipopolysaccharide in this organism. We have now identified the gene encoding the Kdo kinase by using an expression cloning approach. A cosmid library containing random DNA fragments from H. influenzae strain Rd was constructed in Escherichia coli. Extracts of 472 colonies containing individual hybrid cosmids were assayed for Kdo kinase activity. A single hybrid cosmid directing expression of the kinase was found. The kinase gene was identified by activity assays, sub-cloning, and DNA sequencing. When the putative kinase gene was expressed in E. coli behind a T7 promoter, massive overproduction of kinase activity was achieved ( approximately 8000-fold higher than in H. influenzae membranes). The catalytic properties and the product generated by the overexpressed kinase, assayed with Kdo-lipid IV(A) as the substrate, were the same as observed with H. influenzae membranes. Unexpectedly, the kinase gene was identical to a previously characterized open reading frame (orfZ), which had been shown to be important for establishing bacteremia in an infant rat model (Hood, D. W., Deadman, M. E., Allen, T., Masoud, H., Martin, A., Brisson, J. R., Fleischmann, R., Venter, J. C., Richards, J. C., and Moxon, E. R. (1996) Mol. Microbiol. 22, 951-965). However, based solely on the genome sequence of H. influenzae Rd, no biochemical function had been assigned to the product of orfZ, which we now designate kdkA ("Kdo kinase A"). Although Kdo phosphorylation may be critical for bacterial virulence of H. influenzae, it does not appear to be required for growth.     

Mapping of the binding specificity for five monoclonal antibodies recognizing 3-deoxy-D-manno-octulosonic acid in bacterial lipopolysaccharides

Mouse mAb were produced against the deep rough strains Salmonella minnesota R 595, Salmonella typhimurium SL 1102, and Escherichia coli D21f2 and screened by enzyme immunoassay against LPS of several chemotypes. Five antibodies were selected for their ability to bind to chemotype deep rough (Re) LPS which has two 3-deoxy-D-manno-octulosonic acid (Kdo) residues in its nonreducing end. Structurally verified oligosaccharides isolated from rough LPS and synthetic analogues of Kdo were used in an enzyme immunoassay inhibition test to determine the binding epitopes for the antibodies. According to their specificities, the antibodies could be divided into three groups. For two of the groups, the recognized structure was the alpha-Kdo (2----4) Kdo disaccharide and for one group the alpha-Kdo (2----4) alpha-Kdo beta-D-GlcN (1----6) alpha-D-GlcN tetrasaccharide, representing a partial structure of the Re LPS. Inhibition studies with synthetic analogues of Kdo showed that the anomeric configuration and the free carboxyl group of the Kdo residue are important features for antibody binding. Changes in the C-1 to C-6 region of the Kdo molecule do influence the antibody recognition considerably whereas changes in the exocyclic C-7 to C-8 region are of secondary importance. Calculation of the conformation of the inner core region showed that the alpha-Kdo (2----4) alpha-Kdo (2---- disaccharide was free and accessible in chemotype Re LPS, but that linkage of a L-glycero-D-manno-heptose to O-5 of the subterminal Kdo both changes the conformation of the Kdo-disaccharide and covers it thereby making it less accessible.     

Synthesis of analogues of 3-deoxy-D-manno-octulosonic acid (KDO) as potential inhibitors of CMP-KDO synthetase

A series of derivatives of the 2-deoxy analogue of beta-KDO (2,6-anhydro-3-deoxy-D-glycero-D-talo-octonic acid; ammonium salt, 2) has been synthesised as potential inhibitors of CMP-KDO synthetase, starting from methyl 2,6-anhydro-3-deoxy-4,5:7,8-di-O-isopropylidene-D-glycero-D-talo- octonate and replacing the CO2Me group attached to C-2 variously by CONH2, CONHOH, CH2OH, CH2PO(OH)(O-NH4+), COCH2PO(OH)(O-H3N+pheny), CH2CO2-NH4+, CON-HCH2CO2-NH4+, CONHBn, CONHHexyl, CO2Bn, and CO2Hexyl. Of these derivatives, the hydroxamic acid (CONHOH) was the best inhibitor of CMP-KDO synthetase, but was less potent than 2.     

Colorimetric estimation of 3-deoxy-D-manno-octulosonic acid in oligosaccharides with diphenylamine

A method for the colorimetric estimation of 3-deoxy-d-manno-octulosonic acid with diphenylamine is described. The aldulosonic acid can be estimated in the presence of a tenfold excess of other sugars.     

