Division of Biochemical Microbiology, Forschungsinstitut Borstel, Institut fiir Experimentelle Biologie und Medizin,D-23845 Borstel, Germany
Received 11 November 1993/Accepted 9 March 1994
In two strains of Chlamydia psittaci and in Chlamydia trachomatis serotype L,, we have detected aso-far-unknown antigen which (i) is resistant to heat and proteolytic digestion, (ii) can be extracted withphenol-water into the water phase, (iii) gives a ladder-like banding pattern in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (iv) is immunogenic in rabbits and mice, and (v) contains immunoreac-tivity of lipid A, a common and characteristic component of gram-negative lipopolysaccharides (LPS). Thus,chlamydiae contain, in addition to the known rough-type LPS, another LPS type which is phenotypicallysmooth (S-LPS). S-LPS was observed preferentially in chlamydiae grown in the yolk sac of embryonated eggs;it was, however, also detected by immunofluorescence in tissue culture-grown chlamydiae with a monoclonalantibody against S-LPS.
Chlamydia psittaci, Chlamydia pneumoniae, and Chlamydiatrachomatis are pathogenic, obligatory intracellular parasitescausing diseases in animals and humans. The major surfaceantigens of Chlamydia cells are the 39.5-kDa major outermembrane protein and the lipopolysaccharide (LPS) (33). Likethat in other bacteria, chlamydial LPS is composed of lipid Aand a saccharide portion (31). The latter contains a trisaccha-ride of 3-deoxy-D-manno-octulosonic acid (Kdo) of the se-quence aKdo(2->8)-aKdo(2---4)-odKdo, which represents anepitope shared by the whole genus (4, 16). Thus, chlamydialLPS is phenotypically of the rough type (R-LPS). The genus-specific epitope has the following relevant characteristics: (i) itis surface exposed and immunoaccessible on chlamydial ele-mentary bodies (EBs) and reticulate bodies (2), (ii) it has beenchemically synthesized (18), (iii) high-affinity monoclonal an-tibodies (MAbs) have been prepared against it and character-ized for their binding specificity (8), and (iv) the gene respon-sible for its biosynthesis has been cloned (23, 26) andsequenced (3, 23). The gene product is a multifunctionalglycosyltransferase, catalyzing all three glycosylation steps inthe assembly of the Kdo trisaccharide (3). Despite this pro-found knowledge, the biological significance of chlamydial LPSin infection, except for being a diagnostic marker, is unclear.Here we report on the occurrence of a hitherto unknown
antigen in Chlamydia spp. sharing properties with those of LPSfrom enterobacterial wild-type strains (smooth form, S-LPS).
MATERIALS AND METHODS
Chlamydial strains. C. psittaci 6BC and C. trachomatisserotype L2 (strain 434) were obtained from the AmericanType Culture Collection. The ewe abortion strain PK5082 of C.psittaci and C. trachomatis serotype L1 were from our labora-tory in Bratislava, Slovak Republic (32).
Preparation of LPS from yolk sac-grown chlamydiae. C.
* Corresponding author. Mailing address: Forschungsinstitut Bors-tel, Parkallee 22, D-23845 Borstel, Federal Republic of Germany.Phone: 49-4537-10474. Fax: 49-4537-10419.
t Present address: Institute of Virology, Slovak Academy of Sci-ences, 84246 Bratislava, Slovak Republic.
psittaci 6BC and PK5082 and C. trachomatis L, were grown inembryonated eggs as described elsewhere (32). C. psittaci 6BCEBs were purified as follows. Yolk sacs from 1,000 infectedeggs (10 aliquots of 100 eggs) were suspended in 1.5 M sodiumchloride (1 liter for each aliquot) and tested for bacterialgrowth on blood and chocolate agar and on thioglycolate brothUSP (Oxoid). Phenol was then added to a final concentrationof 1%, and the samples were kept at 4°C overnight. The yolksacs were homogenized and centrifuged at 30,000 x g for 1 h.The sediments were homogenized in 0.15 M saline (2 ml peryolk sac), digested with trypsin (1% final concentration, 2 h at37°C), and dialyzed against water. The retentate was extractedwith an equal volume of ether and mixed, and the water phasewas collected from a separating funnel. The ether extractionwas repeated twice. The ether was evaporated from the waterphase under a stream of nitrogen and centrifuged at 30,000 xg for 1 h. The sediments of all 10 batches were combined andlyophilized, yielding 103 mg of purified EBs. This preparationwas used for the gels and Western blots (immunoblots) shownin Fig. 1 and 2 and for the immunization of rabbits and miceand will be referred to as 6BC/#1. Four other batches of C.psittaci 6BC (100 eggs each) were prepared similarly; they willbe referred to as 6BC/#2-5.
