ENDOTOXINS OF PSEUDOMONAS FLUORESCENS
G.M. Zdorovenko, S.N. Veremeychenko, I. Ya. Zakharova and Yu. A. Knire1 Zabo1otny Institute of Microbiology and Virology of the Ukrainian Academy of Sciences. Kiev. USSR
Heterogenous in both phenotype and genotype species. Pseudomonas f1uorescens is presented by 5 biovars in Bergey's Manual. In spite of their belonging to saprophytic organisms the strains of Pseudomonas f1uorescens are frequently isolated from clinical specimens (5). The endotoxins (LPS) of microbial cells are responsible for the mechanisms of interaction between micro- and macroorganisms in the infectious process. Therefore. we studied the LPS's of P. f1uorescens. The strains under study were obtained from the Collection of the Institute of Microbiology and Virology (IMV) and represented the biovars: I (IMV 4125=ATCC 13525). II (IMV 1602). III (IMV 2125). IV (IMV 2111). V (IMV 2763). and biovar i (IMV 2303) initially described in the USSR (2). Bacteria were grown on nutrient agar media. LPS was extracted from the acetone-dried cells by the Westphal method. All methods of analysis were described (3, 6, 7). LPS's were active in homologous reactions of immunoprecipitation (approximate titer was 1:100.000) and in the Ouchter1ony test (1-3 precipitation lines). Weak crossreactions were observed only between strains of biovars I and i. IV and V. We studied separately different parts of the LPS macromolecule (O-chain. core. lipid A) obtained after its cleavage by acetic acid hydrolysis. The fraction of o-specific polysaccharide (PS) was serologically active in each strain (approximate immunoprecipitation titer was 1:500.000). Table 1 shows the results of sugar analysis of the o-chain. The following features of sugar compositions were detected: 1.
Each strain had its characteristic sugar composition.
2.
Rhamnose and amino sugars were the main components in the majority of strains.
3.
Inclusion of rarely occurring sugars such as 3-acetamido-3.6-dideoxy-D-ga1actose. N-acety1-L-fucosamine. 6-deoxy-L-ta1ose. 2.4-dia cetamido-2.4.6-trideoxy-D-g1ucose.
H. Friedman et al. (eds.), Endotoxin © Springer Science+Business Media New York 1990
131
Table 1.
The main sugar components of O-specific PS fwt %).1 (Sugar composition was estimated using GLC. LC, GC-MS, Hand 3C NMRspectroscopy, etc., analysis)
Sugar
4125
1602
2125
49
41
16
Rhamnose 6-deoxy-L ta10se Fucose Mannose Glucose N-acety1-D glucosamine N-acety1-Dgalactosamine N-acety1-Lfucosamine 3-acetamido-3, 6-dideoxy-Dgalactose 2,4-diacetamido2,4,6-trideoxyD-g1ucose Unidentified aminosugar
2111
2303 52
22
2763
20 20
54
18 19
20 21 12
23 11
14
Studies on the core fractions showed their composition usual for pseudomonas (Table 2). The only exception was the strain 2763. We did not succeed to find in its core rhamnose, alanine, phosphorus and KDO but at the same time it contained too much heptose and glucosamine. The fatty acids presented in lipid A of type strain 4125 (Fig 1) were also found in lipid A of 2125, 2111 and 2303. Lipid A of 1602 was similar but it contained 2-0H C12:0. Strain 2763 differed from above strains. 3-0H C14:0 was the major component of its lipid A. Because 2763 was considerably different in its LPS composition from other strains, the structural investigations were performed on its PS. 13 C NMR-spectroscopy of intact PS demonstrated its irregularity, but a strictly regula! po1ym~r (Fig 2) have been received after o-deacety1ation of PS. Using Hand 13C NMR-spectroscopy, methylation, partial hydrolysis, etc., the repeating unit of PS was found to have the structure: 50% OAc
+ 2) -
~np
50% AcO
(1 + 3)-6 -D-GalllAcp (1 + 4)-"
-+-
2) - a - L - 6Ta1p
-D-~
As far as we know only the structure of P. fluorescence LPS was determined earlier (1). The presence of 6-deoxy-L-ta10se is the only similarity between these structures.
