Research Interests: Microbiology, Biology, Medicine, Biological Sciences, Female, and 15 moreAnimals, Spectrum, Lipopolysaccharides, Otitis Media, Rabbits, Moraxella Catarrhalis, Tetanus Toxoid, Enzyme Linked Immunosorbent Assay, Immunoglobulin, Antigen, Bactericidal Activity, immunoglobulin G, epitope, Medical and Health Sciences, and Respiratory Infection
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Research Interests: Chemistry, Organic Chemistry, Mass Spectrometry, Medicine, Cerebrospinal Fluid, and 15 moreGas Chromatography, Phosphorylation, Container Terminal, Inner Core, Lipopolysaccharides, Sialic Acid, Lipid A, Hydrolysis, Carbohydrate, Biochemistry and cell biology, Oligosaccharides, Carbohydrate Sequence, Molecular Sequence Data, Acid hydrolysis, and Neisseria meningitidis
SummaryBacterial exopolysaccharide (EPS) and lipopolysaccharide (LPS) molecules have been shown to play important roles in plant‐bacterium interactions. Here we have demonstrated that the fix‐23 loci, which compensate for exo mutations... more
SummaryBacterial exopolysaccharide (EPS) and lipopolysaccharide (LPS) molecules have been shown to play important roles in plant‐bacterium interactions. Here we have demonstrated that the fix‐23 loci, which compensate for exo mutations during symbiotic nodule development, are involved in the production of a novel polysaccharide that is rich in 3‐deoxy‐D manno‐2‐octulosonic acid (Kdo) but is not the classical LPS. This molecule is likely to be a surface antigen since antiserum to whole Rhizobium meliloti cells reacts strongly with it, and since mutations in fix‐23 result in an inability to produce this polysaccharide and to bind bacteriophage 16‐3. It is likely that this Kdo‐rich polysaccharide is analogous to certain Escherichia coli K‐antigens which are anchored to the membrane via a phospholipid moiety. DNA sequence analysis of one gene cluster of this region revealed that the predicted protein products of six genes exhibit a high degree of homology and similar organization to those of the rat fatty acid synthase multifunctional enzyme domains.
Research Interests: Biochemistry, Biology, Molecular Microbiology, Medicine, Biological Sciences, and 15 moreMolecular, Sequence alignment, Animals, Rhizobium, Gene, Rats, Chickens, Multienzyme complexes, Amino Acid Sequence, Base Sequence, Lipid A, Gene Expression Regulation, Molecular Sequence Data, polysaccharide, and Medical and Health Sciences
Research Interests: Biochemistry, Bacteriology, Biology, Magnetic Resonance Spectroscopy, Medicine, and 13 moreBiological Sciences, Escherichia coli, Rhizobium, High Pressure Liquid Chromatography, Sinorhizobium meliloti, Species Specificity, Disaccharides, Galactose Binding, Size Exclusion Chromatography, Carbohydrate Sequence, Molecular Sequence Data, polysaccharide, and Medical and Health Sciences
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Research Interests: Microbiology, Chemistry, Glycobiology, Medicine, Biological Sciences, and 11 moreLipopolysaccharide, Bacteria, Enterobacteriaceae, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Salmonella, Lipopolysaccharides, Electrophoresis, Gel electrophoresis, Proteus Mirabilis, Proteus, and Medical and Health Sciences
Most nodulatlon genes of the broad host-range Rhizobium sp. NGR234 are located on the 536 kb symbiotic plasmid. Complete sequencing of the replicon revealed over 400 open-reading frames (ORF), many of which correspond to known genes.... more
Most nodulatlon genes of the broad host-range Rhizobium sp. NGR234 are located on the 536 kb symbiotic plasmid. Complete sequencing of the replicon revealed over 400 open-reading frames (ORF), many of which correspond to known genes. Combined physical/genetic analysis of the host specificity of nodulation (hsn) locus lll revealed many nodulation genes, some of which are essential for Nod-factor production (nodABQ and others which modify the Nod-factor structure in specific ways (noeE). When mutated, two other genes, ORF3 and fixF gave Fix⁻ phenotypes on Vigna unguiculata. fixF has a nuclear localisation sequence (NLS) which is functional in nuclear targeting in tobacco protoplasts. Mutations in fixF abolish production of a novel, rhamnose-rich lipopolysaccharide that is produced under conditions of flavonoid induction.
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The first complementation unit of the fix-23 region of Rhizobium meliloti, which comprises six genes (rkpAB-CDEF) exhibiting similarity to fatty acid synthase genes, is required for the production of a novel type of capsular... more
The first complementation unit of the fix-23 region of Rhizobium meliloti, which comprises six genes (rkpAB-CDEF) exhibiting similarity to fatty acid synthase genes, is required for the production of a novel type of capsular polysaccharide that is involved in root nodule development and structurally analogous to group II K antigens found in Escherichia coli (G. Petrovics, P. Putnoky, R. Reuhs, J. Kim, T. A. Thorp, K. D. Noel, R. W. Carlson, and A. Kondorosi, Mol. Microbiol. 8:1083-1094, 1993; B. L. Reuhs, R. W. Carlson, and J. S. Kim, J. Bacteriol. 175:3570-3580, 1993). Here we present the nucleotide sequence for the other three complementation units of the fix-23 locus, revealing the presence of four additional open reading frames assigned to genes rkpGHI and -J. The putative RkpG protein shares similarity with acyltransferases, RkpH is homologous to short-chain alcohol dehydrogenases, and RkpJ shows significant sequence identity with bacterial polysaccharide transport proteins, su...
