FEMS Microbiology Immunology 47 (1988) 151-156 151 Published by Elsevier

F1M00024

Hafnia alvei lipopolysaccharides: Isolation, sugar composition and SDS-PAGE analysis

A n n a Romanowska , Ewa Ka tzene l l enbogen , Ma lgorza t a KuIakowska , Andrzej G am ian , D a n u t a Witkowska, Mar ian Mulczyk and E12bieta R o m a n o w s k a

Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Wroctaw, Poland

Received 22 April 1988 Revision received and accepted 24 June 1988

Key words: Lipopolysaccharide; Hafnei ah, ei; SDS-PAGE analysis

1. SUMMARY

Lipopolysaccharides (LPS) of 33 strains of Hafnia alvei were isolated and purified. LPS con- tent of the dry bacterial mass ranged from 1.2 to 4.5%. All examined lipopolysaccharides contained glucose, glucosamine, heptose, 3-deoxy-oc- tulosonic acid and often galactose. Rhamnose, mannose, galactosamine, mannosamine and un- identified amino sugars were found in some H. alvei strains. Sialic acid was present in LPS of one strain. D-3-Hydroxybutyryl groups also were iden- tified in lipopolysaccharides of 5 strains of this genus.

SDS-PAGE of the lipopolysaccharides was pre- sented in the paper. According to these results two core types exist in H. alvei.

Correspondence to: A. Romanowska, Ludwik Hirszfeld In- stitute of Immunology and Experimental Therapy, 12 Czerska, 53-114 Wroc/aw, Poland.

2. I N T R O D U C T I O N

The name Hafnia alvei was proposed by Moller in 1954 for a new group of Enterobacteriaceae [1]. The numerical taxonomy study by Johnson et al. and DNA reassociation studies by Steigerwalt et al. confirmed the status of Hafnia as a separate genus [2].

According to the serological classification of Sakazaki [2] among H. alvei strains 68 O-antigens and 34 H-antigens exist. Seven O-subgroups have been recognized in this genus. H. alvei strains show numerous serological cross-reactions with Escherichia coli, Enterobacter cloaceae, Citrobacter freundii and Shigella [2].

In general H. alvei occur in stool specimens of healthy persons, however many incidences of nosocomial infections with Hafnia have also been reported [2].

So far no immunochemical data have been obtained for this enterobacterial group. We now report the preliminary chemical characteristics of H. alvei lipopolysaccharides (O-antigens) isolated from 33 strains.

0920-8534/88/$03.50 © 1988 Federation of European Microbiological Societies

152

39

I I

38

1

31

32

37

17

114160

1M

1190

....... 1192

m

I

Ill

1188

1187

1191

1209

1224

1204

1211

1214. 13337

1216

451-L

1203

1221

23

1196

2

1199

1200

1205

1220

1213

3. MATERIALS AND METHODS

3.1 H. alvei organisms." standard strain A TCC 13337

20 strains derived from the collection of Pasteur Institute (Paris) kindly provided by Dr. LeMinor, 3 strains from National Institute of Hygiene (Warsaw) kindly given by Dr. Z. Tyc and 9 strains from the collection of Institute of Immunology and Experimental Therapy (Wrodaw) were used in the experiments.

All the strains were found to possess character- istics typical for H. alvei based on biochemical criteria. They gave positive lysine and ornithine decarboxylase reactions and negative result for the arginine dihydrolase test. All the strains did not ferment raffinose, sorbitol, adonitol and inositol.

Growing the bacteria in liquid medium, isola- tion and purification of the lipopolysaccharides were carried out as described previously [3,4].

3.2 Sugar analysis

For neutral sugars determination the lipopoly- saccharide samples were hydrolyzed with 0.1 M HCI at 100°C for 48 h and monosaccharides identified as alditol acetates [5] by gas-liquid chro- matography (Varian 2100 instrument with flame ionization detector and Gas chrom Q column 3% OV-225 at 200 ° C).

For amino sugars determination the lipopoly- saccharide samples were hydrolyzed with 4 M HCI at 100°C for 12 h. Amino sugars were analyzed according to modified Ludowieg-Benmaman method [6,7] or as alditol acetates by gas-liquid chromatography (see above) at 220 °C. Heptose was determined according to Osborn [8], 3-deoxy- octulosonic acid by the method of Karkhanis et al. [9], 6-deoxyhexose according to Gibbons [10] and sialic acid by the resorcinol method [11]. Neutral glycoses were estimated according to Dubois et al. [12].

Paper chromatography was performed on Whatman no. 1 paper by using 1-butanol/

Fig. 1. Silver-stained SDS-PAGE of purified lipopolysac- charides from Hafnia alvei strains.