The Origin of 8-Amino-3,8-dideoxy-d-manno-octulosonic Acid (Kdo8N) in the Lipopolysaccharide of Shewanella oneidensis

Lipopolysaccharide (LPS; endotoxin) is an essential component of the outer monolayer of nearly all Gram-negative bacteria. LPS is composed of a hydrophobic anchor, known as lipid A, an inner core oligosaccharide, and a repeating O-antigen polysaccharide. In nearly all species, the first sugar bridging the hydrophobic lipid A and the polysaccharide domain is 3-deoxy-d-manno-octulosonic acid (Kdo), and thus it is critically important for LPS biosynthesis. Modifications to lipid A have been shown to be important for resistance to antimicrobial peptides as well as modulating recognition by the mammalian innate immune system. Therefore, lipid A derivatives have been used for development of vaccine strains and vaccine adjuvants. One derivative that has yet to be studied is 8-amino-3,8-dideoxy-d-manno-octulosonic acid (Kdo8N), which is found exclusively in marine bacteria of the genus Shewanella. Using bioinformatics, a candidate gene cluster for Kdo8N biosynthesis was identified in Shewanella oneidensis. Expression of these genes recombinantly in Escherichia coli resulted in lipid A containing Kdo8N, and in vitro assays confirmed their proposed enzymatic function. Both the in vivo and in vitro data were consistent with direct conversion of Kdo to Kdo8N prior to its incorporation into the Kdo8N-lipid A domain of LPS by a metal-dependent oxidase followed by a glutamate-dependent aminotransferase. To our knowledge, this oxidase is the first enzyme shown to oxidize an alcohol using a metal and molecular oxygen, not NAD(P)⁺. Creation of an S. oneidensis in-frame deletion strain showed increased sensitivity to the cationic antimicrobial peptide polymyxin as well as bile salts, suggesting a role in outer membrane integrity.     

Synthesis of C-(beta-D-glycosyl) analogues of 3-deoxy-D-manno-2-octulosonic acid (Kdo) as potential inhibitors of CMP-Kdo synthetase

Eight C-(beta-D-glycosyl) analogues (14-21) of Kdo (3-deoxy-D-manno-2-octulosonic acid) were obtained from isopropylidene-protected ester precursors and tested in vitro for their inhibitory activity on CMP-Kdo synthetase. None had more than minor inhibitory activity.     

Determination of 3-deoxy-D-manno-octulosonic acid (KDO), N-acetylneuraminic acid, and their derivatives by ion-exchange liquid chromatography

A liquid chromatography (1.6 MPa) system for the analysis of 3-deoxy-D-manno-2-octulosonic acid (KDO), N-acetylneuraminic acid (Neu5Ac), methyl alpha- and beta-glycosides of Neu5Ac and KDO, alpha-heptosyl-(1----5)-KDO, various sialyllactoses, alpha-KDO-(2----4)-KDO, alpha-KDO-(2----4)-KDO methyl alpha-glycoside, beta-KDO-(2----4)-KDO methyl beta-glycoside, D-glucuronic acid, D-glucurono-3,6-lactone, and D-galacturonic acid has been developed. Separation was achieved within 10 and 30 min by the use of a small column filled with a strongly basic, anion-exchange resin, Aminex A-29, and 0.75 or 10mM sodium sulfate solutions as mobile phases. This method allowed the determination of KDO and sialic acids in amounts of 100 ng (0.5 nmol) and 200 pg (0.6 pmol), respectively.     

What are toxins to microbes? (the role of toxins in bacterial ecology)

The author attempts to answer two questions: whether the toxins, in particular the toxins having their specificity connected with enzymatic activity, are needed for microbial cell physiology and their significance for bacteria that are not the obligate parasites for warm blooded animals. The analysis of literary data supposes the toxins to be essential cellular metabolites since many of them participate in energy acquiring. Besides that a number of toxins is shown to be relevant to microbial life and to affect the micropredators, especially the monocellular organisms feeding the microbes. In connection with the above mentioned, special attention is paid to extrachromosomal location of many toxins genes relating them to bacteriocins. The possibility is not excluded that in the future the new toxins might come to be found having the enzymatic activities.     

The synthesis of the rhamnogalacturonan II component 3-deoxy-D-manno-2-octulosonic acid (Kdo) is required for pollen tube growth and elongation

