The P. gingivalis Autocitrullinome Is Not a Target for ACPA in Early Rheumatoid Arthritis.

Rheumatoid arthritis (RA), a chronic inflammatory disease affecting primarily the joints, is frequently characterized by the presence of autoimmune anticitrullinated protein antibodies (ACPA) during preclinical stages of disease and accumulation of hypercitrullinated proteins in arthritic joints. A strong association has been reported between RA and periodontal disease, and Porphyromonas gingivalis, a known driver of periodontitis, has been proposed as the microbial link underlying this association. We recently demonstrated P. gingivalis-mediated gut barrier breakdown and exacerbation of joint inflammation during inflammatory arthritis. In the present study, we investigated another potential role for P. gingivalis in RA etiopathogenesis, based on the generation of ACPA through the activity of a unique P. gingivalis peptidylarginine deiminase (PPAD) produced by this bacterium, which is capable of protein citrullination. Using a novel P. gingivalis W50 PPAD mutant strain, incapable of protein citrullination, and serum from disease-modifying antirheumatic drug-naïve early arthritis patients, we assessed whether autocitrullinated proteins in the P. gingivalis proteome serve as cross-activation targets in the initiation of ACPA production. We found no evidence for patient antibody activity specific to autocitrullinated P. gingivalis proteins. Moreover, deletion of PPAD did not prevent P. gingivalis-mediated intestinal barrier breakdown and exacerbation of disease during inflammatory arthritis in a murine model. Together, these findings suggest that the enzymatic activity of PPAD is not a major virulence mechanism during early stages of inflammatory arthritis.

Hemin binding by Porphyromonas gingivalis strains is dependent on the presence of A-LPS.

Porphyromonas gingivalis is a Gram-negative black pigmenting anaerobe that is unable to synthesize heme [Fe(II)-protoporphyrin IX] or hemin [Fe(III)-protoporphyrin IX-Cl], which are important growth/virulence factors, and must therefore derive them from the host. Porphyromonas gingivalis expresses several proteinaceous hemin-binding sites, which are important in the binding/transport of heme/hemin from the host. It also synthesizes several virulence factors, namely cysteine-proteases Arg- and Lys-gingipains and two lipopolysaccharides (LPS), O-LPS and A-LPS. The gingipains are required for the production of the black pigment, µ-oxo-bisheme {[Fe(III)PPIX]2 O}, which is derived from hemoglobin and deposited on the bacterial cell-surface leading to the characteristic black colonies when grown on blood agar. In this study we investigated the role of LPS in the deposition of µ-oxo-bisheme on the cell-surface. A P. gingivalis mutant defective in the biosynthesis of Arg-gingipains, namely rgpA/rgpB, produces brown colonies on blood agar and mutants defective in Lys-gingipain (kgp) and LPS biosynthesis namely porR, waaL, wzy, and pg0129 (α-1, 3-mannosyltransferase) produce non-pigmented colonies. However, only those mutants lacking A-LPS showed reduced hemin-binding when cells in suspension were incubated with hemin. Using native, de-O-phosphorylated and de-lipidated LPS from P. gingivalis W50 and porR strains, we demonstrated that hemin-binding to O-polysaccharide (PS) and to the lipid A moiety of LPS was reduced compared with hemin-binding to A-PS. We conclude that A-LPS in the outer-membrane of P. gingivalis serves as a scaffold/anchor for the retention of µ-oxo-bisheme on the cell surface and pigmentation is dependent on the presence of A-LPS.

Glycosylation of immunoglobulin light chains associated with amyloidosis.

AL amyloidosis is a fatal disease caused by deposition of immunoglobulin light chains in a fibrillarforin (AL) in various organs. By searching the Kabat database of immunoglobulin sequences using the KabatMan software, we have shown that there is a preponderance of the consensus glycosylation sequon (AsnXxxSer/Thr) in the framework regions of amyloid light chains. We have characterised by computer graphics simulations, NMR spectroscopy and carbohydrate biochemistry the structure and conformation of the oligosaccharide from amyloid protein AL MS (lamba1) and from the amyloid associated Bence Jones protein of patient MH (kappa1). These proteins have glycosylation in the hypervariable complementarity-determining region versus framework region, respectively. Both contained a 2-6 sialylated core fucosylated biantennary chain mostly with bisecting GIcNAc. Together our results suggest that light chain glycosylation may be one of several modifications which may render the protein more prone to amyloid formation.