Research Interests: Bacteriology, Biology, Magnetic Resonance Spectroscopy, Medicine, Biological Sciences, and 15 moreMutagenesis, Sequence alignment, Escherichia coli, Rhizobium, Complementation, Gene, Sinorhizobium meliloti, Base Sequence, Open Reading Frame, Recombinant Proteins, Plasmid, Aprotinin, Molecular Sequence Data, Restriction Mapping, and Medical and Health Sciences
The rhizobial production of extracellular polysaccharide (EPS) is generally required for the symbiotic infection of host plants that form nodules with an apical meristem (indeterminate nodules). One exception is Rhizobium meliloti AK631,... more
The rhizobial production of extracellular polysaccharide (EPS) is generally required for the symbiotic infection of host plants that form nodules with an apical meristem (indeterminate nodules). One exception is Rhizobium meliloti AK631, an exoB mutant of Rm41, which is deficient in EPS production yet infects and fixes nitrogen (i.e., is Fix+) on alfalfa, an indeterminate nodule-forming plant. A mutation of lpsZ in AK631 results in a Fix- strain with altered phage sensitivity, suggesting that a cell surface factor may substitute for EPS in the alfalfa-AK631 symbiosis. Biochemical analyses of the cell-associated polysaccharides of AK631 and Rm5830 (AK631 lpsZ) demonstrated that the lpsZ mutation affected the expression of a surface polysaccharide that is analogous to the group II K polysaccharides of Escherichia coli; the polysaccharide contains 3-deoxy-D-manno-2-octulosonic acid or a derivative thereof in each repeating unit. Rm5830 produced a polysaccharide with altered chromatogra...
Research Interests: Biochemistry, Bacteriology, Hydrogen, Biology, Magnetic Resonance Spectroscopy, and 14 moreMedicine, Biological Sciences, Nitrogen Fixation, Mutation, Escherichia coli, Rhizobium, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Gel Permeation Chromatography, Phenotype, Sinorhizobium meliloti, Immunoblotting, polysaccharide, Medical and Health Sciences, and Cystoviridae
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Research Interests: Chemistry, Plant Biology, Medicine, Plant Roots, Glucans, and 9 moreSoybeans, Xylans, Pectins, Cell Wall, Planta, Xyloglucan, Tip Growth, Mannans, and root hair
The bacterial surface is the first line of defense against antimicrobial molecules and stress caused by changes in the environment surrounding the bacterium. In the case of plant- and animal-microbe interactions, many bacterial cell... more
The bacterial surface is the first line of defense against antimicrobial molecules and stress caused by changes in the environment surrounding the bacterium. In the case of plant- and animal-microbe interactions, many bacterial cell surface molecules are important virulence determinants. Thus, in order to understand the molecular basis for bacterial-plant interactions, it is important to characterize the molecular architecture of the bacterial cell surface, and how the bacterium modifies this architecture in response to its different environments, including its in planta environment.
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The cell surface of rhizobia is comprised of a number of polysaccharides; extracellular (EPSs), capsular (KPSs), and lipopolysaccharides (LPSs). Each is important in forming an effective nitrogen-fixing symbiosis. Defective mutants are... more
The cell surface of rhizobia is comprised of a number of polysaccharides; extracellular (EPSs), capsular (KPSs), and lipopolysaccharides (LPSs). Each is important in forming an effective nitrogen-fixing symbiosis. Defective mutants are unable to invade the host root cortical cells in a normal manner, or the infection thread is aborted prior to cortical cell invasion (1,2).
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Move Over, TLR4 The innate immune system senses bacterial lipopolysaccharide (LPS) through Toll-like receptor 4 (TLR4) (see the Perspective by Kagan ). However, Kayagaki et al. (p 1246 , published online 25 July) and Hagar et al. (p. 1250... more
Move Over, TLR4 The innate immune system senses bacterial lipopolysaccharide (LPS) through Toll-like receptor 4 (TLR4) (see the Perspective by Kagan ). However, Kayagaki et al. (p 1246 , published online 25 July) and Hagar et al. (p. 1250 ) report that the hexa-acyl lipid A component of LPS from Gramnegative bacteria is able to access the cytoplasm and activate caspase-11 to signal immune responses independently of TLR4. Mice that lack caspase-11 are resistant to LPS-induced lethality, even in the presence of TLR4.