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Tab le 1

Suga r compos i t i on of the l ipopolysacchar ides isola ted f r o m Hafnia alvei s t ra ins

Source Strain Neu t ra l Sugar c o m p o n e n t s in %

glycoses Glc G a l M a n H e p K D O 6-deoxy G l c N G a l N

Hex

M a n N

Pas teur Ins t i tu te

(Par is )

A T C C d

13337 18.5 8.9 - 7.8 3,1 - 3.5 5.0

1187 d 23.5 9.6 - 8.4 3.6 - 4.5 5.7

1188 29.0 6.8 4.7 2.7 9.4 3,2 2.5 ~ 4.5 -

1190 28.0 8.1 2.9 - 6.3 2.7 6.8 ~ 5.4 -

1191 31.5 12.1 3.l 6.8 3.8 - 6.0 4.5

1192 28.0 6.2 1.2 - 8.3 2.5 5.2 " 7.0 -

1196 b 22.0 7.3 3.3 - 6.4 5.8 - 9.7 -

1199 27.0 7.7 0.7 - 7.2 3.0 - 7.8

1200 23.0 nd nd nd 8.0 3.1 - 8.6

1203 24.0 10.6 4.8 - 8.4 3.8 - 10.0

1204 8 25.5 5.0 4.3 7.4 8.7 1.6 - 3.8 2.2

1205 24.0 7.7 3.5 - 9.8 1.6 - 8.6

1209 29.0 11.7 2.8 - 10.2 3.0 5.3 ~ 2.5 3.0

1211 b'a 26.0 17.8 1.2 - 6.2 2.2 - 6.3 5.2

1213 33.0 21.9 1.7 - 8.0 2.2 - 1.5 7.8

1214 29.0 8.9 12.5 - 8.4 1.7 - 7.7

1216 8'd 23.0 9.0 5.6 - 9.0 3.2 - 11.6

1220 b 43.0 nd nd nd 7.2 2.9 - 8.2

1221 d 31.0 nd nd nd 8.8 3.8 3.8 8.0 6.0

1222 42.0 nd nd nd 7.5 nd 7.8 8.0 1.5

1224 29.0 11.4 2.6 - 7.0 2.7 - 5.5 6.0

N a t i o n a l Ins t i tu te 481-L 18.5

of Hyg ien e ( W a r s a w ) 1M 18.5

114-60 22.0

6.8 3.7 - 8.0 3.0 - 2.3 7.7

5.2 0.5 - 10.2 3.6 - 4.8

7.6 - 12.6 3.0 - 5.2 -

Ins t i tu te of

I m m u n o l o g y and

Exper imen ta l

T h e r a p y (Wroclaw) .

1 29.0 12.6 3.0 - 8.8 2.0 - 6.8 - 4.2

2 c 31.0 14.9 7.7 - 6.0 3.0 - 3.2 3.8 -

17 20.0 7.5 - - 6.7 1.1 - 14.5 - 12.0

23 27.5 nd nd nd 5.5 2.0 10.0 6.0 -

31 18.0 nd nd nd 4.5 1.4 12.0 - 7.0

32 18.5 nd nd nd 5.2 1.1 6.5 3.0 3.0

37 15.5 nd nd nd 5.2 1.0 9.0 - 11.0

38 21.0 nd nd nd 6.0 1.7 4.5 10.0 - 1 1 . 5

39 27.5 nd nd nd 5.5 2.2 10.0 6.0 7.5

- = absent c o m p o n e n t ; nd = not de t e rmined ; ~ 6 -deoxyhexose was iden t i f i ed as r h a m n o s e ; b u n k n o w n a m i n o sugar present ; ~ sialic

acid present ; d D - 3 - h y d r o x y b u t y r i c acid present ; Gal , ga lac tose ; Glc, g lucose; Hep , heptose ; M a n , m a n n o s e ; 6 -deoHex , 6 -deoxyhe-

xose; G lcN , g lucosamine ; GAIN, ga l ac tosamine ; M a n N , m a n n o s a m i n e ; K D O , 3 -deoxy-oc tu loson ic acid.

154

pyridine/water (6 :4 : 3, v / v / v ) as the irrigant and Trevelyan spray or ninhydrin (0.25% in acetone) reagent as the detectors.

Determination of D-3-hydroxybutyric acid was performed using D-3-hydroxybutyrate dehydro- genase [13]. Lipopolysaccharide samples (1 mg) were hydrolyzed with 4 M HC1 at 100 °C for 2.5 h prior to the analysis.

