Shinichi Yokota

Variable cross-reactivity of Pseudomonas aeruginosa lipopolysaccharide- core-specific monoclonal antibodies and its possible relationship with serotype

The core region of Pseudomonas aeruginosa lipopolysaccharide (LPS) was analysed by four LPS-core-specific human monoclonal antibodies (mAbs; FK-2E7, MH-4H7, OM-1D6 and NM-3G8). Reactivity of these mAbs to about 180 P. aeruginosa strains was tested. FK-2E7 bound to strains of Homma serotype E and I at a frequency of about 90%, to strains of serotype M at about 50%, and to strains of serotype A and G at about 30%. MH-4H7 bound to P. aeruginosa strains of serotype A, F, G, H, K and M at a high frequency (45-87%), but did not bind to any strains of serotype B, C, E and I. OM-1D6 and NM-3G8 bound to P. aeruginosa strains in a nearly serotype-specific manner. OM-1D6 reacted with all strains of serotype G so far tested, and a few strains of serotype M. Furthermore, L-rhamnose in the LPS core of serotype G was an immunodominant sugar recognized by OM-1D6 as an epitope. NM-3G8 bound to only a few strains of serotype B and M. The variable reactivity of these mAbs suggests that antigenic heterogeneity of the LPS core is somewhat related with (O-polysaccharide-based) serotype. Among these mAbs, MH-4H7 and OM-1D6 showed a high level of protective activity against P. aeruginosa in an experimental infection model using normal mice. In vivo protective activity was shown to be closely related to in vitro binding activity to whole cells as determined by agglutination and flow cytometry, but not ELISA.

Structure of the O-polysaccharide chain of the lipopolysaccharide of Vibrio anguillarum v-123

The O-polysaccharide chain (PS-1), released by mild acidic treatment of the LPS of V. anguillarum V-123 (serogroup JO-2), a pathogenic bacterium of marine and estuarine fish, consists of 2-amino-2-deoxy-D-galacturonic acid, 2-amino-2,6-dideoxy-D-glucose (D-quinovosamine), and 4-amino-4,6-dideoxy-D-glucose (D-viosamine) N-acylated with 2,4-dihydroxy-3,3,4-trimethylpyroglutamic acid. Strong-acid hydrolysis of PS-1 afforded alpha-GalNA-(1----4)-alpha-GalNA-(1----3)-QuiN (A1) and alpha-GalNA-(1----3)-QuiN (A2), and hydrolysis with hydrogen fluoride gave N-acetylated A1 and 4-amino-4,6-dideoxy-D-glucose N-acylated by 2,4-dihydroxy-3,3,4-trimethylpyroglutamic acid. Mild treatment of PS-1 with alkali removed the N-formyl substituents and Smith degradation of the product gave alpha-QuiNAc-(1----3)-beta-VioNAcyl-(1----3)-alpha-GalNAc A-(1----3)-2,3,4- trihydroxybutanoic acid (S1) and S2 in which the carboxyl group of the GalNAcA residue was amidated. Thus, the repeating unit of the O-polysaccharide is----3)-alpha-GalNAcA(amino)-(1----4)-alpha-GalNFoA-(1----3 )- alpha-QuiNAc-(1----3)-beta-VioNAcyl-(1----in which the N-Acyl group is 2,4-dihydroxy-3,3,4-trimethylpyroglutamic acid and Fo is formyl.

Novel O-polysaccharide expression, as a lipid A-core-free form, in a lipopolysaccharide-core-defective mutant of Pseudomonas aeruginosa

Pseudomonas aeruginosa PML14e is a mutant strain, isolated from strain PML14 (Homma serotype I), that is resistant to all types of R-pyocins. PML14e completely lacked glucose and rhamnose as components of the lipopolysaccharide (LPS) outer core region. Whereas the O-polysaccharide attachment site on the LPS core was considered to be absent, PML14e was agglutinable with anti-serotype-I antibodies. The O-polysaccharide of PML14e was recovered in the supernatant after ultracentrifugation of the aqueous layer from a hot phenol/water extraction. Chromatographic behaviour and chemical analysis indicated that the PML14e O-polysaccharide was not linked to the lipid A. 1H-NMR spectroscopy indicated that the structure of the PML14e O-polysaccharide was the same as that of the O-polysaccharide from PML14. The above evidence indicated that the O-polysaccharide is expressed on the cell surface of the mutant strain PML14e as the lipid A-free form. To examine the nature of the cell surface, the accessibility of monoclonal antibodies (mAbs) against cell surface antigens was tested by enzyme-linked immunosorbent assay. An anti-lipid A mAb and an anti-outer-membrane protein mAb, the epitopes for which are considered to be exposed on rough strains, bound to a greater extent to the PML14e cells than to two other LPS-core-defective rough mutants, PML14b and PML14d. Whereas these mutants appeared to have lesser defects in the LPS core, they expressed less O-polysaccharide than PML14e. The results indicated that the epitopes exposed on rough strains, such as lipid A and outer-membrane proteins, were mainly hindered by covalently linked core oligosaccharide rather than by the O-polysaccharide chain.

Epitopes for human monoclonal antibodies and serotyping antisera against the O-specific polysaccharide of Pseudomonas aeruginosa O11

Epitopes for Pseudomonas aeruginosa O11-specific human monoclonal antibodies (mAbs) and O11 serotyping antisera have been characterized. These mAbs recognized the O-polysaccharide portion of the lipopolysaccharide. The structure of the O-polysaccharide of O11 has been reported to be comprised of trisaccharide repeating-units as follows: -->3)-alpha-L-FucpNAc-(1-->1)-beta-D-FucpNAc-(1--> 2)-beta-D-Glcp-(1-->. (FucpNAc, 2-acetamido-2,6-dideoxygalactopyranoside.) Data from inhibition studies of binding in enzyme-linked immunosorbent assays and cell-agglutination assays, using monosaccharides and periodate-oxidized O-polysaccharide showed that the glucose residue, especially the C-3-C-6 segment and the beta-anomeric configuration, in the polysaccharide is essential for the epitopes of all anti-O11 mAbs; however, the detailed epitope specificities were different from one another. Furthermore, epitopes for serotyping antisera of O11 seemed to be similar to those for the human mAbs.