Structure of a neutral O-specific polysaccharide of the bacterium Proteus mirabilis O24

Lipopolysaccharide of the bacterium Proteus mirabilis O24 was found to have a neutral O-specific polysaccharide chain containing D-galactose, 2-acetamido-2-deoxy-D-glucose, and 2-acetamido-2-deoxy-D-galactose in ratios 1:2:1. On the basis of 1H- and 13C-NMR spectroscopy, including two-dimensional correlation spectroscopy (COSY), H-detected 1H, 13C heteronuclear multiple-quantum coherence (HMQC), and nuclear Overhauser effect spectroscopy (NOESY), the following structure of the branched tetrasaccharide repeating unit of the O-specific polysaccharide was established: -->3)-beta-D-GlcpNAc-(1-->4)-beta-D-GalpNAc-(1-->4)-beta-D-GlcpNAc-(1--> [formula: see text] beta-D-Galp.     

Structure and epitope characterisation of the O-specific polysaccharide of Proteus mirabilis O28 containing amides of D-galacturonic acid with L-serine and L-lysine

The O-specific polysaccharide of Proteus mirabilis O28 was found to contain D-galactose, D-galacturonic acid (GalA), 2-acetamido-2-deoxy-D-glucose, L-serine, L-lysine, and O-acetyl groups in molar ratios 1:2:1:1:1:1, the amino acids being linked via their alpha-amino group to the carboxyl group of GalA. The polysaccharide was studied using 1H- and 13C-NMR spectroscopy, including selective spin-decoupling, one-dimensional total correlation spectroscopy, two-dimensional homonuclear correlation spectroscopy (COSY), heteronuclear 13C,1H COSY, one-dimensional NOE, and two-dimensional rotating-frame NOE spectroscopy and partial acid hydrolysis followed by borohydride reduction, methylation, and GLC/MS analysis of the derived glycosyl alditols. The following structure of the repeating unit was established: [formula: see text] Epitope specificity of the P. mirabilis O28 polysaccharide was analysed using a homologous rabbit polyclonal antiserum in quantitative precipitation, passive immunohemolysis, and inhibition of passive immunohemolysis. Study with related synthetic glycopolymers (2-acrylamidoethyl glycosides of amides of alpha-D-GalA with amino acids copolymerised with acrylamide) showed the importance of D-GalA(L-Lys) for manifesting serological specificity of the O-antigen. Serological cross-reactions between P. mirabilis O28, S1959, and R14/S1959 (a transient-like form) are discussed.     

Structure of a new neutral O-specific polysaccharide of Proteus penneri 34

Since Pneumocystis carinii cannot be cultured in vitro, the introduction of polymerase chain reaction (PCR) has been an enormous advantage for research purposes. It is now possible to detect P. carinii in specimens containing low numbers of organisms where conventional detection methods using microscopic examination of histochemical stains has been insufficient. PCR has been used to detect P. carinii in bronchoalveolar lavage, induced sputum, spontaneous expectorates, oropharyngeal gargles, nasopharyngeal aspirates, serum, blood and in environmental samples. The use of PCR will enable the study of the epidemiology of P. carinii infection by detecting the organism in environmental samples, permitting molecular typing and thereby the study of the transmission of the organism. Furthermore PCR will facilitate studies on the response to therapy, studies monitoring for the emergence of drug resistant strains of P. carinii and in the diagnosis of P. carinii pneumonia in noninvasive specimens, in patients unable to undergo more invasive diagnostic procedures.     

Structural and immunologic studies of Proteus mirabilis 033 O-specific polysaccharide

A variety of adrenal imaging agents have been used in nuclear medicine, but no agent has been developed for magnetic resonance (MR) imaging. The authors have previously observed accumulation of aminated macromolecules in adrenal glands. They now report the synthesis of a model polymeric aminated contrast agent for enhanced MR imaging of the adrenal glands. The model agent consisted of a poly-L-lysine conjugate (molecular weight, 245 kd) that had 70% free epsilon amino groups and 30% diethylenetriaminepentaacetic acid (DTPA)-derivatized amino groups to bind indium-111 or gadolinium. One hour after intravenous administration of this compound, adrenal uptake was 10.1% +/- 0.7 of injected dose per gram of tissue. When all free epsilon amino groups of the polylysine were completely substituted with DTPA, adrenal uptake was 3.4 times lower, indicating the importance of free amino groups for adrenal uptake. MR imaging in rats showed that a dose of 0.08 mmol of gadolinium per kilogram of the agent was sufficient to enhance the signal intensity of adrenal glands. There hours after intravenous administration of the agent, signal intensity of the adrenal glands was 186% of precontrast values (liver, 165%; kidney, 91%). Fluorescence microscopy showed that the agent accumulated primarily in the cortical zona glomerulosa and in the adrenal medulla. These initial studies demonstrate the feasibility of designing contrast agents for MR imaging of the adrenal glands.     