Research Interests: Biology, Macrophages, Cell Signaling, Innate immunity, Medicine, and 15 moreMultidisciplinary, Lipopolysaccharide, Caspases, Mutation, Escherichia coli, Mice, Animals, Salmonella Typhimurium, Inflammasome, Inflammasomes, Lipid A, Cytoplasm, Escherichia Coli Infections, Innate Immune System, and Cholera toxin
Lysophosphatidic acid (LPA) and phosphatidic acid (PA) are critical phospholipid intermediates in the biosynthesis of cell membranes. In Escherichia coli, LPA acyltransferase (1‐acyl‐sn‐glycerol‐3‐phosphate acyltransferase; EC 2.3.1.51)... more
Lysophosphatidic acid (LPA) and phosphatidic acid (PA) are critical phospholipid intermediates in the biosynthesis of cell membranes. In Escherichia coli, LPA acyltransferase (1‐acyl‐sn‐glycerol‐3‐phosphate acyltransferase; EC 2.3.1.51) catalyses the transfer of an acyl chain from either acyl‐coenzyme A or acyl–acyl carrier protein onto LPA to produce PA. While E. coli possesses one essential LPA acyltransferase (PlsC), Neisseria meningitidis possesses at least two LPA acyltransferases. This study describes the identification and characterization of nlaB (neisserial LPA acyltransferase B), the second LPA acyltransferase identified in N. meningitidis. The gene was located downstream of the Tn916 insertion in N. meningitidis mutant 469 and differed in nucleotide and predicted amino acid sequence from the previously characterized neisserial LPA acyltransferase homologue nlaA. NlaB has specific LPA acyltransferase activity, as demonstrated by complementation of an E. coli plsC(Ts) mutan...
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Phosphoethanolamine (PEA) on Neisseria gonorrhoeae lipid A influences gonococcal inflammatory signaling and susceptibility to innate host defenses in in vitro models. Here, we evaluated the role of PEA-decorated gonococcal lipid A in... more
Phosphoethanolamine (PEA) on Neisseria gonorrhoeae lipid A influences gonococcal inflammatory signaling and susceptibility to innate host defenses in in vitro models. Here, we evaluated the role of PEA-decorated gonococcal lipid A in competitive infections in female mice and in male volunteers. We inoculated mice and men with mixtures of wild-type N. gonorrhoeae and an isogenic mutant that lacks the PEA transferase, LptA. LptA production conferred a marked survival advantage for wild-type gonococci in the murine female genital tract and in the human male urethra. Our studies translate results from test tube to animal model and into the human host and demonstrate the utility of the mouse model for studies of virulence factors of the human-specific pathogen N. gonorrhoeae that interact with non-host-restricted elements of innate immunity. These results validate the use of gonococcal LptA as a potential target for development of novel immunoprophylactic strategies or antimicrobial trea...
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Pseudomonas aeruginosa in the lungs of cystic fibrosis (CF) patients is characterized by a series of genotypic and phenotypic changes that reflect the transition from acute to chronic infection. These include the overproduction of the... more
Pseudomonas aeruginosa in the lungs of cystic fibrosis (CF) patients is characterized by a series of genotypic and phenotypic changes that reflect the transition from acute to chronic infection. These include the overproduction of the exopolysaccharide alginate and the loss of complete lipopolysaccharide (LPS). LPS is a major component of the Gram-negative outer membrane and is composed of lipid A, core oligosaccharide, and O antigen. In this report, we show that the LPS defect of the P. aeruginosa chronic infection isolate 2192 is temperature sensitive. When grown at 25°C, 2192 expresses serotype O1 LPS with a moderate chain length and in reduced amounts relative to those of a wild-type serotype O1 laboratory strain (stO1). In contrast, 2192 expresses no LPS O antigen when grown at 37°C. This is the first time that a temperature-sensitive defect in O-antigen production has been reported. Using complementation analyses with a constructed wbpM deletion mutant of stO1, we demonstrate ...
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Research Interests: Chemistry, Organic Chemistry, Structure, Mass Spectrometry, Medicine, and 15 moreHumans, Stereochemistry, Inner Core, Lipopolysaccharides, Gas Chromatography/mass Spectrometry, Sialic Acid, Glycolipid, Hydrolysis, Carbohydrate, Biochemistry and cell biology, Oligosaccharides, Myristic acid, Carbohydrate Sequence, Molecular Sequence Data, and Neisseria meningitidis
Bacillus anthracis (Ba) and human infection-associated Bacillus cereus (Bc) strains Bc G9241 and Bc 03BB87 have secondary cell wall polysaccharides (SCWPs) comprising an aminoglycosyl trisaccharide repeat:... more
Bacillus anthracis (Ba) and human infection-associated Bacillus cereus (Bc) strains Bc G9241 and Bc 03BB87 have secondary cell wall polysaccharides (SCWPs) comprising an aminoglycosyl trisaccharide repeat: →4)-β-d-ManpNAc-(1→4)-β-d-GlcpNAc-(1→6)-α-d-GlcpNAc-(1→, substituted at GlcNAc residues with both α- and β-Galp. In Bc G9241 and Bc 03BB87, an additional α-Galp is attached to O-3 of ManNAc. Using NMR spectroscopy, mass spectrometry and immunochemical methods, we compared these structures to SCWPs from Bc biovar anthracis strains isolated from great apes displaying "anthrax-like" symptoms in Cameroon (Bc CA) and Côte d'Ivoire (Bc CI). The SCWPs of Bc CA/CI contained the identical HexNAc trisaccharide backbone and Gal modifications found in Ba, together with the α-Gal-(1→3) substitution observed previously at ManNAc residues only in Bc G9241/03BB87. Interestingly, the great ape derived strains displayed a unique α-Gal-(1→3)-α-Gal-(1→3) disaccharide substitution at som...