Structural studies on the acidic exopolysaccharide from Haloferax denitrificans ATCC 35960.

The structure of a linear, acidic exopolysaccharide isolated from the Archaeon Haloferax denitrificans ATCC 35960 has been determined using NMR spectroscopy. The sugar residues in the repeating unit of the polysaccharide were identified as Gal and GlcA2,3NAc after the assignment of the 1H and 13C resonances using COSY, HOHAHA, HMQC and HMQC-TOCSY experiments. The sequence of the residues in the polysaccharide was established from the inter-residue connectivities observed in the HMQC-NOESY plot. The only sugar released on acid hydrolysis was shown to be D-Gal by GLC analysis, while the absolute configuration of the acidic sugars was shown to be D by comparison of the carbon chemical shifts with those of model compounds. Partial acid hydrolysis yielded a tetrasaccharide, terminated by D-Gal at the reducing end, whose structure confirmed that of the repeating unit of the polysaccharide as-->4)-beta-D-GlcpA2,3NAc-(1-->4)-beta-D-GlcpA2, 3NAc-(1-->4)-alpha-D-GlcpA2,3NAc-(1-->3)-alpha-D-Galp- (1-->, where D-GlcpA2,3NAc is 2,3-diacetamido-2,3-dideoxy-D-glucopyranosiduronic acid.

Structure and serological specificity of a new acidic O-specific polysaccharide of Proteus vulgaris O45.

The following structure of the O-specific polysaccharide (OPS) of Proteus vulgaris O45 lipopolysaccharide (LPS) was established using 1H- and 13C-NMR spectroscopy, including two-dimensional NOESY and H-detected 1H, 13C heteronuclear multiple-quantum coherence ( HMQC) experiments: [structure: see text text] Immunochemical studies, using rabbit polyclonal anti-P. vulgaris O45 serum and LPS, OPS and Smith-degraded OPS of P. vulgaris O45, showed the importance of beta-D-GlcA in manifesting the serological specificity of the O-antigen studied.

Structural and serological studies on a new acidic O-specific polysaccharide of Proteus vulgaris O32.

The following structure of the O-specific polysaccharide chain (O-antigen) of the Proteus vulgaris 032 lipopolysaccharide (LPS) was established by 1H-NMR and 13C-NMR spectroscopy, including two-dimensional NOESY and H-detected 1H,13C heteronuclear multiple-quantum coherence (HMQC) experiments: -->2)-alpha-L-RhapI-(1-->2)-alpha-L-RhapII-(1-->4)-beta-D-++ +GalpA(I)-(1-->3)-beta-D-GlcpNAc-(1-->4)-alpha-D-GalpA(II)-(1-- >. In addition, an O-acetyl group was detected, which, most probably, is located at position 3 of a part of RhapI residues. Serological studies, using rabbit polyclonal anti-(P. vulgaris 032) serum, homologous and heterologous Proteus O-antigens and related artificial antigens, revealed the importance of an a-D-GalA-associated epitope in manifesting the immunospecificity of P. vulgaris 032 and substantiated serological relationships between the O-antigen studied and those of some other Proteus strains.

Somatic antigens of pseudomonads: structure of the O-specific polysaccharide of Pseudomonas fluorescens biovar B, strain IMV 247.