C. psittaci PK5082 and C. trachomatis L, were processeddifferently. The values given in the following are from the latterpreparation; they were also representative of other experi-ments. Yolk sacs from 100 eggs were combined, exposed tophenol (1% final concentration) at 4°C overnight, homoge-nized in saline (1,000 ml), and lyophilized (106 g). The dryproduct was washed with ethanol, acetone, and ether (200 mleach), dried (30.4 g), suspended in saline (500 ml), digestedwith trypsin (1%, 37°C, 2 h), and dialyzed against water. Aftercentrifugation (2,860 x g for 30 min), the sediment waslyophilized (7.6 g) and the supernatant was ultracentrifuged(100,000 x g for 4 h), yielding a supernatant (5.2 g) and asediment (640 mg), the latter containing EBs. The two high-speed fractions and the low-speed sediment were separatelylyophilized and subjected to hot phenol-water extraction,yielding a water phase and phenol phase each. After dialysisagainst water, the samples were lyophilized. The yields of thewater phases obtained from the low-speed sediment, the
high-speed sediment, and the supernatant were 23, 12, and 585mg, respectively, and those for the phenol phases were 4,000,330, and 3,100 mg, respectively. Aliquots of the extracts weredigested with proteinase K and analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Pu-rified R-LPS has been described previously (6).
Rabbit antisera. Antisera were raised against EBs of C.psittaci 6BC (preparation 6BC/# 1) and C. trachomatis L2grown in L929 cells. New Zealand White rabbits (4 to 6animals), free of chlamydial antibodies as determined byimmunofluorescence and Western blot of preimmune sera,were injected intravenously with 50, 100, 100, 200, and 500 ,ugof EBs on days 0, 4, 7, 11, and 51, respectively, and were bled7 days later. Rabbit antisera against C. psittaci PK5082 werethose described earlier (6).MAbs. BALB/c mice were immunized with EBs of C. psittaci
6BC (preparation 6BC/#1) according to the following modi-fication of a protocol described by Stahli et al. (34). Four micewere injected with an emulsion of 50 p.g of EBs in 125 ,ul ofphosphate-buffered saline (PBS) mixed with an equal volumeof Freund's complete adjuvant (Difco). On day 0, four aliquotsof the mixture (50 [L1 each) were injected subcutaneously and50 p.1 was injected intraperitoneally. On day 28, mice receiveda single intraperitoneal injection of 50 pLg of EBs in 50 p.1 ofPBS emulsified with an equal volume of Freund's incompleteadjuvant (Difco). Eight days later, the mice were bled from thetail vein and the serum was tested for the presence ofantibodies to the immunizing antigen in Western blot. Themouse with the highest titer received three booster injectionsof 200 p.g each in 200 p.l of PBS on days 56, 57, and 58. The firstinjection was intravenous, and the others were intraperitoneal.Two days after the last injection, the animal was exsanguinatedand the spleen was removed. Spleen cells were prepared andfused at a ratio of 1:1 with mouse myeloma cells X63Ag8 withpolyethylene glycol 1500 (Boehringer Mannheim) according toconventional protocols. Screening of hybridoma supernatantsfor initial characterization of MAbs was evaluated by Westernblot with proteinase K-digested whole-cell lysates of 6BC/# 1EBs. Relevant hybridomas were cloned twice by limitingdilution, adapted to serum-free medium (Ultroser Gibco,Eggenstein, Federal Republic of Germany), and isotyped.Culture supernatants were purified on protein G-Sepharose.MAb S25-23 (immunoglobulin GI [IgG 1]) (8), recognizing theKdo trisaccharide as the minimal structure, was used to detectchlamydial R-LPS.SDS-PAGE and Western blots. Proteinase K-digested
whole-cell lysates (14) of EBs or phenol-water extracts wereseparated by SDS-PAGE with a 5% stacking and a 10 or 15%separating gel followed by silver staining. For Western blots,gels were electrotransferred to nitrocellulose (0.45 p.m) anddeveloped with polyclonal antibodies or MAbs as previouslydescribed (8). Protein profiles in whole-cell lysates withoutproteinase K digestion were visualized by staining of 15% SDSgels with Coomassie brilliant blue.