SDS-polyacrylamide gel electrophoresis (SDS- PAGE) was carried out by the method of Laemmli [14] with the following modifications: (a) slab gels (10 × 15 × 0.1 cm) contained the linear 10-20% acrylamide gel gradient and 5% acrylamide stack- ing gel; (b) LPS suspension (0.5 mg/ml) in 50 mM Tris-HCl-10 mM EDTA buffer, pH 6.8 con- taining 4% SDS, 10% glycerol and 0.005% bromophenol blue was boiled for 20 min and then 2 /~1 portion was applied to the gel. Electrophore- sis was run with a constant current of 30 mA per slab gel. LPS in the gel was visualized by silver stain procedure of Tsai and Frash [15].

RESULTS AND DISCUSSION

Lipopolysaccharides of H. aluei after phenol- water extraction of bacterial cells were isolated from aqueous phase and purified by gel filtration on Sepharose 2B. LPS containing fractions, free of RNA contamination showed A 2 6 0 / A 2 4 0 lower than 1.0 (see reference 4). LPS yield ranged from 1.2 to 4.5% of the dry bacterial mass.

The sugar compositions of lipopolysaccharides obtained from 33 strains are shown in Table 1. All lipopolysaccharides contained glucose, gluco- samine, heptose and 3-deoxy-octulosonic acid; galactose was a frequent component of Hafnia lipopolysaccharides. Furthermore, the following sugars were identified in Hafnia genus: galac- tosamine (13 strains), 6-deoxyhexose (9 strains), mannosamine (7 strains), mannose (2 strains), sialic acid (1 strain) and unknown amino sugars (5 strains). In general, amino sugars of Hafnia lipopolysaccharides were N-acetylated, but in 5 strains N-3-D-hydroxybutyryl residues also were found. The LPS content of D-3-hydroxybutyric acid ranged from 1.05 to 2.5% in these strains.

Lipopolysaccharides from Hafnia strains were

analyzed by SDS-PAGE and silver staining for molecular heterogeneity. The majority of them showed high molecular weight ladderlike pattern of bands up the gel (Fig. 1). These bands are characteristic for S-type lipopolysaccharides. There is a group of the lipopolysaccharides (strains: 2, 23, 1199, 1203, 1205, 1213, 1221) whose O-specific polysaccharide chains are rather short and occur in markedly lower amount by comparison with the core oligosaccharide region. Finally, the LPSs of some strains: 1 M, 114-60, 1191, 1196 and 1200, showed R-type character by the presence of fast migrating bands only. These bands were formed by low molecular weight core lipopolysaccharide. It should be emphasized however, that R-type character of a lipopolysaccharide observed in SDS-PAGE because of the limited sensitivity of the method, could not exclude the presence of minute O-specific polysaccharide portion in this LPS preparation. The fractionation on BioGel P-4 of the carbohydrate material isolated from the lipopolysaccharides of strains 1191, 1196 and 114- 60 proved that besides the main core fraction small amounts of O-specific material also were present (unpublished results).

REFERENCES

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[5] Sawardeker, J.S., Sloneker, J.N. and Jeanes, A. (1965) Quantitative determination of monosaccharides as their alditol acetates by gas liquid chromatography. Anal. Chem. 37, 1602.

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[7] Romanowska, E. and Reinhold, V. (1973) 2-Amino-2-de- oxyhexuronic acid: a constituent of Shigella sonnei phase I lipopolysaccharide. Eur. J. Biochem. 36, 160.

[8] Osborn, M.J. (1963) Studies on the gram-negative cell wall. I. Evidence for the role of 2-keto-3-deoxyoctonate in the lipopolysaccharide of Salmonella typhimurium. Proc. Natl. Acad. Sci. USA 50, 499.

[9] Karkhanis, Y.D., Zeltner, J.Y., Jackson, J.J. and Carlo, D.J. (1978) A new and improved microassay to determine 2-keto-3-deoxyoctonate in lipopolysaccharide of Gram- negative bacteria. Anal. Biochem. 85, 595.

[10] Gibbons, M.N. (1962) Reaction of 6-deoxyhexoses with thioglycolic acid and sulfuric acid. Methods Carbohydr. Res. 1. 502.

[11] Svennerholm, L. (1957) Quantitative estimation of sialic acids lI. A colorimetric resorcinol-hydrochloric acid method. Biochem. Biophys. Acta 24, 604.

[12] Dubois, H., Gilles, K.A., Hamilton, J.K., Rebers, P. and

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Smith, F. (1956) Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350.

[13] Williamson, D.H. and Mellanby, J. (1974) D-(-)-3-Hy- droxybutyrat in H.U. Bergmeyer (ed.), Methoden der Enzymatische Analyse, Verlag Chemie, Weinheim. F.R.G.

[14] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680.

[15] Tsai, C.M. and Frash, C.E. (1982) A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal. Biochem. 119, 115.