Research Interests: Biochemistry, Evolutionary Biology, Genetics, Microbiology, Developmental Biology, and 15 moreImmunology, Molecular Biology, Computational Biology, Biotechnology, Cancer, Biology, Infectious Diseases, Medicine, Multidisciplinary, Animals, Nmr, Bacillus Cereus, Cell Wall, Pathogenicity, and Bacillus Anthracis
A lipooligosaccharide (LOS) mutant of Neisseria meningitidis serogroup B strain NMB (immunotype L3,7,9) was identified in a Tn916 (tetM) mutant bank by loss of reactivity with monoclonal antibody 3F11, which recognizes the terminal... more
A lipooligosaccharide (LOS) mutant of Neisseria meningitidis serogroup B strain NMB (immunotype L3,7,9) was identified in a Tn916 (tetM) mutant bank by loss of reactivity with monoclonal antibody 3F11, which recognizes the terminal Galbeta1-->4GlcNAc epitope in the lacto-N-neotetraose moiety of the wild-type LOS structure. The mutant, designated 559, was found to express a truncated LOS of 3.0 kDa. Southern and PCR analyses demonstrated that there was a single intact Tn916 insertion (class I) in the mutant 559 chromosome. Linkage of the LOS phenotype and the Tn916 insertion was confirmed by transformation of the wild-type parent. Nucleotide sequence analysis of the region surrounding the transposition site revealed a 1,065-bp open reading frame (ORF). A homology search of the GenBank/EMBL database revealed that the amino acid sequence of this ORF had 46.8% similarity and 21.2% identity with the alpha1,2 N-acetylglucosamine transferase (RfaK) from Salmonella typhimurium. Glycosyl ...
Research Interests: Bacteriology, Biology, Medicine, Gene expression, Sequence Analysis, and 13 moreBiological Sciences, Mutation, Lipopolysaccharides, Glucosamine, Amino Acid Sequence, Base Sequence, Serotyping, Carbohydrate Sequence, Molecular Sequence Data, Monosaccharides, epitope, Neisseria meningitidis, and Medical and Health Sciences
We have characterized an operon required for inner-core biosynthesis of the lipooligosaccharide (LOS) of Neisseria meningitidis. Using Tn916 mutagenesis, we recently identified the alpha-1,2-N-acetylglucosamine (GlcNAc) transferase gene... more
We have characterized an operon required for inner-core biosynthesis of the lipooligosaccharide (LOS) of Neisseria meningitidis. Using Tn916 mutagenesis, we recently identified the alpha-1,2-N-acetylglucosamine (GlcNAc) transferase gene (rfaK), which when inactivated prevents the addition of GlcNAc and alpha chain to the meningococcal LOS inner core (C. M. Kahler, R. W. Carlson, M. M. Rahman, L. E. Martin, and D. S. Stephens, J. Bacteriol. 178:1265-1273, 1996). During the study of rfaK, a second open reading frame (lgtF) of 720 bp was found upstream of rfaK. An amino acid sequence homology search of the GenBank and EMBL databases revealed that the amino terminus of LgtF has significant homology with a family of beta-glycosyltransferases involved in the biosynthesis of polysaccharides and O antigen of lipopolysaccharides. The chromosomal copy of lgtF was mutagenized with a nonpolar antibiotic resistance cassette to minimize potential polar effects on rfaK. Tricine sodium dodecyl sulf...
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Rhizobium bacteria live in soil and plant environments, are capable of inducing symbiotic nodules on legumes, invade these nodules, and develop into bacteroids that fix atmospheric nitrogen into ammonia. Rhizobial lipopolysaccharide (LPS)... more
Rhizobium bacteria live in soil and plant environments, are capable of inducing symbiotic nodules on legumes, invade these nodules, and develop into bacteroids that fix atmospheric nitrogen into ammonia. Rhizobial lipopolysaccharide (LPS) is anchored in the bacterial outer membrane through a specialized lipid A containing a very long-chain fatty acid (VLCFA). VLCFA function for rhizobial growth in soil and plant environments is not well understood. Two genes, acpXL and lpxXL, encoding acyl carrier protein and acyltransferase, are among the six genes required for biosynthesis and transfer of VLCFA to lipid A. Rhizobium leguminosarum mutant strains acpXL, acpXL−/lpxXL−, and lpxXL− were examined for LPS structure, viability, and symbiosis. Mutations in acpXL and lpxXL abolished VLCFA attachment to lipid A. The acpXL mutant transferred a shorter acyl chain instead of VLCFA. Strains without lpxXL neither added VLCFA nor a shorter acyl chain. In all strains isolated from nodule bacteria, ...