Immunochemical studies of O-specific polysaccharide from Proteus penneri 14 lipopolysaccharide

OBJECTIVE: To assess the safety of Norplant contraceptive implant use by women with mild-moderate homozygous sickle cell disease (HbSS). METHOD: Prospective observation of women pre- and post-insertion of Norplant, with each woman serving as her own control. Participants: 25 women 18-40 years of age who attended a hospital sickle cell clinic; post-insertion data were available for 23 women. Outcome measures: Changes in hematologic parameters including PCV, MCV, reticulocytes, ISCs, HbF and bilirubin; changes in biochemical parameters including HDL cholesterol, aspartate transaminase, alkaline phosphate, serum creatinine and serum albumin. RESULT: With a mean follow-up of 12.4 months (range 1-29 months), there were no clinically or statistically significant group or individual changes in the hematologic or biochemical parameters after Norplant insertion. CONCLUSION: Norplant appears to be a safe and appropriate contraceptive for women with mild-moderate HbSS disease.     

Structure of the O-specific polysaccharide of Citrobacter freundii O3a,3b,1c

The following structure of the O-specific polysaccharide of Citrobacter freundii O3a,3b,1c containing D-mannose and D-rhamnose was established using sugar analysis and NMR spectroscopy, including computer-assisted analysis of the 13C NMR spectrum, 2D COSY, H,H-relayed COSY, heteronuclear 13C, 1H correlation (HETCOR), and rotating-frame NOE spectroscopy (ROESY):-->4)-alpha-D-Manp-(1-->3)-beta-D-Rhap-(1-->4) -beta-D-Rhap-(1-->.     

A new tetracycline resistance determinant cloned from Proteus mirabilis

The chromosomal inducible Tcr determinant from Proteus mirabilis was cloned and the nucleotide sequence of both the structural and repressor genes determined. The deduced amino acid sequence of the structural protein shows the highest similarity to TetA(H) from Pasteurella multocida (78.4%), followed by TetA(B) from Tn10 (50.9%). Based on this analysis, we suggest that this new determinant can be assigned to a new class, TetJ.     

Structural and immunochemical studies on the O-specific polysaccharide of Proteus penneri strain 15

On the basis of sugar analysis and 1H- and 13C-NMR spectroscopy, it was shown that the O-specific polysaccharide of Proteus penneri strain 15 has a trisaccharide repeating unit, including an acetal-linked pyruvic acid residue, and is structurally identical to the capsular polysaccharide of Proteus vulgaris strain ATCC 49990. Serological studies supported this conclusion and demonstrated the presence in the homological antiserum of both anti-core and anti-O chain antibodies reacting with a lipopolysaccharide (LPS) epitope containing N-acetylglucosamine and galactose residues.     

Structure of a highly acidic O-specific polysaccharide of lipopolysaccharide of Pseudoalteromonas haloplanktis KMM 223 (44-1) containing L-iduronic acid and D-QuiNHb4NHb

An acidic O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide isolated by phenol-water extraction of Pseudoalteromonas haloplanktis strain KMM 223 (44-1). L-Iduronic acid (IdoA) was found to be a component of the polysaccharide and identified by NMR spectroscopy and after carboxyl-reduction followed by acid hydrolysis and acetylation, by GLC-MS as 2,3,4-tri-O-acetyl-1,6-anhydroidose. On the basis of 1H and 13C NMR spectroscopic studies, including 1D NOE, 2D NOESY, HSQC and HMBC experiments, the following structure of the branched pentasaccharide repeating unit of the polysaccharide was established: -->4)-beta-D-GlcpAI-(1-->4)-beta-D-GlcpAII-(1-->3)-beta-D-++ +QuipNHb4NHbII- (1-->2)-alpha-L-IdopA-(-->4 increases 1 alpha-D-QuipNAc4NAcI where QuiNAc4NAc and QuiNHb4NHb are 2,4-diacetamido-2,4,6-trideoxyglucose and 2,4,6-tri-deoxy-2,4- di[(S)-3-hydroxybutyramido]glucose, respectively. This is the first report of L-iduronic acid in a lipopolysaccharide and of D-QuiNHb4NHb in nature.     