132
Composition of core oligosaccharide
Table 2. 4125
Strain Rhamnose Fucose Arabinose Mannose Glucose Galactose Heptose *D-g1ucosamine *D-ga1actosamine *2-keto-3-deoxyoctu1osonic acid *A1 ani ne *Phosphorus
2125
1602
19.8
21.0
18.0 61.1 tr tr 1.0 0.5 0.6
14.8 18.5 33.3 12.3 tr tr 0.4 7.5
2.0 3.6
1.1 2.8
II
2303
52.0 39.0
3.8
38.0
1.2 3.6 1.2 3.0 tr 6.7 4.6
17.3 57.7 19.2 tr tr 0.7 2.7
tr 41.4 tr 19.8 7.2 tr 0,2
1.8 3.7
2.8 5.2
3.5 5.0
2763
tr 1.2 37.6 19.5 41.8 14.4 1.0
*(wt %)
(Peak area % GLC-ana1ysis)
4125
2111
2
1602
2163 9
2
6 4
4
2
1
,)
7
5
1
,
10
6
)
,- ---Fig 1.
GL chromatograms of fatty acid components of lipid A fractions released by acid (1% CH3 COOH. 100 o C. 1.5 hr) hydrolysis from LPS. Methyl esters received by using 1.5N methano1ic HC1 were separated on column (0.3 x 120 cm) with 5% SE-30 on Chromaton N-AW-DMCS. 80-100 mesh at 130-230 oC. 3 0 C/min. Identification of peaks was made by co-chromatography with authentic standards and by GC-MS. The peaks correspond to: 3-0H C10:0«1). C12:0(2). 2-0H C12:0(3). 3-0H C12:0(4). C16:1(5). C16:0(6). ct8:1(7). C18:0(8). 3-0H C14:0(9). 3-0H C1 5 :O(10).
133
Biovar V is a heterogenous group of bacteria (5) which lack some peculiarities of pseudomonas. We may speculate that unusual compositions of these LPS may provide additional characteristics for this group. There is a correlation between diversity of the strains by biological properties and their LPS composition. By inclusion of 6-deoxyhexoses and aminosugars into the o-chain, as well as lipid A composition of P. fluorescence resembles corresponding products from some serotypes of P. aeruginosa and some enteric bacteria (4).
.- ...- . • .00.0
Fig 2.
i
•• 0
0,
~ .'t\. ~~ . 'I
•••
•
i
7•• '
....
.,'
..,
--a..
'.u, '0'
PI'N
SI.'
.,.,'
••• 0
J.
•
10.0
-
• %0.'
~
•
10. C
13 C NMR-spectrum of o-specific polysaccharide of P. fluorescence IMV 2763.
REFERENCES
1.
Khomenko, V. A., Naberezhnych, G. A., Isakov, V. V., Solov'eva, T. F., Ovodov, Y. S., Knire1, Y. A., Vinogradov, E. V., 1986, Structural study of o-specific polysaccharide chain of Pseudomonas f1uorescens 1ipopo1~ saccharide. Bioorg.~. 12: 1641.
2.
Kiprianova, E. A., Panichev, A. V., Boyko, O. I., and Garagu1ya, A. D., 1979, Numerical taxonomy of bacteria belonging to the genus Pseudomonas. Microbiology, XLVIII: 1023.
3.
Knire1, Yu. A., Zdorovenko, G. M., Dashunin, V. M., Yakov1eva, L. M., Shashkov~ A. S., Zakharova, I. Ya., Gvozdyac, R. I., Kochetkov, N. K., 1986, Antigenic polysaccharides of bacteria. 15. Structure of the repeating unit of o-specific polysaccharide chain of Pseudomonas wieringae lipopolysaccharide. Bioorg.~. 12: 1253.
4.
Knire1, Yu. A., 1985, Lipopo1ysaccharides of gram-negative bacteria, in: itprogress in chemistry of carbohydrates, it I.V. Torgov, ed., Science, Moskow.
5.
Pa11eroni, N. J., 1984, Family I. Pseudomonadaceae, in: i~ergey's Manual of Systematic Bacteriology," J. G. Holt, ed.~ Wi11ia;; &Wilkins, Baltimore, MD.
134
6.
Veremeychenko. S. N•• 1986. Core oligosaccharides of Pseudomonas f1uorescens. Microbio1. J. 49: 18.
7.
Zdorovenko. G. M•• Veremeychenko. S. N•• Zakharova. I. Ya •• 1986. Comparative characteristic of 1ipopo1ysaccharides from different Pseudmonas f1uorescens strains. Microbio1. J. 49: 12.