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Lipooligosaccharide (LOS) is a critical virulence factor of Neisseria meningitidis. A Tn916 insertion mutant, designated 469, was found to exhibit a markedly truncated LOS of 2.9 kDa when compared by Tricine/SDS-PAGE to the parental LOS... more
Lipooligosaccharide (LOS) is a critical virulence factor of Neisseria meningitidis. A Tn916 insertion mutant, designated 469, was found to exhibit a markedly truncated LOS of 2.9 kDa when compared by Tricine/SDS-PAGE to the parental LOS (4.6 kDa). Electrospray mass spectrometry analysis of 469 LOS revealed that it consisted of the deep rough, heptose-deficient structure, Kdo(2)-lipid A. Sequencing of chromosomal DNA flanking the Tn916 insertion in mutant 469 revealed that the transposon had inserted into an ORF predicted to encode a 187 aa protein with sequence homology to the histidinol-phosphate phosphatase domain of Escherichia coli HisB and to a family of genes of unknown function. The gene, designated gmhX, is part of a polycistronic operon (ice-2) containing two other genes, nlaB and orfC. nlaB encodes a lysophosphatidic-acid acyltransferase and orfC is predicted to encode a N-acetyltransferase. Specific polar and non-polar gmhX mutations in the parental strain, NMB, exhibited...
Research Interests: Microbiology, Mass Spectrometry, Biology, Helicobacter pylori, Medicine, and 14 moreMultidisciplinary, Lipopolysaccharide, Mutation, Escherichia coli, Gene, Enzyme, Inner Core, Lipopolysaccharides, Virulence factor, Homologous Recombination, Lipid A, Lysophosphatidic Acid, Molecular Sequence Data, and Neisseria meningitidis
The phospholipids of Neisseria meningitidis and Neisseria gonorrhoeae were characterized by fast atom bombardment (FAB)-MS and GLC-MS. The major phospholipids were phosphatidylethanolamine (PE), followed by phosphatidylglycerol (PG), with... more
The phospholipids of Neisseria meningitidis and Neisseria gonorrhoeae were characterized by fast atom bombardment (FAB)-MS and GLC-MS. The major phospholipids were phosphatidylethanolamine (PE), followed by phosphatidylglycerol (PG), with minor amounts of phosphatidic acid (PA) and trace levels of cardiolipin (DPG). All of the phospholipid preparations were variable in their fatty acyl substituents, which included C16:1, C16:0, C18:1, C14:0, C14:1 and C12:0. By MS/MS analysis, all pathogenic Neisseria spp. phospholipids contained a saturated fatty acyl substituent and either a saturated or unsaturated fatty acyl substituent in the sn-1 and sn-2 positions, respectively. Compared with enteric bacterial species, the phospholipids of N. meningitidis and N. gonorrhoeae have increased levels of phospholipids with short-chain fatty acyl residues (i.e. increases in C12:0, C14:1 and C14:0) and variable amounts of C18:1. The percentage of total PE and PG molecules with the shorter-chain fatty...
Research Interests: Biochemistry, Genetics, Microbiology, Chemistry, Mass Spectrometry, and 15 morePhospholipids, Medicine, Multidisciplinary, Humans, Fatty acids, Mutation, Escherichia coli, Membrane Lipids, Fatty Acid, Species Specificity, Phosphatidic acid, Phosphatidylglycerol, Neisseria gonorrhoeae, Cardiolipin, and Neisseria meningitidis
There is no licensed vaccine available against Moraxella catarrhalis, an exclusive human pathogen responsible for otitis media in children and respiratory infections in adults. We previously developed conjugate vaccine candidates based on... more
There is no licensed vaccine available against Moraxella catarrhalis, an exclusive human pathogen responsible for otitis media in children and respiratory infections in adults. We previously developed conjugate vaccine candidates based on lipooligosaccharides (LOSs) of M. catarrhalis serotypes A, B, and C, each of which was shown to cover a portion of the clinical strains. To generate conserved LOS antigens and eliminate a potential autoimmune response to a similar epitope between M. catarrhalis LOS moiety Galα1-4Galβ1-4Glc and human P(k) antigen, two LOS mutants from strain O35E were constructed. Mutant O35Elgt5 or O35EgalE revealed a deletion of one or two terminal galactose residues of wild type O35E LOS. Each LOS molecule was purified, characterized, detoxified, and coupled to tetanus toxoid (TT) to form conjugates, namely dLOS-TT. Three subcutaneous immunizations using dLOS-TT from O35Elgt5 or O35EgalE elicited significant increases (a 729- or 1263-fold above the preimmune seru...