Structure of the O-specific polysaccharide of an Aeromonas trota strain cross-reactive with Vibrio cholerae O139 Bengal

The O-specific polysaccharide of an Aeromonas trota strain was isolated by hydrolysis of the lipopolysaccharide at pH 4.5 followed by gel-permeation chromatography and found to consist of hexasaccharide repeating units containing D-galactose, L-rhamnose, 3,6-dideoxy-L-xylo-hexose (colitose, Col), 2-acetamido-2-deoxy-D-glucose and 2-acetamido-2-deoxy-D-galactose in the ratios 1:1:2:1:1. Partial hydrolysis of the polysaccharide with 48% hydrofluoric acid resulted in selective removal of colitose to give a modified polysaccharide containing the other four sugar constituents. On the basis of methylation analysis and NMR spectroscopic studies of the initial and modified, colitose-free polysaccharide, it was concluded that the repeating unit of the O-specific polysaccharide has the following structure [sequence: see text] The known cross-reactivity between the strain studied and Vibrio cholerae O139 Bengal is substantiated by the presence of a common colitose-containing epitope shared by the O-specific polysaccharide of A. trota and the capsular polysaccharide of V. cholerae, which is thought to carry determinants of O-specificity.     

Structural studies of the O-specific polysaccharide of Hafnia alvei strain PCM 1206 lipopolysaccharide containing D-allothreonine

The structure of the O-specific side-chain of the Hafnia alvei strain PCM 1206 lipopolysaccharide has been investigated. Methylation analysis, partial acid hydrolysis, FAB-MS/MS and 1H-NMR and 13C-NMR spectroscopy were the principal methods used. D-Allothreonine (D-aThr), amide-linked to the D-galacturonic acid, was identified as a constituent in the polysaccharide and the following structure of a pentasaccharide repeating unit was established: [structure: see text].     

Structural studies of the putative O-specific polysaccharide of Acinetobacter baumannii O2 containing 3,6-dideoxy-3-N-(D-3-hydroxybutyryl)amino-D-galactose

A polysaccharide containing D-galactose, 2-deoxy-2-N-acetylamino-D-galactose and 3,6-dideoxy-3-N-(D-3-hydroxybutyryl)amino-D-galactose, probably corresponding to the lipopolysaccharide side chain, was obtained from an aqueous phenol extract of isolated cell walls from Acinetobacter baumannii strain O2. By means of NMR studies and chemical degradations, the repeating unit of the polymer was identified as a branched hexasaccharide of the structure shown, where Fuc3N represents 3-amino-3,6-dideoxygalactose and R represents D-3-hydroxybutyryl. Serological tests indicated that the polymer corresponded to the O2 antigen.     

Chromosomally encoded ampC-type beta-lactamase in a clinical isolate of Proteus mirabilis

A clinical strain of Proteus mirabilis (CF09) isolated from urine specimens of a patient displayed resistance to amoxicillin (MIC >4,096 microg/ml), ticarcillin (4,096 microg/ml), cefoxitin (64 microg/ml), cefotaxime (256 microg/ml), and ceftazidime (128 microg/ml) and required an elevated MIC of aztreonam (4 microg/ml). Clavulanic acid did not act synergistically with cephalosporins. Two beta-lactamases with apparent pIs of 5.6 and 9.0 were identified by isoelectric focusing on a gel. Substrate and inhibition profiles were characteristic of an AmpC-type beta-lactamase with a pI of 9.0. Amplification by PCR with primers for ampC genes (Escherichia coli, Enterobacter cloacae, and Citrobacter freundii) of a 756-bp DNA fragment from strain CF09 was obtained only with C. freundii-specific primers. Hybridization results showed that the ampC gene is only chromosomally located while the TEM gene is plasmid located. After cloning of the gene, analysis of the complete nucleotide sequence (1,146 bp) showed that this ampC gene is close to blaCMY-2, from which it differs by three point mutations leading to amino acid substitutions Glu --> Gly at position 22, Trp --> Arg at position 201, and Ser --> Asn at position 343. AmpC beta-lactamases derived from that of C. freundii (LAT-1, LAT-2, BIL-1, and CMY-2) have been found in Klebsiella pneumoniae, E. coli, and Enterobacter aerogenes and have been reported to be plasmid borne. This is the first example of a chromosomally encoded AmpC-type beta-lactamase observed in P. mirabilis. We suggest that it be designated CMY-3.     

