Tài liệu Báo cáo khoa học: The structure and biological characteristics of the Spirochaeta aurantia

The structure and biological characteristics of the

Spirochaeta aurantia outer membrane glycolipid LGLB

Evgeny Vinogradov1

, Catherine J. Paul2

, Jianjun Li1

, Yuchen Zhou2

, Elizabeth A. Lyle3

, Richard I. Tapping3

,

Andrew M. Kropinski2 and Malcolm B. Perry1

1

Institute for Biological Sciences, National Research Council, Ottawa, ON, Canada; 2

Queen’s University, Kingston, ON, Canada; 3

University of Illinois, Urbana, IL, USA

In an attempt to isolate lipopolysaccharide from Spirocha￾eta aurantia, Darveau-Hancock extraction of the cell mass

was performed.While no lipopolysaccharide was found, two

carbohydrate-containing compounds were detected. They

were resolved by size-exclusion chromatography into high

molecular mass (LGLA) and low molecular mass (LGLB)

fractions. Here we present the results of the analysis of the

glycolipid LGLB. Deacylation of LGLB with hydrazine and

separation of the products by using anion-exchange chro￾matography gave two major products. Their structure was

determined by using chemical methods, NMR and mass

spectrometry. All monosaccharides had the D-configuration,

and aspartic acid had the L-configuration. Intact LGLB

contained two fatty groups at O-2 and O-3 of the glycerol

residue. Nonhydroxylated C14 to C18 fatty acids were

identified, which were predominantly unsaturated or bran￾ched. LGLB was able to gel Limulus amebocyte lysate, albeit

at a lower level than that observed for Escherichia coli O113

lipopolysaccharide. However, even large amounts of LGLB

were unable to stimulate any Toll-like receptor (TLR)

examined, including TLR4 and TLR2, previously shown

to be sensitive to lipopolysaccharide and glycolipids from

diverse bacterial origins, including other spirochetes.

Keywords: glycolipid; Spirochaeta aurantia; structure.

Spirochetes are a group of bacteria unified by spiral or

flattened-waveform cell morphology and periplasmic endo￾flagella; Spirochaeta is one of the six genera within this

phylum [1]. This bacterium is a free-living nonpathogenic

spirochete, originally isolated from pond mud and able to

fix atmospheric nitrogen [2–4]. Other members of this

phylum include the human pathogens Borrelia burgdorferi

(Lyme disease), the Leptospira (leptospiroses), Treponema

pallidum (syphilis), and T. denticola, T. brennaborense, and

T. maltophilum, which are implicated in periodontal disease

[5–7]. Although classified as Gram-negative, controversy

exists over the existence of lipopolysaccharide (LPS) in the

outer membranes of spirochetes. Clear genetic and bio￾chemical evidence exists for the presence of LPS in

Leptospira [8] and for its absence in T. pallidum and Borrelia

[9,10]. Limited structural analysis suggests that several oral

treponemes (T. brennaborense and T. maltophilium [6],

T. medium [11], and T. denticola [12]) possess a surface

glycolipid similar to the lipotechoic acid of Gram-positive

bacteria. Recently, several small surface glycolipids were

identified in B. burgdorferi [13,14].

Toll-like receptors (TLR) are an important component of

the host response to invading bacteria, with TLR4 required

for signal transduction and the inflammatory response

following exposure of cells to LPS derived from Gram￾negative enteric bacteria [15–17]. Although LPS derived

from enteric bacteria is a potent agonist for TLR4, other

nonenteric bacterial LPS, such as that derived from

Legionella pneumophila, Leptospira interrogans and at least

one strain of Porphyromonas gingivalis can act as agonists

for TLR2 [8,18,19].

The glycolipids isolated from T. denticola, T. brennabo￾rense, and T. maltophilum appear to have functional

similarity to LPS in that they possess some ability to gel

Limulus amebocyte lysate (LAL) [12,20], a standard assay

for endotoxin activity. In addition, while glycolipid derived

from T. brennaborense stimulates immune cells through

TLR4, the glycolipids from T. denticola and T. maltophilum

stimulate cells through TLR2 [5,6,20]. The strict correlation

between the structure of the LPS molecule with that of TLR

specificity remains undefined but it is clear that TLR2 is

capable of recognizing a wider range of potential lipid A

structures than TLR4 [21].

S. aurantia has simple growth requirements that facilitate

studies otherwise limited by the amount of cell mass, a

problem often limiting studies on other spirochetes [2]. We

describe here the structural characterization of the carbo￾hydrate skeleton and fatty acids of one of its glycolipids,

LGLB. In addition we present evidence which suggests that

Correspondence to E. Vinogradov, Institute for Biological Sciences,

National Research Council, 100 Sussex Dr., Ottawa, ON, Canada

K1A 0R6. Fax: +1 613 952 9092, Tel.: +1 613 990 0832,

E-mail: [email protected]

Abbreviations: EU, endotoxin units; FAME, fatty acid methyl esters;

GalNAcA, N-acetylgalactosaminuronic acid; GSL, glycosphinogo￾lipids; Fuc3N, 3-amino-3,6-dideoxygalactose; Kdo, 2-keto-3-deoxy￾D-manno-oct-2-ulosonic acid; LAL, Limulus amebocyte lysate; LBP,

LPS-binding protein; LPS, lipopolysaccharide; SGM, spirochaete

growth medium; TLR, Toll-like receptor; TNF-a, tumour necrosis

factor-a.

(Received 9 August 2004, revised 30 September 2004,

accepted 13 October 2004)

Eur. J. Biochem. 271, 4685–4695 (2004)
FEBS 2004 doi:10.1111/j.1432-1033.2004.04433.x