Research Interests: Microbiology, Biology, Transmission Electron Microscopy, Medicine, Biological Sciences, and 15 moreFemale, Animals, Vaccination, Spectrum, Lipopolysaccharides, Otitis Media, Rabbits, Moraxella Catarrhalis, Tetanus Toxoid, Enzyme Linked Immunosorbent Assay, Immunoglobulin, Antigen, Bactericidal Activity, immunoglobulin G, and Medical and Health Sciences
Lipo-oligosaccharide (LOS) is a major surface component and virulence factor of the human respiratory pathogen Moraxella catarrhalis. Two late acyltransferase genes, lpxX and lpxL, have been identified involved in the incorporation of... more
Lipo-oligosaccharide (LOS) is a major surface component and virulence factor of the human respiratory pathogen Moraxella catarrhalis. Two late acyltransferase genes, lpxX and lpxL, have been identified involved in the incorporation of acyloxyacyl-linked secondary acyl chains into lipid A during M. catarrhalis LOS biosynthesis. In this study, a double mutant with a deletion of both the lpxX and lpxL genes in M. catarrhalis strain O35E was constructed and named O35ElpxXL. Structural analysis of lipid A showed that the O35ElpxXL mutant lacked two decanoic acids (10 : 0) and one dodecanoic (lauric) acid (12 : 0). In comparison with the O35E parental strain and the single mutants O35ElpxX and O35ElpxL, the double mutant O35ElpxXL displayed prominently decreased endotoxin content, reduced resistance to normal human serum and accelerated bacterial clearance at 0, 3 and 6 h after an aerosol challenge in a mouse model of bacterial pulmonary clearance. These results indicate that these two ge...
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Research Interests: Chemistry, Kinetics, Mass Spectrometry, Polysaccharides, Biological Chemistry, and 15 moreMagnetic Resonance Spectroscopy, Medicine, Biological Sciences, Biological, CHEMICAL SCIENCES, Secondary Cell Wall, Bacillus Cereus, Cell Wall, Bacillus Anthracis, Ions, Hexosamines, Carbohydrate Sequence, Molecular Sequence Data, polysaccharide, and Medical and Health Sciences
Rhizobium leguminosarum is a Gram-negative bacterium that forms nitrogen-fixing symbioses with compatible leguminous plants via intracellular invasion and establishes a persistent infection within host membrane-derived subcellular... more
Rhizobium leguminosarum is a Gram-negative bacterium that forms nitrogen-fixing symbioses with compatible leguminous plants via intracellular invasion and establishes a persistent infection within host membrane-derived subcellular compartments. Notably, an unusual very-long-chain fatty acid (VLCFA) is found in the lipid A of R. leguminosarum as well as in the lipid A of the medically relevant pathogens Brucella abortus , Brucella melitensis , Bartonella henselae , and Legionella pneumophila , which are also able to persist within intracellular host-derived membranes. These bacterial symbionts and pathogens each contain a homologous gene region necessary for the synthesis and transfer of the VLCFA to the lipid A. Within this region lies a gene that encodes the specialized acyl carrier protein AcpXL, on which the VLCFA is built. This study describes the biochemical and infection phenotypes of an acpXL mutant which lacks the VLCFA. The mutation was created in R. leguminosarum bv. phase...
Research Interests: Microbiology, Electron Microscopy, Bacteriology, Mass Spectrometry, Symbiosis, and 15 moreBiology, Scanning Electron Microscopy, Medicine, Biological Sciences, Virulence, Gas Chromatography, Phaseolus, Anti-Bacterial Agents, Detergents, Lipid A, Acyl carrier protein, Sodium Dodecyl Sulfate, Rhizobium leguminosarum, Hydroxy acids, and Medical and Health Sciences
The induction of an intense inflammatory response by Neisseria gonorrhoeae and the persistence of this pathogen in the presence of innate effectors is a fascinating aspect of gonorrhea. Phosphoethanolamine (PEA) decoration of lipid A... more
The induction of an intense inflammatory response by Neisseria gonorrhoeae and the persistence of this pathogen in the presence of innate effectors is a fascinating aspect of gonorrhea. Phosphoethanolamine (PEA) decoration of lipid A increases gonococcal resistance to complement-mediated bacteriolysis and cationic antimicrobial peptides (CAMPs), and recently we reported that wild-type N. gonorrhoeae strain FA1090 has a survival advantage relative to a PEA transferase A ( lptA ) mutant in the human urethral-challenge and murine lower genital tract infection models. Here we tested the immunostimulatory role of this lipid A modification. Purified lipooligosaccharide (LOS) containing lipid A devoid of the PEA modification and an lptA mutant of strain FA19 induced significantly lower levels of NF-κB in human embryonic kidney Toll-like receptor 4 (TLR4) cells and murine embryonic fibroblasts than wild-type LOS of the parent strain. Moreover, vaginal proinflammatory cytokines and chemokine...