An antigenic polysaccharide from Campylobacter coli serotype O:30. Structure of a teichoic acid-like antigenic polysaccharide associated with the lipopolysaccharide

A water-soluble antigenic polysaccharide of high M(r) associated with the lipopolysaccharide has been isolated from phenol-water extraction of cells of Campylobacter coli serotype O:30. The polysaccharide and oligosaccharide degradation products formed on O-dephosphorylation and by periodate oxidation followed by reduction have been investigated by one- and two-dimensional 1H, 13C, and 31P NMR. It is concluded that the antigenic polysaccharide has a teichoic acid-like structure with a poly-Ribitol phosphate, [5-Ribitol-1-P]n, backbone with side chains at O-2 of O-(6-deoxy-beta-D-talo-heptopyranosyl)-(1-->4)-(2-acetylamino-2-deoxy-beta-D- glucopyranosyl) units. The structure is unusual in Gram-negative bacteria and is unique in possessing 6-deoxy-D-talo-heptose as a constituent sugar. Evidence for the relationship of the antigenic polysaccharide to the lipopolysaccharide of low M(r) is discussed.     

Complete amino acid sequence of Proteus mirabilis PR catalase. Occurrence of a methionine sulfone in the close proximity of the active site

The catalase of Proteus mirabilis PR, a peroxide-resistant (PR) mutant of Proteus mirabilis, binds strongly NADPH, which is a unique property among known bacterial catalases. The enzyme subunit consists of 484 amino acid residues for a mass of 55,647 daltons. The complete amino acid sequence was resolved through the combination of protein sequencing, mass spectrometry, and nucleotide sequencing of a PCR fragment. The sequence obtained was compared with that of other known catalases. Amino acids of the active site are all conserved as well as essential residues involved in NADPH binding. Among the amino acids interacting with the heme, a methionine sulfone was found at position 53, in place of a valine in most other catalases. The origin of oxidation of this methionine is unknown, but the presence of this modification could change iron accessibility by large substrates or inhibitors. This posttranslational modification was also demonstrated in the wild-type P. mirabilis catalase.     

Structure of the repeating unit of acid polysaccharide from Alteromonas sp. 4MS17

An acidic polysaccharide from Alteromonas sp. 4MC17 is built up of trisaccharide repeating units containing D-glucose, D-mannose and D-galacturonic acid residues. On the basis of methylation studies, 1H and 13C NMR-spectroscopy data, including two-dimensional homonuclear correlation spectroscopy and nuclear Overhauser effects, the following structure was suggested for the polysaccharide repeating unit: -->4)-beta-D-Glcp-(1-->4)-beta-D-GalpA-(1-->4)-beta-D-Manp-( 1-->.     

The O-specific polysaccharide chain of Campylobacter fetus serotype A lipopolysaccharide is a partially O-acetylated 1,3-linked alpha-D-mannan

A polysaccharide fraction liberated from Campylobacter fetus subsp. fetus serotype A lipopolysaccharide by mild acid hydrolysis followed by gel-permeation chromatography contained a partially O-acetylated D-mannan chain, as an O-specific polysaccharide, with a core oligosaccharide attached. The structure of the polysaccharide was studied by O-deacetylation, methylation, and 1H- and 13C-NMR spectroscopy, including computer-assisted analysis of the 13C-NMR spectrum. A structure of -->3)-alpha-D-Manp2Ac-(1--> was established as the structure of the O-specific polysaccharide, the degree of O-acetylation of the mannose residues at position 2 being estimated as 80-90%. As judged by the ratio of mannose to core constituents, the D-mannan chain consists on average of 10-12 monosaccharide units.