Characterization of lipids A of Ralstonia solanacearum lipopolysaccharides

The analysis of fatty acid profiles of lipopolysacharid.es has shown that R. solanacearum strains tested may be divided into two groups. The first group is represented by R. solanacearum strains (5712, 7945, 7955 and 8110) the lipids A of which contained hydroxylated fatty acids with long chains: 3-hydroxy­ tetradecanoic, 2-hydroxyhexadecanoic and 2-hydroxyoctadecanoic. The second group was represented by R. solanacearum strains the lipids A of which contained hydroxylated fatty acids with short chains: 3-hydroxydecanoic, 2-hydroxydodecanoic and 3-hydroxydodecanoic. 3-hydroxytetradecanoic acid was observed in a small amount. A comparative analysis of the fatty acid composition and biological activity gives a possibility to suppose that 3-hydroxytetradecanoic, 2-hydroxyhexadecanoic and 2-hydroxy­ octadecanoic acids may be responsible for the toxicity and pyrogenicity of the lipopolysaccharides tested.

Introduction.There is a number of problems in classification of Ralstonia solanacearum which is a heterogeneous species.Up to 1992 the representatives of R. solanacearum were attributed to genus Pseudomonas.But during next 4 years Yabuuchi and coworkers twice reclassified species solanacearum.At first they transferred it to new genus Burkholderia [1] and then, in 1995 -to genus Ralstonia [2].Whether this classification is the last, it is unknown.R. solanacearum is a very complex species.The heterogeneity of its strains remained to be solved taxonomically.The generally accepted criteria useful in taxonomy of gram-negative bacteria are the compo sition and structure of lipopolysaccharides (LPS), the components of outer membrane.Each of LPS com ponents: O-specific polysaccharides (O-PS), oligo saccharide core and lipid A, is characterised by specific composition, displays different biological acti vity and has a various taxonomy significance.While fine O-PS structures are used as a basis for intraspecies classification schemes of gram-negative bac teria, lipid A is an endotoxic centre which is respon sible for a majority of biological effects of LPS.So far as lipid A is the most conservative part of LPS V. VINARSKAYA, I. V.