Detection of lipid A immunoreactivity in chlamydial S-LPS.S-LPS was hydrolyzed for 1 h at 100°C in 1% acetic acid or 0.1M HCI, neutralized with triethylamine, and used in a passivehemolysis assay as inhibitor for a lipid A MAb (19).Immunomicroscopy. L929 monolayers infected with C.
psittaci 6BC were fixed 28 h after infection in 2% paraformal-dehyde in PBS and stained with MAbs and fluorescein isothio-cyanate- or alkaline phosphatase-conjugated goat anti-mouseIgG (heavy plus light chains) (Dianova, Hamburg, FederalRepublic of Germany) as described earlier (2). For doublelabeling, S-LPS was stained with MAb G3-77 (IgG2b) andalkaline phosphatase-conjugated goat anti-mouse IgG (heavy
plus light chains). The preparations were examined, and thepositive ones were then stained for R-LPS with MAb S25-23(IgGI) and subtype-specific fluorescein isothiocyanate-conju-gated goat anti-mouse Ig (medac, Hamburg, Federal Republicof Germany). A Zeiss photomicroscope with optics for light,phase contrast, and incident fluorescence microscopy was used.PCR. The single-stranded oligonucleotide primers CT
GCTTT'AGCTATGGTCGTGC and TCCGACCTT[GGGTTATGAG, which are specific for the gseA gene of Chlamydiapsittaci 6BC (23), were synthesized on an automated DNAsynthesizer (Cyclone Plus; MilliGen/Biosearch) by the phos-phoramidite method. EBs of C. psittaci 6BC (5 p.g each) wereboiled for 10 min in 50 p.1 of lysis buffer (10 mM Trishydrochloride, pH 8.0, 1 mM EDTA, 20 mM dithiothreitol),chilled on ice, and subjected to PCR with the DNA Mastercy-cler (Eppendorf). Reaction mixtures (100 pl) contained 5 p.1 ofthe lysate, the primer pair (0.5 p.M each), all four deoxynucleo-side triphosphates (0.2 mM each), Tris hydrochloride (10 mM,pH 8.3), KCl (50 mM), MgCl (2.5 mM), gelatin (0.0(1%), andTaq DNA polymerase (2.5 U; Pharmacia). The samples weresubjected to 40 cycles consisting of a denaturation period of 45s at 94°C, an annealing period of 45 s at 57°C, and an extensionperiod of 2 min at 72°C. The PCR products were run on a 0.8%agarose gel alongside the amplified region of 1,694 residues byuse of 10 ng of purified chromosomal DNA of C. psittaci 6BC.
RESULTS
Detection of S-LPS in yolk sac-grown C. psittaci 6BC. Duringthe isolation of LPS from yolk sac-grown EBs, we obtained apreparation which exhibited, upon SDS-PAGE, a bandingpattern suggestive of wild-type LPS from enterobacteria (Fig.IA). The staining was not particularly sensitive, requiring 25p.g of EBs for visible patterns to emerge, and varied inreproducibility. Besides the ladder-like banding, a fast-migrat-ing band was identified as chlamydial R-LPS, as shown byWestern blots with MAb S25-23 against the genus-specific LPSepitope (8). Aliquots of the infected yolk sacs had been testedbefore the addition of phenol for growth of aerobic andanaerobic bacteria by use of blood and chocolate agar andthioglycolate broth. The purified EBs were subjected to SDS-PAGE, and the protein profile, which was similar to thatpublished for C. psittaci 6BC (13), was visualized with Coom-assie blue (Fig. 2, lane 2). Upon electron microscopy, only thetypical morphology and size of chlamydial EBs were seen (notshown). Finally, PCR with primers for the chlamydial Kdotransferase gene gseA of 6BC (23) yielded the same amplifica-tion products from the purified EBs as from an authenticculture obtained from the American Type Culture Collection(data not shown). Thus, gross contamination of the yolk sacswas excluded.