135
G.M. Zdorovenko, S.N. Veremeychenko, I. Ya. Zakharova and Yu. A. Knire1 Zabo1otny Institute of Microbiology and Virology of the Ukrainian Academy of Sciences. Kiev. USSR
Heterogenous in both phenotype and genotype species. Pseudomonas f1uorescens is presented by 5 biovars in Bergey's Manual. In spite of their belonging to saprophytic organisms the strains of Pseudomonas f1uorescens are frequently isolated from clinical specimens (5). The endotoxins (LPS) of microbial cells are responsible for the mechanisms of interaction between micro- and macroorganisms in the infectious process. Therefore. we studied the LPS's of P. f1uorescens. The strains under study were obtained from the Collection of the Institute of Microbiology and Virology (IMV) and represented the biovars: I (IMV 4125=ATCC 13525). II (IMV 1602). III (IMV 2125). IV (IMV 2111). V (IMV 2763). and biovar i (IMV 2303) initially described in the USSR (2). Bacteria were grown on nutrient agar media. LPS was extracted from the acetone-dried cells by the Westphal method. All methods of analysis were described (3, 6, 7). LPS's were active in homologous reactions of immunoprecipitation (approximate titer was 1:100.000) and in the Ouchter1ony test (1-3 precipitation lines). Weak crossreactions were observed only between strains of biovars I and i. IV and V. We studied separately different parts of the LPS macromolecule (O-chain. core. lipid A) obtained after its cleavage by acetic acid hydrolysis. The fraction of o-specific polysaccharide (PS) was serologically active in each strain (approximate immunoprecipitation titer was 1:500.000). Table 1 shows the results of sugar analysis of the o-chain. The following features of sugar compositions were detected: 1.
Each strain had its characteristic sugar composition.
2.
Rhamnose and amino sugars were the main components in the majority of strains.
3.
Inclusion of rarely occurring sugars such as 3-acetamido-3.6-dideoxy-D-ga1actose. N-acety1-L-fucosamine. 6-deoxy-L-ta1ose. 2.4-dia cetamido-2.4.6-trideoxy-D-g1ucose.
H. Friedman et al. (eds.), Endotoxin © Springer Science+Business Media New York 1990
131
Table 1.
The main sugar components of O-specific PS fwt %).1 (Sugar composition was estimated using GLC. LC, GC-MS, Hand 3C NMRspectroscopy, etc., analysis)
Sugar
4125
1602
2125
49
41
16
Rhamnose 6-deoxy-L ta10se Fucose Mannose Glucose N-acety1-D glucosamine N-acety1-Dgalactosamine N-acety1-Lfucosamine 3-acetamido-3, 6-dideoxy-Dgalactose 2,4-diacetamido2,4,6-trideoxyD-g1ucose Unidentified aminosugar
2111
2303 52
22
2763
20 20
54
18 19
20 21 12
23 11
14
Studies on the core fractions showed their composition usual for pseudomonas (Table 2). The only exception was the strain 2763. We did not succeed to find in its core rhamnose, alanine, phosphorus and KDO but at the same time it contained too much heptose and glucosamine. The fatty acids presented in lipid A of type strain 4125 (Fig 1) were also found in lipid A of 2125, 2111 and 2303. Lipid A of 1602 was similar but it contained 2-0H C12:0. Strain 2763 differed from above strains. 3-0H C14:0 was the major component of its lipid A. Because 2763 was considerably different in its LPS composition from other strains, the structural investigations were performed on its PS. 13 C NMR-spectroscopy of intact PS demonstrated its irregularity, but a strictly regula! po1ym~r (Fig 2) have been received after o-deacety1ation of PS. Using Hand 13C NMR-spectroscopy, methylation, partial hydrolysis, etc., the repeating unit of PS was found to have the structure: 50% OAc
+ 2) -
~np
50% AcO
(1 + 3)-6 -D-GalllAcp (1 + 4)-"
-+-
2) - a - L - 6Ta1p
-D-~
As far as we know only the structure of P. fluorescence LPS was determined earlier (1). The presence of 6-deoxy-L-ta10se is the only similarity between these structures.