GOGOMAN, 2004
molecule, its composition of fatty acids, in particular, hydroxylated ones, may be used as one of the additional taxonomic criteria.The purpose of present research was to investigate the fatty acid composition of lipids A of R. solanacearum strains and to study biological activity (toxicity and pyrogenicity) of the native and deacylated LPS.Materials and Methods.R. solanacearum strains were kindly given by Dr. J. Young, curator of ICMP (New Zealand) (Table 1).
The bacterial cultures were grown on a synthetic medium [3 ] at 28 °С with shaking for 48 h.
The LPS were extracted from acetone-and ether-dried cells with 45 % aqueous phenol at 65-68 °С [4].The aqueous layers were dialyzed against distilled water, nucleic acids were removed by pre cipitation with trichloroacetic acid and the solution was liophylized.For isolation of lipid A, the LPS were treated with 1 % acetic acid (100 °С, 2.0 h) and lipids A were obtained by ultracentrifugation (25000& 40 min).The LPS were hydrolyzed in 1.5 % acetyl chloride in methanol (100 °С, 4 h) and methyl esters of fatty acids were analyzed by gas-liquid chromatography/mass spectrometry (Hewlett Packard, USA), equipped with computer assistance.
The O-deacylated LPS were obtained by alkaline hydrolysis (0.2 M NaOH in 99.0 % ethanol, 50 °С, 18 h).The hydrolyzate was neutralized, dialysed against water to remove the salt, and liophylized.The N-, O-deacylated LPS were obtained by treating with anhydrous hydrazine (1 ml) in a sealed tube for 40 h at 100 °С.After the reaction, excess hydrazine was diluted with water.The mixture was neutralized with HC1 and the fatty acid hydrazides liberated were removed by extraction with chloroform.The deacylated LPS were obtained after eliminating the salt by gel-filtration [5].
The toxicity was studied by estimation of lethal toxicity (LD 50 ), using the galactosamine-sensitized mice, by injection of different concentrations (from 50 to 300 //g/ml) of native and modified LPS.The pyrogenicity was estimated by taking rabbits tem perature after injection of different concentrations (from 0.5 10~2 to 1.0-10" 2 mg/ml) of the native and modified LPS [6 ].
Results and Discussion.Lipid A is the most conservative part of the LPS molecule.It consists of l,4'-biphosphorylated /2-1,6-interlinked glucosaminedisaccharide with 4 residues of amide and esterlinked (R)-3-hydroxylated fatty acids which carry 2 or 4 nonhydroxylated acyl groups.Lipids A of different bacterial species vary from each other in the com position of fatty acids, in particular, hydroxylated ones, which is a stable index and therefore may be used as one of the chemotaxonomic criteria.
An analysis of fatty acid profiles of LPS showed that R. solanacearum strains tested may be divided into two groups (Table 2).The first group was represented by R. solanacearum strains (5712, 7945, 7955 and 8110) lipids A of which contained the hydroxylated fatty acids with long chains: 3-hydroxy tetradecanoic, 2-hydroxy hexadecanoic and 2-hydroxyoctadecanoic.The second group was represented by R. solanacearum strains lipids A of which contained the hydroxylated fatty acids with short chains: 3hydroxydecanoic, 2-hydroxydodecanoic and 3-hydroxydodecanoic. 3-hydroxytetradecanoic acid was ob served in a small amount.
Wilkinson and coworkers [7 ] studied a number of strains of genus Burkholderia cepacia, to which R. solanacearum was applied up to 1995, and two Ralstonia species: eurotropha and pickettii.The com parative analysis of their fatty acids profiles indicates that R. solanacearum is closely relates to B. cepacia and contains 3-hydroxytetradecanoic and 3-hydroxyhexadecanoic acids in their lipids A. The investigated LPS from either R. pickettii or R. eurotropha strains, don't contain 3-hydroxydecanoic acid and 2-hydroxyoctadecenic acid (observed by some authors in R. solanacearum [8]).The presence of 3-hydroxydodecanoic acids is characteristic of lipids A of such phytopathogenic species as Pseudomonas syringae and P. fluorescens.The results obtained on heterogeneity of lipids A of R. solanacearum strains coincides with the data on heterogeneity of O-specific polysaccharide structures, on the basis of which the R. solanacearum strains tested were disributed into 5 serogroups (Table 3).
R. solanacearum is one of the most destructive bacterial pathogens, damaging a wide range of econo mically important plants such as potato, tomato, eggplants, banana, sweet pepper etc. Via the agricul tural products R. solanacearum get into the warm blooded organisms and may display toxicity.There fore we studied the toxicity and pyrogenicity of the LPS of R. solanacearum strains investigated.Accor ding to the results obtained, the LPS may be divided into two groups one of which includes nontoxic and nonpyrogenic LPS (ICMP 767, 7944, 8089 and 4157), and the other one contains toxic and pyrogenic LPS (ICMP 5712, 8110, 7945 and 7955).To establish the chemical groups responsible for toxicity and pyro genicity, the modified LPS, dephosphorylated and deacylated, were obtained from R. solanacearum 5712.It was shown that the LPS tested have lost the toxicity and pyrogenicity.These data indicate the lipid A acyl and phosphate groups are responsible for R. solanacearum LPS toxicity and pyrogenicity.The comparative analysis of fatty acid composition and biological activity gives a possibility to suppose that 3-hydroxytetradecanoic, 2-hydroxyhexadecanoic and 2-hydroxyoctadecanoic acids may be responsible for the toxicity and pyrogenicity of the LPS tested.The literature data on the correlation between the LPS biological activity and length of lipid A fatty acids are in favour of such suggestion.In particular, the LPS containing short chain fatty acids are less pyrogenic and toxic in comparison to long chain ones [9 ].Conclusions.Thus it has been shown the hete rogeneity of R. solanacearum strains tested in the composition of their LPS fatty acids.The strains differ by the presence of hydroxylated fatty acids with short or long of chain.
Acknowledgements.We are deeply grateful to Dr. J. Young, curator of ICMP (New Zealand), who has provided us by the Ralstonia solanacearum strains.