Polyclonal antisera against S-LPS from yolk sac-grown C.psittaci 6BC. The EBs so obtained were used to immunizerabbits, and the antisera were tested by Western blots. Theresult of one antiserum out of six (rabbit K220), tested withvarying amounts of antigen, showed that it contained antibod-ies against the high-molecular-weight bands (Fig. IB). Thetiter of the antiserum was 3,200 in Western blots, whcreas thepreimmune serum was negative at a dilution of 1:50. As seen inFig. IB, the minimal amount of EBs detected with an anti-serum dilution of 1:400 was 200 ng. Thus, the sensitivity wasmore than 100-fold higher than that obtained with silver-stained gels. In addition, Western blots were reproducible.Assuming in analogy to other gram-negative bacteria thatS-LPS can be obtained in a 1 to 5% yield of the dry bacterialmass, Western blots detected LPS in a range of 2 to 10 ng. This
a n c aFIG. 1. SDS-PAGE (A) and Western blot (B) of S-LPS and R-LPS from yolk sac-grown C. psittaci 6BC/# 1. (A) Enterobacterial LPS from
Salmonella abortus eqti (lane a, 1 jig) and Citrobacterfreundii (lane b, 1 [ig), chlamydial R-LPS (lane c, I jig) (6), and a proteinase K-digestedwhole-cell lysate from C. psittaci 6BC EBs (lane d, 50 [ig) were separated on a 15% gel and stained with silver nitrate. (B) The same chlamydialantigen preparation as in panel A lane d was applied in various amounts (twofold dilutions from 50 ,ug to 200 ng), blotted, and developed withrabbit antiserum K220 against 6BC EBs.
further indicated that the banding pattern could not be due toa contamination.The polyclonal antiserum also contained antibodies against
the already-known R-LPS of chlamydiae (see the lower part ofFig. IB). These antibodies were also detected by a passivehemolysis assay with sheep erythrocytes coated with isolatedR-LPS of C. psittaci in which the antiserum had a titer of 1,280(data not shown).MAbs against S-LPS of yolk sac-grown C. psittaci 6BC.
MAbs were obtained after immunization of BALB/c mice with6BC EBs. From 480 primary hybridomas, three clones wereselected to bind in Western blots to the high-molecular-weightbands of S-LPS. Two of these antibodies (G3-29 and G3-95)were of the IgGI isotype and one (G3-77) was an IgG2b. Thebinding of MAb G3-95 to S-LPS in Western blot is shown inFig. 3. None of the three MAbs reacted with R-LPS, and noneof our MAbs recognizing the Kdo trisaccharide or partialstructures thereof (8) reacted with the high-molecular-weightbands (data not shown).
Variation of S-LPS expression. Four additional batches ofyolk sac-grown EBs of C. psittaci 6BC (samples 6BC/#2 to
66-45S
31
14~~~~~~~~~~~~~~-
mol wt 1 2 3 4 5
FIG. 2. Protein profiles of five different preparations of EBs of C.psittaci 6BC. Whole-cell lysates of EBs (50 ,ug each) were separated ona 15% gel and stained with Coomassie brilliant blue. The sampleshown in lane 2 is from the same preparation (6BC/#1) as those in Fig.lB and 3. Molecular weights (in thousands) are shown at the left.
#5), which after SDS-PAGE and staining with Coomassie blueshowed a comparable protein profile, were prepared. Thebands seen at 62, 40, and 12 kDa correspond to those reportedby others for C. psittaci 6BC (13). The samples were thentested by Western blots for the presence of S-LPS. As shown inFig. 4A, development with the polyclonal antiserum revealed aregular banding pattern for all samples. However, only two ofthese patterns (lanes 1 and 5 in Fig. 4A) were identical. Whenthe same samples were developed with MAb G3-95, two of thefive samples were stained (Fig. 4B). This indicated that the 0side chain was variably expressed in these LPSs; the antiserumrecognized an epitope present in all five preparations, whereasthe MAb bound to an epitope present only in two samples.