The O-specific polysaccharide of Pseudomonas fluorescens biovar B, strain IMV 247, was studied by acid hydrolysis, GLC-MS and 1H and 13C NMR spectroscopy, including 1D and 2D NOE, 2D hybrid TOCSY and ROESY (TORO), and 2D H-detected heteronuclear multiple-bond correlation (HMBC) experiments. The polysaccharide was found to contain L-rhamnose, 3.6-dideoxy-3-[(S)-3-hydroxybutyramido]-D-glucose (D-Qui3NHb), 2-acetamido- 2,4,6-trideoxy-4-[(S)-3-hydroxybutyramido-D-glucose (D-QuiNAc4NHb) and 2-acetamido-2- deoxy-D-galacturonic acid (D-GalNAcA). Partial acid hydrolysis of the polysaccharide resulted in a non-reducing GalNAcA-->QuiNAc4NHb disaccharide with the 3-hydroxybutyryl group glycosylated intramolecularly by the QuiN4N residue. The following structure of the tetrasaccharide repeating unit of the polysaccharide was established:-->4) -alpha-D-GalpNAcA-(1-->3)- alpha-D-QuipNAc4NHb-(1-->2)-beta-D-Quip3NHb-(1-->2)-alpha-L- Rhap(1-->.

The structure of the exocellular polysaccharide produced by the Archaeon Haloferax gibbonsii (ATCC 33959).

The structure of the neutral exocellular polysaccharide isolated from the Archaeon Haloferax gibbonsii (ATCC 33959) has been determined using acid hydrolysis, methylation analysis and NMR spectroscopy. The polysaccharide contained D-Man, D-Glc, D-Gal and L-Rha in the ratios 2:1:3:1. The substitution patterns of the sugar residues were deduced from the methylation analysis which indicated the polymer to be composed of a heptasaccharide repeating unit containing two branches. The 1H and 13C NMR resonances of the component sugars were assigned using COSY, HOHAHA, HMQC, and HMQC-TOCSY 2D NMR experiments and the sequence of the sugars in the repeating unit was determined from NOESY and HMBC experiments. The structure can be written as: [formula: see text]

Structure of the O-specific polysaccharide of Proteus penneri strain 41 from a new proposed serogroup O62.

O-specific polysaccharide of Proteus penneri strain 41 was studied using 1H- and 13C-NMR spectroscopy, including two-dimensional COSY, heteronuclear 13C,1H-correlation (HETCOR) and one-dimensional NOE spectroscopy, and the following structure of a non-stoichiometrically O-acetylated hexasaccharide repeating unit was established:[structure: see text] where RGlcNAc is 2-acetamido-4-O-[(S)-1-carboxyethyl]-2-deoxyglucose. Cross-reactivity of anti-P. penneri 41 O-serum with other P. penneri strains is discussed, and a new, separate O62 serogroup is proposed which is the next Proteus O-serogroup containing P. penneri strains only.

Structural and immunochemical studies of two cross-reactive Proteus mirabilis O-antigens, O6 and O23, containing beta1-->3-linked 2-acetamido-2-deoxy-D-glucopyranose residues.

A marked serological cross-reactivity was observed by ELISA and a precipitation test between anti-Proteus mirabilis O23 serum and the lipopolysaccharide as well as the O-specific polysaccharide from the Proteus mirabilis strain belonging to serogroup O6. The structures of the O-specific polysaccharides were elucidated using chemical and NMR spectroscopic analyses, and the only common component, 2-acetamido-2-deoxy-beta-D-glucopyranose (beta-D-GlcNAc), was revealed, which was suggested to be responsible for the cross-reactivity observed. Both anti-O23 and anti-O6 sera were shown to react with 1, 3-Linked beta-D-GlcNAc-containing O-antigen from Salmonella enterica ssp. arizonae O59 also. The lack of reactivity of Smith-degraded P. mirabilis O6 O-specific polysaccharide with homologous antiserum indicated the crucial role of alpha-D-glucuronic acid in specific antibody binding.

Structural heterogeneity in the O polysaccharide of Pseudomonas syringae pv. coriandricola GSPB 2028 (NCPPB 3780, W-43).