132
Composition of core oligosaccharide
Table 2. 4125
Strain Rhamnose Fucose Arabinose Mannose Glucose Galactose Heptose *D-g1ucosamine *D-ga1actosamine *2-keto-3-deoxyoctu1osonic acid *A1 ani ne *Phosphorus
2125
1602
19.8
21.0
18.0 61.1 tr tr 1.0 0.5 0.6
14.8 18.5 33.3 12.3 tr tr 0.4 7.5
2.0 3.6
1.1 2.8
II
2303
52.0 39.0
3.8
38.0
1.2 3.6 1.2 3.0 tr 6.7 4.6
17.3 57.7 19.2 tr tr 0.7 2.7
tr 41.4 tr 19.8 7.2 tr 0,2
1.8 3.7
2.8 5.2
3.5 5.0
2763
tr 1.2 37.6 19.5 41.8 14.4 1.0
*(wt %)
(Peak area % GLC-ana1ysis)
4125
2111
2
1602
2163 9
2
6 4
4
2
1
,)
7
5
1
,
10
6
)
,- ---Fig 1.
GL chromatograms of fatty acid components of lipid A fractions released by acid (1% CH3 COOH. 100 o C. 1.5 hr) hydrolysis from LPS. Methyl esters received by using 1.5N methano1ic HC1 were separated on column (0.3 x 120 cm) with 5% SE-30 on Chromaton N-AW-DMCS. 80-100 mesh at 130-230 oC. 3 0 C/min. Identification of peaks was made by co-chromatography with authentic standards and by GC-MS. The peaks correspond to: 3-0H C10:0«1). C12:0(2). 2-0H C12:0(3). 3-0H C12:0(4). C16:1(5). C16:0(6). ct8:1(7). C18:0(8). 3-0H C14:0(9). 3-0H C1 5 :O(10).
133
Biovar V is a heterogenous group of bacteria (5) which lack some peculiarities of pseudomonas. We may speculate that unusual compositions of these LPS may provide additional characteristics for this group. There is a correlation between diversity of the strains by biological properties and their LPS composition. By inclusion of 6-deoxyhexoses and aminosugars into the o-chain, as well as lipid A composition of P. fluorescence resembles corresponding products from some serotypes of P. aeruginosa and some enteric bacteria (4).
.- ...- . • .00.0
Fig 2.
i
•• 0
0,
~ .'t\. ~~ . 'I
•••
•
i
7•• '
....
.,'
..,
--a..
'.u, '0'
PI'N
SI.'
.,.,'
••• 0
J.
•
10.0
-
• %0.'
~
•
10. C
13 C NMR-spectrum of o-specific polysaccharide of P. fluorescence IMV 2763.
REFERENCES
1.
Khomenko, V. A., Naberezhnych, G. A., Isakov, V. V., Solov'eva, T. F., Ovodov, Y. S., Knire1, Y. A., Vinogradov, E. V., 1986, Structural study of o-specific polysaccharide chain of Pseudomonas f1uorescens 1ipopo1~ saccharide. Bioorg.~. 12: 1641.
2.
Kiprianova, E. A., Panichev, A. V., Boyko, O. I., and Garagu1ya, A. D., 1979, Numerical taxonomy of bacteria belonging to the genus Pseudomonas. Microbiology, XLVIII: 1023.
3.
Knire1, Yu. A., Zdorovenko, G. M., Dashunin, V. M., Yakov1eva, L. M., Shashkov~ A. S., Zakharova, I. Ya., Gvozdyac, R. I., Kochetkov, N. K., 1986, Antigenic polysaccharides of bacteria. 15. Structure of the repeating unit of o-specific polysaccharide chain of Pseudomonas wieringae lipopolysaccharide. Bioorg.~. 12: 1253.
4.
Knire1, Yu. A., 1985, Lipopo1ysaccharides of gram-negative bacteria, in: itprogress in chemistry of carbohydrates, it I.V. Torgov, ed., Science, Moskow.
5.
Pa11eroni, N. J., 1984, Family I. Pseudomonadaceae, in: i~ergey's Manual of Systematic Bacteriology," J. G. Holt, ed.~ Wi11ia;; &Wilkins, Baltimore, MD.
134
6.
Veremeychenko. S. N•• 1986. Core oligosaccharides of Pseudomonas f1uorescens. Microbio1. J. 49: 18.
7.
Zdorovenko. G. M•• Veremeychenko. S. N•• Zakharova. I. Ya •• 1986. Comparative characteristic of 1ipopo1ysaccharides from different Pseudmonas f1uorescens strains. Microbio1. J. 49: 12.
135