Despite a very complex structure, the sugar composition of the rhamnogalacturonan II (RG-II) pectic fraction is extremely conserved. Among its constituting monosaccharides is the seldom-observed eight-carbon sugar 3-deoxy-D-manno-octulosonic acid (Kdo), whose phosphorylated precursor is synthesized by Kdo-8-P synthase. As an attempt to alter specifically the RG-II structure in its sugar composition and assess the consequences on the function of RG-II in cell wall and its relationship with growth, Arabidopsis null mutants were sought in the genes encoding Kdo-8-P synthase. Here, the isolation and characterization of one null mutant for the isoform 1 (AtkdsA1-S) and two distinct null mutants for the isoform 2 of Arabidopsis Kdo-8-P synthase (AtkdsA2-V and AtkdsA2-S) are described. Evidence is provided that AtkdsA2 gene expression is preferentially associated with plantlet organs displaying a meristematic activity, and that it accounts for 75% of the mRNAs to be translated into Kdo-8-P synthase. Furthermore, this predominant expression of AtKDSA2 over AtKDSA1 was confirmed by quantification of the cytosolic Kdo content in the mutants, in a variety of ecotypes. The inability to identify a double knockout mutant originated from pollen abortions, due to the inability of haploid pollen of the AtkdsA1- AtkdsA2- genotype to form an elongated pollen tube properly and perform fertilization.     

Plasma membrane association of three classes of bacterial toxins is mediated by a basic-hydrophobic motif

Plasma membrane targeting is essential for the proper function of many bacterial toxins. A conserved fourhelical bundle membrane localization domain (4HBM) was recently identified within three diverse families of toxins: clostridial glucosylating toxins, MARTX toxins and Pasteurella multocida-like toxins. When expressed in tissue culture cells or in yeast, GFP fusions to at least one 4HBM from each toxin family show significant peripheral membrane localization but with differing profiles. Both in vivo expression and in vitro binding studies reveal that the ability of these domains to localize to the plasma membrane and bind negatively charged phospholipids requires a basic-hydrophobic motif formed by the L1 and L3 loops. The different binding capacity of each 4HBM is defined by the hydrophobicity of an exposed residue within the motif. This study establishes that bacterial effectors utilize a normal host cell mechanism to locate the plasma membrane where they can then access their intracellular targets.     

3-Deoxy-D-manno-oct-2-ulosonic acid (Kdo) transferase (WaaA) and kdo kinase (KdkA) of Haemophilus influenzae are both required to complement a waaA knockout mutation of Escherichia coli

The lipopolysaccharide (LPS) of the deep rough mutant Haemophilus influenzae I69 consists of lipid A and a single 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) residue substituted with one phosphate at position 4 or 5 (Helander, I. M., Lindner, B., Brade, H., Altmann, K., Lindberg, A. A., Rietschel, E. T., and Z?hringer, U. (1988) Eur. J. Biochem. 177, 483-492). The waaA gene encoding the essential LPS-specific Kdo transferase was cloned from this strain, and its nucleotide sequence was identical to H. influenzae DSM11121. The gene was expressed in the Gram-positive host Corynebacterium glutamicum and characterized in vitro to encode a monofunctional Kdo transferase. waaA of H. influenzae could not complement a knockout mutation in the corresponding gene of an Re-type Escherichia coli strain. However, complementation was possible by coexpressing the recombinant waaA together with the LPS-specific Kdo kinase gene (kdkA) of H. influenzae DSM11121 or I69, respectively. The sequences of both kdkA genes were determined and differed in 25 nucleotides, giving rise to six amino acid exchanges between the deduced proteins. Both E. coli strains which expressed waaA and kdkA from H. influenzae synthesized an LPS containing a single Kdo residue that was exclusively phosphorylated at position 4. The structure was determined by nuclear magnetic resonance spectroscopy of deacylated LPS. Therefore, the reaction products of both cloned Kdo kinases represent only one of the two chemical structures synthesized by H. influenzae I69.     

Biosynthesis of endotoxins. Purification and catalytic properties of 3-deoxy-D-manno-octulosonic acid transferase from Escherichia coli.

The enzyme 3-deoxy-D-manno-octulosonic acid (Kdo) transferase is encoded by the kdtA gene of Escherichia coli and plays a key role in lipopolysaccharide biosynthesis. It transfers Kdo from CMP-Kdo to lipid A or its tetraacyldisaccharide-1,4'-bisphosphate precursor, lipid IVA. Using a strain that overproduces the transferase approximately 500-fold, we have purified the enzyme to near homogeneity. The subunit molecular mass is approximately 43 kDa. Activity is stimulated by Triton X-100, is maximal at pH 7, but does not require Mg2+. The apparent Km values for lipid IVA and CMP-Kdo are 52 and 88 microM, respectively. Vmax is 15-18 mumol/min/mg when both substrates are added near saturation at pH 8. The purified enzyme transfers 2 Kdo residues to lipid A precursors or analogs bearing four to six fatty acyl chains and a 4'-monosphosphate moiety. Activity is inhibited by polymixin B and Re endotoxin. At low Kdo concentrations small amounts of the intermediate, (Kdo)1-IVA, accumulate. When this substance is isolated and incubated with purified enzyme in the presence of CMP-Kdo, it is converted to (Kdo)2-IVA. Formation of (Kdo)1-IVA is also observed when purified enzyme is incubated with (Kdo)2-IVA and 5 mM CMP, demonstrating that Kdo transfer is reversible. In summary, Kdo transferase consists of a single bifunctional polypeptide that incorporates the 2 innermost Kdo residues common to all lipopolysaccharide molecules in E. coli.     

