Tài liệu Báo cáo khoa học: Endotoxic activity and chemical structure of lipopolysaccharides from

Endotoxic activity and chemical structure of lipopolysaccharides

from Chlamydia trachomatis serotypes E and L2

and Chlamydophila psittaci 6BC

Holger Heine, Sven Mu¨ ller-Loennies, Lore Brade, Buko Lindner and Helmut Brade

Research Center Borstel, Center for Medicine and Biosciences, Borstel, Germany

The lipopolysaccharide (LPS) of Chlamydia trachomatis

serotype E was isolated from tissue culture-grown element￾ary bodies and analyzed structurally by mass spectrometry

and 1

H, 13C and 31P nuclear magnetic resonance. The LPS

is composed of the same pentasaccharide bisphosphate

aKdo-(2–8)-aKdo-(2–4)-aKdo-(2–6)-bGlcN-4P-(1–6)-

aGlcN-1P (Kdo is 3-deoxy-a-D-manno-oct-2-ulosonic acid)

as reported for C. trachomatis serotype L2 [Rund,S.,Lind￾ner,B.,Brade,H. and Holst,O. (1999) J. Biol. Chem. 274,

16819–16824]. The glucosamine disaccharide backbone is

substituted with a complex mixture of fatty acids with ester

or amide linkage whereby no ester-linked hydroxy fatty

acids were found. The LPS was purified carefully (with

contaminations by protein or nucleic acids below 0.3%) and

tested for its ability to induce proinflammatory cytokines in

several readout systems in comparison to LPS from C. tra￾chomatis serotype L2 and Chlamydophila psittaci strain 6BC

as well as enterobacterial smooth and rough LPS and syn￾thetic hexaacyl lipid A. The chlamydial LPS were at least 10

times less active than typical endotoxins; specificity of the

activities was confirmed by inhibition with the LPS anta￾gonist,B1233,or with monoclonal antibodies against

chlamydial LPS. Like other LPS,the chlamydial LPS used

toll-like receptor TLR4 for signalling,but unlike other LPS

activation was strictly CD14-dependent.

Keywords: toll-like receptors; innate immunity; MALDI￾TOF MS; ESI-FT-IR MS.

Chlamydia are obligatory intracellular bacteria [1] causing

acute and chronic infections in animals and humans [2,3].

Little is known about the pathogenic mechanisms involved

in chlamydial infections but chronic inflammation is

observed in classical chlamydial diseases such as ocular

trachoma,that is the world’s leading cause of preventable

blindness,or chronic salpingitis,that is the major cause of

secondary female infertility in developed nations. Athero￾sclerosis is a chronic inflammatory disease of the arterial

walls which is presently considered to be associated with

infection by Chlamydophila pneumoniae [4]. Members of the

family Chlamydiaceae are Gram-negative bacteria contain￾ing,in their outer membrane,a lipopolysaccharide (LPS)

harbouring a surface-exposed,family specific epitope that

has been well characterized [5]. It is well known that LPS of

Gram-negative bacteria in general is one of the most potent

stimulators of innate immunity and that the lipid A moiety

of LPS is responsible for this activity [6,7]. Detailed studies

on the structure–function relationships of lipid A have

indicated that the number,type and distribution of fatty

acids in lipid A determine whether it exhibits weak or strong

agonist or antagonist activities [8]. Analytical data have

shown that the fatty acids in LPS of Chlamydiae have acyl

chains with up to 22 carbon atoms and also that 3-hydroxy

fatty acids occur only with amide-linkage [9,10]. Studies on

the biological activity of chlamydial LPS have shown that it

possesses significant lower activity than enterobacterial LPS

in terms of pyrogenicity or Schwartzman-reactivity in

rabbits,lethality in galactosamine-sensitized mice,anti￾complementary activity in guinea-pigs and induction of

proinflammatory cytokines in human peripheral blood

monocytes [10,11]. It was,however,mitogenic for mouse

B-cells and activated mouse peritoneal macrophages,thus,

producing prostaglandin E2 [10]. These reported data were

obtained on LPS of unknown chemical structure and of ill￾defined purity. Therefore,in this study we have compared

the endotoxic activities of three chlamydial LPS of known

structure and defined purity. Whereas the chemical struc￾tures of the LPS from C. trachomatis serotype L2 and of

Chl. psittaci 6BC have been reported previously [12,13], that

of the LPS from C. trachomatis serotype E,which is the

Correspondence to H. Brade,Research Center Borstel,Center for

Medicine and Biosciences,D-23845 Borstel,Germany.

Fax: + 49 4537 188 419,Tel.: + 49 4537 188 474,

E-mail: [email protected]

Abbreviations: CSD,capillary skimmer dissociation; COSY,

correlation spectroscopy; EB,elementary body; ESI-FT-ICR MS,

electrospray ionization Fourier-transform ion cyclotron resonance

mass spectrometry; FBS,fetal bovine serum; HEK,human epithelial

kidney; HMBC,heteronuclear multiple bond correlation; HPAEC,

high-performance anion-exchange chromatography; HSQC,hetero￾nuclear single quantum coherence; Kdo,3-deoxy-a-D-manno-oct-2-

ulopyranosonic acid; LPS,lipopolysaccharide(s); mAb,monoclonal

antibody; MALDI-TOF MS,matrix assisted laser desorption/ion￾ization time-of-flight mass spectrometry; MNC,mononuclear cells;

NOE,nuclear Overhauser effect; NOESY,nuclear Overhauser effect

spectro-scopy; TLR,Toll-like receptor; TNF,tumor necrosis factor

alpha; ROESY,rotating frame nuclear Overhauser effect spectro￾scopy; TOCSY,total correlation spectroscopy.

Note: H. H. and S. M. L. contributed equally to this study.

(Received 11 July 2002,revised 11 November 2002,

accepted 26 November 2002)

Eur. J. Biochem. 270,440–450 (2003)
FEBS 2003 doi:10.1046/j.1432-1033.2003.03392.x