Lipopolysaccharides (LPS) extracted from Escherichia coli

Details Lipopolysaccharides (LPS) extracted from Escherichia coli Lipopolysaccharides (LPS) extracted from Escherichia coli

2002-12-23

2004-12-21

Barts, Samuel (Department: 1623)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S053000, C536S117000, C514S054000, C424S241100, C435S084000

Reexamination Certificate

active

06833451

ABSTRACT:

This is the national phase Application of PCT EP01/03153 filed, Mar. 20, 2001.
BACKGROUND OF THE INVENTION
The invention concerns new lipopolysaccharides extracted from
E. coli.
Endotoxins are bacterial structural components, which, unlike exotoxins, are not secreted, but rather are released, especially following autolysis. The classic endotoxins are heat-stable lipopolysaccharides (LPS) from the outer cell membrane of gram-negative bacteria. LPS consists of lipid A, which is responsible for the toxic effect of LPS, a core oligosaccharide and an O-specific chain.
In macroorganisms, endotoxins stimulate the production of immune system mediators, such as interleukin-1 (IL-1) and tumor necrosis factor (TNF&agr;).
Many studies have already been conducted on the composition of the endotoxins of enterobacteria, especially
E. coli
, in which it was determined that S/R mutants generally contain only one repeating unit of their O specific chain (cf. FIG.
1
). It is assumed that in these cases, the gene that codes for the polymerizing enzyme of the O-specific chain is defective, and therefore only one repeating unit it transferred to the core oligosaccharide. LPS structures of a similar type but different structure are also commonly found in bacteria that are pathogenic in man, such as Neisseria, Vibrio, Campylobacter, Helicobacter, etc. These bacteria have an LPS which allows them to evade the immune defense of the host by means of a special molecular mimicry, including the presence of sialic acid and oligosaccharides that contain sialic acid, which resemble glycoproteins and glycolipids in mammals. The 06 serotype was determined for
E. coli
DSM 6601. This structure was studied and published by P. E. Jansson et al.,
Carbohydr. Res
. 131 (1984) 277-283. The structure corresponds to the formula shown in FIG.
2
.
FIG. 2
is a representation of the structure of the O antigen of
E. coli
06. All sugars are present in the D-pyranose form. In contrast to the structure of the O-specific chain of
E. coli
06 published by Jannson et al., the first repeating unit of the S/R mutants of
E. coli
DSM 6601 is linked by a &bgr;-glycosidic bond and was determined as such for the first time in accordance with the present invention, together with the site of substitution at the side-chain glucose (Glc
III
) of the core oligosaccharide. (See
FIG. 4.
)
The lipid A of the coli bacteria has also been investigated by various research groups, and it was found that the structure of the lipid A generally has the hexaacyl form and is consistent for all serotypes of
E. coli
(FIG.
3
). The structure of the hexaacyl compound was published in 1984 by T. Rietschel et al., Structure and Conformation of the Lipid A Component of Lipopolysaccharides.
Handbook of Endotoxins
(Proctor, R., ed.), Vol. 1
, Chemistry of Endotoxin
(E. T. Rietschel, ed.), Elsevier, Amsterdam (1984), pp. 187-220. The structure is shown in FIG.
3
.
FIG. 3
is a representation of the structure of the hexaacyl lipid A of
E. coli
. (Zähringer, U., Lindner, B. and E. T. Rietschel, Molecular Structure of Lipid A, the Endotoxic Center of Bacterial Lipopolysaccharides,
Adv. Carbohydr. Chem. Biochem
., 50 (1994) 211-276). The numbers in the circles indicate the number of carbon atoms in the given fatty acid. The free hydroxyl group of the GlcN (II) represents the bonding site for the Kdo (I) of the core oligosaccharide.
The O-specific chain and the lipid A are linked by the core oligosaccharide. There are five previously known core oligosaccharides of
E. coli
; see O. Holst et al., Chemical Structure of the Core Region of Lipopolysachharide, IN:
Bacterial Endotoxic Lipopolysaccharides
, Vol. 1, Morrison, D. C. and Ryan, J. L. (eds.), Boca Raton, Fla., USA (1992) pp. 135-170 (cf. FIG.
4
).
FIG. 4
is a representation of the structure of the carbohydrate skeleton of the principal fraction in the core oligosaccharide of
E. coli
R1. (Vinogradov, E. V., van der Drift, K., Thomas Oates, J. E., Meshkov, S., Brade, H-. and O. Holst (1999)
Eur. J. Biochem
., 261, 629-639.) The O-specific chain substitution at the side-chain glucose (Glc
III
) and its anomerism were determined for the first time in accordance with the present invention. All sugars are present in the D-pyranose form. (L, D-Hep, L-glycero-D-manno-heptose; Kdo D-manno-oct-2-ulosonic acid; P, phosphate.)
SUMMARY OF THE INVENTION
The invention relates to lipopolysaccharides, for example, lipopolysaccharides that are extracted from
E. coli
DSM 6601. In one aspect of the present invention, a lipopolysaccharide is provided comprising a lipid A portion, a core oligosaccharide portion, and an O-specific chain having a single repeating unit of serotype 06. Preferably, the O-specific chain is linked to the core oligosaccharide portion. More preferably, the O-specific chain is linked to the core oligosaccharide by a &bgr;-glycosidic bond.
In accordance with another aspect of the invention, the linkages within the O-specific chain are linked by &agr;-glycosidic bonds.
In another aspect of the invention, the lipid A portion of the lipopolysaccharide is linked to the core oligosaccharide portion of the lipopolysaccharide.
In another aspect of the invention, the lipopolysaccharide has eight (8) phosphate groups per molecule of lipopolysaccharide.
In another aspect of the invention, the lipopolysaccharide has a phosphate substituent P-Etn in a concentration of 0.5 moles per mole of lipopolysaccharide.
In accordance with another aspect of the invention, a process for producing lipopolysaccharides is provided. An
E. coli
bacterial mass is washed and dried, the washed and dried bacterial mass is subjected to a phenol/water extraction, and the phenol/water extract is treated with RNases, DNases and proteinase K. Preferably, the
E. coli
bacterial mass is derived from
E. coli
strain DSM 6601.
In yet another aspect of the invention, a process for the use of the lipopolysaccharide for microbiological, bioengineering, analytical, diagnostic or medical purposes is provided. Preferably, the lipopolysaccharide is
E. coli
strain DSM 6601 lipopolysaccharide.


REFERENCES:
patent: 19844191 (2000-03-01), None
Jansson et al., Carbohydrate Research, 131 (1984) 277-283.*
P.-E. Jansson et al., Structural Studies of theEscherichia coliO-Antigen 6, Carbohydrate Research, 1984, 277-283, Elsevier Science Publishers, The Netherlands.
J.A. Yethon et al., Involvement of waaY, waaQ, and waaP in the Modification ofEscherichia coliLipopolysaccharide and Their Role in the Formation of a Stable Outer Membrane, The Journal of Biological Chemistry, 1998, 26310-26316, The American Society for Biochemistry and Molecular Biology, Inc.
Structure and Conformation of the Lipid A Component of Lipopolysaccharides, Handbook of Endotoxin, 1984, 187-220, vol. 1, Elsevier, Amsterdam.
Chemical Structure of the Core Region of Lipopolysaccharides, Bacterial Endotoxic Lipopolysaccharides, 135-170, vol. I, CRC Press, Boca Raton.

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