Research Interests: Microbiology, Biology, Cytokines, Medicine, Toll like receptor signaling, and 15 moreBiological Sciences, Infection and immunity, Humans, Mice, Female, Animals, Reproductive Tract Infections, Microbial Sensitivity Tests, Chemokines, Lipid A, Neisseria gonorrhoeae, fibroblasts, TLR, Medical and Health Sciences, and gonorrhea
The lipooligosaccharide (LOS) of Neisseria meningitidis can be decorated with phosphoethanolamine (PEA) at the 4′ position of lipid A and at the O-3 and O-6 positions of the inner core of the heptose II residue. The biological role of PEA... more
The lipooligosaccharide (LOS) of Neisseria meningitidis can be decorated with phosphoethanolamine (PEA) at the 4′ position of lipid A and at the O-3 and O-6 positions of the inner core of the heptose II residue. The biological role of PEA modification in N. meningitidis remains unclear. During the course of our studies to elucidate the pathogenicity of the ST-2032 (invasive) meningococcal clonal group, disruption of lptA, the gene that encodes the PEA transferase for 4′ lipid A, led to a approximately 10-fold decrease in N. meningitidis adhesion to four kinds of human endothelial and epithelial cell lines at an multiplicity of infection of 5,000. Complementation of the lptA gene in a ΔlptA mutant restored wild-type adherence. By matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis, PEA was lost from the lipid A of the ΔlptA mutant compared to that of the wild-type strain. The effect of LptA on meningococcal adhesion was independent of other adhesins ...
Research Interests: Microbiology, Biology, Transmission Electron Microscopy, Medicine, Time-of-flight mass spectrometry, and 15 moreWestern blotting, Biological Sciences, Infection and immunity, Cell line, Humans, Endothelial Cells, Infection, Inner Core, Lipopolysaccharides, Epithelial cells, Lipid A, Bacterial Adhesion, Multiplicity of Infection (MOI), Neisseria meningitidis, and Medical and Health Sciences
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ABSTRACTTheBacillus anthracisexosporium protein BclA contains an O-linked antigenic tetrasaccharide whose terminal sugar is known as anthrose (J. M. Daubenspeck et al., J. Biol. Chem. 279:30945–30953, 2004). We hypothesized that serologic... more
ABSTRACTTheBacillus anthracisexosporium protein BclA contains an O-linked antigenic tetrasaccharide whose terminal sugar is known as anthrose (J. M. Daubenspeck et al., J. Biol. Chem. 279:30945–30953, 2004). We hypothesized that serologic responses to anthrose may have diagnostic value in confirming exposure to aerosolizedB. anthracis. We evaluated the serologic responses to a synthetic anthrose-containing trisaccharide (ATS) in a group of five rhesus macaques that survived inhalation anthrax following exposure toB. anthracisAmes spores. Two of five animals (RM2 and RM3) were treated with ciprofloxacin starting at 48 hours postexposure and two (RM4 and RM5) at 72 h postexposure; one animal (RM1) was untreated. Infection was confirmed by blood culture and detection of anthrax toxin lethal factor (LF) in plasma. Anti-ATS IgG responses were determined at 14, 21, 28, and 35 days postexposure, with preexposure serum as a control. All animals, irrespective of ciprofloxacin treatment, moun...
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Huanglongbing (HLB) disease, also known as citrus greening disease, was first reported in the US in 2005. Since then, the disease has decimated the citrus industry in Florida, resulting in billions of dollars in crop losses and the... more
Huanglongbing (HLB) disease, also known as citrus greening disease, was first reported in the US in 2005. Since then, the disease has decimated the citrus industry in Florida, resulting in billions of dollars in crop losses and the destruction of thousands of acres of citrus groves. The causative agent of citrus greening disease is the phloem limited pathogen Candidatus Liberibacter asiaticus. As it has not been cultured, very little is known about the structural biology of the organism. Liberibacter are part of the Rhizobiaceae family, which includes nitrogen-fixing symbionts of legumes as well as the Agrobacterium plant pathogens. To better understand the Liberibacter genus, a closely related culturable bacterium (Liberibacter crescens or Lcr) has attracted attention as a model organism for structural and functional genomics of Liberibacters. Given that the structure of lipopolysaccharides (LPS) from Gram-negative bacteria plays a crucial role in mediating host-pathogen interactio...