Niche heterogeneity determines bacterial community structure in the termite gut (Reticulitermes santonensis)

Differences in microenvironment and interactions of microorganisms within and across habitat boundaries should influence structure and diversity of the microbial communities within an ecosystem. We tested this hypothesis using the well characterized gut tract of the European subterranean termite Reticulitermes santonensis as a model. By cloning and sequencing analysis and molecular fingerprinting (terminal restriction fragment length polymorphism), we characterized the bacterial microbiota in the major intestinal habitats - the midgut, the wall of the hindgut paunch, the hindgut fluid and the intestinal protozoa. The bacterial community was very diverse (> 200 ribotypes) and comprised representatives of several phyla, including Firmicutes (mainly clostridia, streptococci and Mycoplasmatales-related clones), Bacteroidetes, Spirochaetes and a number of Proteobacteria, all of which were unevenly distributed among the four habitats. The largest group of clones fell into the so-called Termite group 1 (TG-1) phylum, which has no cultivated representatives. The majority of the TG-1 clones were associated with the protozoa and formed two phylogenetically distinct clusters, which consisted exclusively of clones previously retrieved from the gut of this and other Reticulitermes species. Also the other clones represented lineages of microorganisms that were exclusively recovered from the intestinal tract of termites. The termite specificity of these lineages was underscored by the finding that the closest relatives of the bacterial clones obtained from R. santonensis were usually derived also from the most closely related termites. Overall, differences in diversity between the different gut habitats and the uneven distribution of individual phylotypes support conclusively that niche heterogeneity is a strong determinant of the structure and spatial organization of the microbial community in the termite gut.     

Biosynthesis of lipopolysaccharide in Escherichia coli. Cytoplasmic enzymes that attach 3-deoxy-D-manno-octulosonic acid to lipid A

Previous studies in our laboratory led to the elucidation of the covalent structure of a tetraacyldisaccharide 1,4'-bisphosphate precursor of lipid A (designated lipid IVA), that accumulates at 42 degrees C in temperature-sensitive mutants defective in 3-deoxy-D-manno-octulosonic acid (KDO) biosynthesis (Raetz, C. R. H., Purcell, S., Meyer, M. V., Qureshi, N., and Takayama, K. (1985) J. Biol. Chem. 260, 16080-16088). Using [4'-32P]lipid IVA as the probe, we now demonstrate the existence of cytoplasmic KDO-transferases in Escherichia coli capable of attaching 2 KDO residues, derived from CMP-KDO, to lipid IVA. A partial purification has been developed to obtain a cytoplasmic subfraction that adds these 2 KDO residues with a 90% yield. The product is shown to have the stoichiometry of (KDO)2-IVA by fast atom bombardment mass spectrometry and NMR spectroscopy. The partially purified enzyme can utilize alternative lipid-disaccharide cosubstrates bearing five or six fatty acyl chains, but it has an absolute requirement for a monophosphate residue at position 4' of the lipid acceptor. When reincubated with a crude cytoplasmic fraction, a nucleoside triphosphate and Mg2+, (KDO)2-IVA is rapidly metabolized to more polar substances, the identity of which is unknown. The KDO-transferase(s) described in the present study should be very useful for the semisynthetic preparation of complex lipopolysaccharide substructures and analogs.     

Structure of the hexose region of Shigella sonnei phase II lipopolysaccharide with 3-deoxy-D-manno-octulosonic acid as possible immunodeterminant and its relation to Escherichia coli R1 core.

Comparative chemical analysis (methylation, gas chromatography/mass spectrometry, periodate oxidation, etc.) of the lipopolysaccharides and degraded polysaccharides derived from Shigella sonnei phase I, phase II and galactose-deficient R mutants revealed a structure as shown: (formula: see text) 3-Deoxy-D-manno-octulosonic acid (dOclA) as an immunodeterminant was observed in the passive hemolysis inhibition test by (a) selective inhibition of the phase II system by dOclA; (b) the kinetics of the change of serological activity during mild acid treatment: 1% acetic acid abolished serological activity; (c) a lack of activity in galactose-less R mutants and reactivity with Re mutants including Salmonella minnesota Re. An enhanced sensitivity of phase II lipopolysaccharide to galactose oxidase after prolonged treatment with 1% acetic acid suggests that dOclA is linked to C-6 of the terminal or subterminal galactose. dOclA as immunodeterminant could explain some different polysaccharide structures described for Escherichia coli R1 core.