The O polysaccharide (OPS) of the lipopolysaccharide of Pseudomonas syringae pv. coriandricola GSPB 2028 (NCPPB 3780, W-43) was studied by Smith degradation and 1H-NMR and 13C-NMR spectroscopy, including two-dimensional COSY, TOCSY, NOESY, and H-detected 1H,13C heteronuclear multiple-quantum coherence (HMQC) experiments. The OPS was shown to consist of pentasaccharide O repeats of two types both containing four L-rhamnose and one 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc) residue. Structure 1 of the major O repeat which had been established earlier [Das, S., Ramm, M., Kochanowski, H. & Basu, S. (1994) J. Bacteriol. 176, 6550-6557], was confirmed by our data, and a new structure 2 was elucidated for the minor O repeat and found to differ from the structure 1 only in the position of substitution of one of the rhamnose residues in the main chain. [structures: see text] A role of structural and immunochemical features of the LPS for defining the taxonomical position of the bacterium studied is discussed.

Structure of the O-specific polysaccharide of Proteus vulgaris O25 containing 3-O-[(R)-1-carboxyethyl]-D-glucose.

The O-specific polysaccharide of Proteus vulgaris O25 was studied by acid hydrolysis and by 1H-NMR and 13C-NMR spectroscopies, including one-dimensional NOE and two-dimensional COSY and heteronuclear 13C,1H correlation (HETCOR) spectroscopy. The polysaccharide was found to contain 3-O-[(R)-1-carboxyethyl]-D-glucose (D-RGlc), and the following structure of the pentasaccharide repeating unit was established: [structure in text]

Structure of the O-specific polysaccharide of the bacterium Proteus mirabilis O13 containing a novel component: an amide of D-galacturonic acid with N(epsilon)-(1-carboxyethyl)lysine.

An acidic O-specific polysaccharide was obtained by mild degradation of the lipopolysaccharide of the bacterium Proteus mirabilis O13 and found to contain D-galactose, 2-acetamido-2-deoxy-D-glucose, and N(epsilon)-(1-carboxyethyl)-N(alpha)-(D-galacturonoyl)lysine. On the basis of full acid hydrolysis and 1H- and 13C-NMR spectroscopy, including two-dimensional correlation spectroscopy (COSY), H-detected heteronuclear 1H, 13C multi-quantum coherence (HMQC), and rotating-frame nuclear Overhauser effect spectroscopy (ROESY), the following structure of the branched trisaccharide repeating unit of the polysaccharide was established [structure: see text]

Structure of the capsular polysaccharide from Alteromonas nigrifaciens IAM 13010T containing 2-acetamido-2,6-dideoxy-L-talose and 3-deoxy-D-manno-octulosonic acid.

A capsular polysaccharide was obtained from Alteromonas nigrifaciens IAM 13010T by saline extraction. On the basis of 1H and 13C NMR spectroscopy, including one-dimensional (1D) NOE spectroscopy, 2D rotating-frame NOE spectroscopy (ROESY), and 1H-detected heteronuclear 1H,13C multiple-quantum coherence (HMQC), it was concluded that the polysaccharide contained inter alia an acidic sugar, 3-deoxy-D-manno-octulosonic acid (Kdo), and a rare amino sugar, 2-acetamido-2,6-dideoxy-L-talose (L-6dTalNAc, N-acetylpneumosamine), and has a pentasaccharide repeating unit of the following structure: [equation: see text]

Structural and serological studies of the O-antigen of the bacterium Proteus vulgaris OX2 (serogroup O2) used in the Weil-Felix test.