Research Interests: Genetics, Microbiology, Biology, Medicine, Bacteria, and 4 morePathogen, Organism, Molecular sciences, and Rhizobiaceae
Granulibacter bethesdensis can infect patients with chronic granulomatous disease, an immunodeficiency caused by reduced phagocyte NADPH oxidase function. Intact G. bethesdensis (Gb) is hypostimulatory compared to Escherichia coli, i.e.,... more
Granulibacter bethesdensis can infect patients with chronic granulomatous disease, an immunodeficiency caused by reduced phagocyte NADPH oxidase function. Intact G. bethesdensis (Gb) is hypostimulatory compared to Escherichia coli, i.e., cytokine production in human blood requires 10–100 times more G. bethesdensis CFU/mL than E. coli. To better understand the pathogenicity of G. bethesdensis, we isolated its lipopolysaccharide (GbLPS) and characterized its lipid A. Unlike with typical Enterobacteriaceae, the release of presumptive Gb lipid A from its LPS required a strong acid. NMR and mass spectrometry demonstrated that the carbohydrate portion of the isolated glycolipid consists of α-Manp-(1→4)-β-GlcpN3N-(1→6)-α-GlcpN-(1⇿1)-α-GlcpA tetra-saccharide substituted with five acyl chains: the amide-linked N-3′ 14:0(3-OH), N-2′ 16:0(3-O16:0), and N-2 18:0(3-OH) and the ester-linked O-3 14:0(3-OH) and 16:0. The identification of glycero-d-talo-oct-2-ulosonic acid (Ko) as the first constit...
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In the symbiosis formed between Mesorhizobium loti strain R7A and Lotus japonicus Gifu, rhizobial exopolysaccharide (EPS) plays an important role in infection thread formation. Mutants of strain R7A affected in early exopolysaccharide... more
In the symbiosis formed between Mesorhizobium loti strain R7A and Lotus japonicus Gifu, rhizobial exopolysaccharide (EPS) plays an important role in infection thread formation. Mutants of strain R7A affected in early exopolysaccharide biosynthetic steps form nitrogen-fixing nodules on L. japonicus Gifu after a delay, whereas mutants affected in mid or late biosynthetic steps induce uninfected nodule primordia. Recently it was shown that a plant receptor-like kinase, EPR3, binds low molecular mass exopolysaccharide from strain R7A to regulate bacterial passage through the plant's epidermal cell layer (Kawaharada, Y. et al. (2015) Nature 523, 308-312). In this work, we define the structure of both high and low molecular mass exopolysaccharide from R7A. The low molecular mass exopolysaccharide produced by R7A is a monomer unit of the acetylated octasaccharide with the structure (2,3/3-OAc)β-D-RibfA-(1→4)-α-D-GlcpA-(1→4)-β-D-Glcp-(1→6)-(3OAc)β-D-Glcp-(1→6)-*[(2OAc)β-D-Glcp-(1→4)-(2/...
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During infection, the sexually transmitted pathogen Neisseria gonorrhoeae (the gonococcus) encounters numerous host-derived antimicrobials including cationic antimicrobial peptides (CAMPs) produced by epithelial and phagocytic cells.... more
During infection, the sexually transmitted pathogen Neisseria gonorrhoeae (the gonococcus) encounters numerous host-derived antimicrobials including cationic antimicrobial peptides (CAMPs) produced by epithelial and phagocytic cells. CAMPs have both direct and indirect killing mechanisms and help link the innate and adaptive immune responses during infection. Gonococcal CAMP resistance is likely important for avoidance of host nonoxidative killing systems expressed by polymorphonuclear granulocytes (e.g., neutrophils) and intracellular survival. Previously studied gonococcal CAMP resistance mechanisms include modification of lipid A with phosphoethanolamine by LptA and export of CAMPs by the MtrCDE efflux pump. In the related pathogen, Neisseria meningitidis, a two-component regulatory system (2CRS) termed MisR-MisS has been shown to contribute to the capacity of the meningococcus to resist CAMP killing. We report that the gonococcal MisR response regulator, but not the MisS sensor ...
Research Interests: Microbiology, Medical Microbiology, Biology, Antimicrobial Peptides, Medicine, and 12 moreHumans, Aminoglycosides, Antimicrobial, Anti-Bacterial Agents, Antimicrobial Cationic Peptides, Efflux, Lipid A, Neisseria gonorrhoeae, Innate Immune System, Neisseria meningitidis, Pharmacology and pharmaceutical sciences, and gonorrhea
Rhizobial surface polysaccharides are required for nodule formation on the roots of at least some legumes but the mechanisms by which they act are yet to be determined. As a first step to investigate the function of exopolysaccharide... more
Rhizobial surface polysaccharides are required for nodule formation on the roots of at least some legumes but the mechanisms by which they act are yet to be determined. As a first step to investigate the function of exopolysaccharide (EPS) in the formation of determinate nodules, we isolated Mesorhizobium loti mutants affected in various steps of EPS biosynthesis and characterized their symbiotic phenotypes on two Lotus spp. The wild-type M. loti R7A produced both high molecular weight EPS and lower molecular weight (LMW) polysaccharide fractions whereas most mutant strains produced only LMW fractions. Mutants affected in predicted early biosynthetic steps (e.g., exoB) formed nitrogen-fixing nodules on Lotus corniculatus and L. japonicus ‘Gifu’, whereas mutants affected in mid or late biosynthetic steps (e.g., exoU) induced uninfected nodule primordia and, occasionally, a few infected nodules following a lengthy delay. These mutants were disrupted at the stage of infection thread (I...