Bacteria of the genus Proteus are a common use of urinary tract infections which may result in severe complications, such as pyelonephrits and formation of bladder and kidney stones. The genus includes four species, most medically important of which are P. mirabilis, P. vulgaris and P. penneri (formerly P. vulgaris biogroup I). Strains of Proteus are antigenically heterogeneous and divided into more than 60 O-serogroups. A number of strains, mostly those of P. penneri, remain unclassified. The major surface antigen of Proteus is the outer-membrane lipopolysaccharide (O-antigen). Each serologically distinguishable strain possesses a unique O-antigen structure that allowes the use of the structural data as the basis for serological classification.
For 1996-2002 I've been working in the N.D. Zelinsky Institute of Organic Chemistry NMR center as glycopolymer structural researcher in the group of Dr. Knirel (partly as a post-graduate student supervised by Dr. Shashkov). Our systematic chemical and immunochemical studies are aimed at correlation between the O-antigen structure and the immunospecificity of strains, which is important for obtaining synthetic diagnosticums, vaccines and immunostimulators. These studies include the elucidation of structure of new O-specific polysaccharide chains of natural lipopolysaccharides from P. mirabilis, P. vulgaris and P. penneri.
Together with widespread carbohydrate residues common for Proteus (Glc, Gal, GlcNAc, GalNAc, GlcA, GalA), several unusual components have been identified, two of them (L-AltpA and L-Rhap2NAc3NAc) for the first time in polymers of gram-negative bacteria. Of unusual neutral monomers L-6dTalp, L-QuipNAc, L-Quip3NAc, D-Fucp3NAc, D-Fucp4NAc and L-Rhap2NAc3NAc are worth mentioning, of charged are L-AltpA, D-ManpANAc, amides of uronic acids with L-Lys, L-Ala and AlaLys, pyruvic acid residue R-Pyr, phosphate-containing P-EtN and P-Rbo. Three ribitol-containing O-antigens (P.mirabilis O16, P.mirabilis O33 and P. penneri 103) were found to resemble teichoic acids of gram-positive bacteria. Two other phosphorylated polysaccharides (P. vulgaris OX19 and O21) were built of oligosaccharide-phosphate repeating units and, as a result, were highly acid-labile. As well as ribitol and ethanolamine phosphates, glycosyl phosphates have not been found in Proteus O-antigens ever before.
My part of work were structural studies of these glycopolymers and glycoconjugates using NMR spectroscopy, which is the main approach to biopolymer structure elucidation. Click here to view the approximate plan of glycopolymer NMR research. The NMR experiments were carried out on Bruker NMR instruments (WM250, AM300, DRX500) and included the following:
|NMR experiment||Its result|
|1D 13C NMR||preliminary data, glycosylation effects (=> substitution pattern, absolute configurations)|
|1D 1H NMR||HH-coupling constants analysis (=> monomeric composition, anomeric configurations)|
|1D 31P NMR||revealing non-sugar substituents|
|1D H-Gated 13C NMR||anomeric configuration, cycle sizes|
|1D Attached Proton Test||methylen groups assignment|
|2D 1H,1H COSY||vicinal proton-proton interactions (=> 1H NMR spectrum assignment)|
|2D 1H,1H Double Quantum Filter COSY||simplifying the COSY spectrum by the removal of diagonal line|
|2D H,H-relayed COSY (RCT, RCT2)||far proton-proton interactions (=> 1H NMR spectrum assignment)|
|2D TOtal Correlation SpectroscopY||excluding the spin system subsets (of residues) from 1H NMR spectrum|
|1D 1H difference mode double resonance||signal shape analysis (=> thorough 1H NMR spectrum assignment )|
|2D 31P,1H COSY||localization of phospho-containing substituents|
|2D 13C,1H COSY||direct heteronuclear coupling constants (=> 13C NMR spectrum assignment)|
|2D 1H,13C Heteronuclear Multiple-Quantum Coherence||direct heteronuclear coupling constants (=> 13C NMR spectrum assignment, constant measurement)|
|2D Heteronuclear Multiple-Bond Correlation||far heteronuclear interactions, including transglycosidic (=> residue sequence)|
|2D NOE SpectroscopY||revealing spatial H-H contacts in oligomers, including transglycosydic (=> substitution pattern, residue sequence)|
|2D Rotating frame nuclear Overhauser Effect SpectroscopY||revealing spatial H-H contacts, including transglycosydic (=> substitution pattern, residue sequence)|
|1D difference mode NOE spectroscopy||thorough analysis of H-H spatial contacts|
Structural studies of the Proteus O-antigens were supported by RFBR grants N93-03-5839, N96-04-50460, N96-15-97380, N99-04-48279 and N00-15-97373.