Based on monosaccharide analysis and 1H- and 13C-NMR spectroscopy, the following structure of the O-specific polysaccharide chain of Proteus vulgaris OX2 lipopolysaccharide (LPS), which defines the O2 specificity of Proteus, was established: [formula: see text] where L-QuiNAc is N-acetyl-L-quinovosamine (2-acetamido-2,6-dideoxy-L-glucose). Various strains of P. vulgaris OX2 used in the Weil-Felix test for serodiagnosis of rickettsiosis (spotted fevers, except for Rocky Mountain spotted fever) were shown to produce LPS with the same O-specific polysaccharide, which differs structurally and serologically from LPS of P. vulgaris OX19 used as antigen for serodiagnosis of typhus and Rocky Mountain spotted fever. O-Acetyl groups present in the polysaccharide are not important for manifesting the immunospecificity. ELISA confirmed that the epitope responsible for the cross-reactivity between sera from patients with Japanese spotted fever and P. vulgaris OX2 cells is located on the P. vulgaris LPS. At the same time, no cross-reaction was observed between rabbit anti-P. vulgaris OX2 antibodies and the spotted fever group (SFG) rickettsial cells. Therefore, human anti-SFG rickettsial antibodies and rabbit anti-P. vulgaris OX2 antibodies may bind to distinct epitopes on P. vulgaris OX2 LPS, and no epitope recognized by rabbit anti-P. vulgaris OX2 antibodies is present on the LPS or any other surface antigen of SFG rickettsiae.

Characterization of O-antigens from different strains of Pseudomonas syringae pv. tabaci.

O-Antigens (lipopolysaccharides, LPS) were isolated by NaCl extraction from microbial biomass of Pseudomonas syringae pv. tabaci and purified by ultracentrifugation. Individual structural components of the LPS macromolecule (O-specific polysaccharide (O-PS), core oligosaccharide, and lipid A) were obtained and characterized. Fatty acids 3-OH-C10:0, C12:0, 2-OH-C12:0, 3-OH-C12:0, C16:1, C16:0, C18:1, and C18:0 were identified in the lipid A composition. Glucosamine, ethanolamine, and phosphoethanolamine were found in the hydrophilic part of the lipid A macromolecule in all strains tested. Lipid A preparations contained phosphorus and amino acids. Rhamnose, glucose, glucosamine, 2-keto-3-deoxyoctulosonic acid, heptose, alanine, and phosphorus were identified as the main core components. The strains differed in O-PS structure. We describe the O-chain of LPS in strain P-28. It contains repeating units of the following structure: [formula: see text] The O-PS structures of LPS from strains P-28 and 225 are identical, however, they differ substantially from that of strain 223. Both structures from strains 223 and 225 were reported previously. Antibodies to antigenic epitopes of O-PS, core, and lipid A were revealed in O-serum against the whole bacterial cells. Correlation of O-PS structure with the serological grouping of strains was observed.

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.

Structural and serological studies of the O-specific polysaccharide of the bacterium Proteus mirabilis O10 containing L-altruronic acid, a new component of O-antigens.

An acidic O-specific polysaccharide from the lipopolysaccharide of Proteus mirabilis O10 contains 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose, D-galacturonic acid, and L-altruronic acid, the last-named sugar having not been found hitherto in O-antigens. Structure of a branched tetrasaccharide repeating unit of the polysaccharide was established by 1H and 13C NMR spectroscopy, including two-dimensional COSY and rotating-frame NOE spectroscopy. The lateral L-altruronic acid residue plays the immunodominant role in manifestation of the O10 specificity of Proteus, whereas a disaccharide fragment of the main chain in common with the O-specific polysaccharide of P. mirabilis O43 provides the one-way serological cross-reactivity between anti-O10 serum and O43-antigen.

[Structure of O-specific polysaccharide from Proteus mirabilis O10, containing a new component of O-antigens--L-altruronic acid]. / Struktura O-spetsificheskogo polisacharida bakterii Proteus mirabilis O10, soderzhashchego novyi komponent O-antigenov--L-altruronovuiu kislotu.

An acidic O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Proteus mirabilis O10 and studied after full acid hydrolysis and carboxyl reduction by 1H- and 13C-NMR spectroscopy, including two-dimensional correlation spectroscopy (COSY), H-detected heteronuclear 1H,13C multi-quantum coherence (HMQC), and rotating-frame nuclear Overhauser effect spectroscopy (ROESY). It was found that the polysaccharide contains 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose, D-galacturonic acid, and L-altruronic acid, and the following structure of the branched tetrasaccharide repeating unit of the polysaccharide was established: [sequence: see text]