Biological Activity of (Lipo)Polysaccharides of the Exopolysaccharide-Deficient Mutant Rt120 Derived from Rhizobium leguminosarum bv. trifolii Strain TA1*

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J. Kutkowska¹, A. Turska-Szewczuk¹, M. Janczarek¹, R. Paduch², T. Kamińska², and T. Urbanik-Sypniewska^1**

¹Department of Genetics and Microbiology, M. Curie-Skłodowska University, Akademicka 19 St., 20-033 Lublin, Poland; fax: +48(81)537-5959; E-mail: turbanik@hektor.umcs.lublin.pl

²Department of Immunology and Virology, M. Curie-Skłodowska University, Akademicka 19 St., 20-033 Lublin, Poland

^* This paper is based on a presentation made at the 4th Baltic Conference on Microbial Carbohydrates, Hyytiälä Forestry Field Station, Finland, September 19-22, 2010.

^** To whom correspondence should be addressed.

Received January 4, 2011; Revision received February 2, 2011

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Lipopolysaccharides (LPS) from Rhizobium leguminosarum biovar trifolii TA1 (RtTA1) and its mutant Rt120 in the pssBpssA intergenic region as well as degraded polysaccharides (DPS) derived from the LPS were elucidated in terms of their chemical composition and biological activities. The polysaccharide portions were examined by methylation analysis, MALDI-TOF mass spectrometry, and ¹H NMR spectroscopy. A high molecular mass carbohydrate fraction obtained from Rt120 DPS by Sephadex G-50 gel chromatography was composed mainly of L-rhamnose, 6-deoxy-L-talose, D-galactose, and D-galacturonic acid, whereas that from RtTA1 DPS contained L-fucose, 2-acetamido-2,6-dideoxy-D-glucose, D-galacturonic acid, 3-deoxy-3-methylaminofucose, D-glucose, D-glucuronic acid, and heptose. Relative intensities of the major ¹H NMR signals for O-acetyl and N-acetyl groups were 1 : 0.8 and 1 : 1.24 in DPS of Rt120 and RtTA1, respectively. The intact mutant LPS exhibited a twice higher lethal toxicity than the wild type LPS. A higher in vivo production of TNFα and IL-6 after induction of mice with Rt120 LPS correlated with the toxicity, although the mutant LPS induced the secretion of IL-1β and IFNγ more weakly than RtTA1 LPS. A polysaccharide obtained by gel chromatography on Bio-Gel P-4 of the high molecular mass material from Rt120 had a toxic effect on tumor HeLa cells but was inactive against the normal human skin fibroblast cell line. The polysaccharide from RtTA1 was inactive against either cell line. The potent inhibitory effect of the mutant DPS on tumor HeLa cells seems to be related with the differences in sugar composition.

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KEY WORDS: lipopolysaccharide, Rhizobium leguminosarum, 3-deoxy-3-methylaminofucose, lethal toxicity, TNFα

DOI: 10.1134/S0006297911070157

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