Detection by immunomicroscopy of S-LPS and R-LPS inL929 cells infected with C. psittaci 6BC. The first attempts todetect S-LPS in infected monolayers by immunofluorescencewith MAbs against S-L-PS and fixation with acetone or meth-anol failed. When, however, fixation with aldehydes was used(2), positive staining of a few chlamydial inclusions was ob-served. To ascertain the specificity of the staining, doublelabeling experiments were performed with MAbs G3-77 andS25-23 against S-LPS and R-LPS, respectively. Figure 5A to C
FIG. 3. Reactivity in Western blot of MAbs against S-LPS fromyolk sac-grown C. psittaci 6BC. Various amounts (twofold dilutionsfrom 25 jig to 40)0 ng) of proteinase K-digested whole-cell lysates from6BC EBs were separated by SDS-PAGE on a 10% gel, blotted, anddeveloped with MAb G3-95. The same pattern was observed in use ofMAbs G3-29 and G3-77 (data not shown).
1 2 3 4 5 1 2 3 4 5FIG. 4. S-LPS profiles of five different preparations of EBs of C.
psittaci 6BC. Whole-cell lysates of EBs (10 ,ug each) were digested withproteinase K and separated on a 10% gel, electrotransferred tonitrocellulose, and stained with polyclonal rabbit serum K220 (thesame as in Fig. IB) (A) or with MAb G3-95 (B). The sample shown inlanes 3 is from the same preparation (6BC/# 1) as those in Fig. lB and3.
shows that some inclusions were stained with both antibodieswhereas others were positive for only one of them. Figure 5Dand E shows a cell with three inclusions, two of which werepositive for R-LPS and one of which was positive for S-LPS. Itshould be noted that the occurrence of cells positive for S-LPSwas a rare event; usually fewer than 10 cells per cm2 gave astaining for S-LPS. It thus seemed that in tissue culture moreR-LPS than S-LPS was made. S-LPS could not be visualized intissue culture-grown chlamydiae by Western blots even whenthe bacteria were first extracted with phenol-water. Obviously,
TABLE 1. Detection of lipid A immunoreactivity in S-LPS
Inhibition (,ug) of MAb againstbInhibitor Hydrolysis'
HAc, 1% acetic acid; HCI, 0.1 M hydrochloric acid; both for 1 h at 100°C."Inhibition was determined in a passive hemolysis assay with sheep erythro-
cytes coated with either synthetic Escherichia coli-type 4'-monophosphoryl lipidA (19) and or with chlamydial R-LPS (3).
the amount of S-LPS was below the detection limit of ourWestern blot protocol.
Detection of lipid A immunoreactivity in S-LPS (Table 1).When yolk sac-grown EBs of C. psittaci 6BC were treated withhot phenol-water, S-LPS was extracted into the water layer(approximately 10% of the dry bacterial weight) and did notcontain R-LPS, as shown by Western blot with the R-LPS-specific MAb S25-23 (data not shown). In addition, the ab-sence of R-LPS was shown by inhibition assays in which theantigen failed to inhibit MAbs against the genus-specificepitope of R-LPS. The water layer was hydrolyzed in acetic orhydrochloric acid, conditions known to release the 1,4'-bis- and4'-monophosphorylated lipid A moiety, respectively, fromLPS, and the products were tested for inhibition of lipid Aantibodies in a passive hemolysis assay (19). The results shownin Table 1 show that lipid A immunoreactivity was released
FIG. 5. Double labeling of S-LPS and R-LPS of C. psittaci 6BC grown in L929 cells by use of MAb G3-77 (A and D; alkaline phosphataselabeling) and MAb S25-23 (B and E; green fluorescence), respectively. The upper three pictures show the same area of a monolayer as seen bylight (A), fluorescence (B), and phase-contrast (C) microscopy. Note that the inclusion (arrow) is positive for S-LPS and negative for R-LPS, andnote the second inclusion in the same cell with an inverted staining pattern. The lower two pictures show a cell (arrow) containing three inclusions,of which two are positive for S-LPS (D) whereas all three are positive for R-LPS (E). Magnification, x400.
(30). At this time, we began to speculate that another LPSpopulation which may have been overlooked existed.Now, we have detected an antigen in chlamydiae which has
not been described to date. The antigen is a phase-variantS-LPS since it contains immunoreactive lipid A, is resistant to
N ~~~~heat and proteolytic digestion, is extractable by phenol-waterinto the water layer, exhibits a ladder-like banding pattern likethat of wild-type LPS of enterobacteria in PAGE, and isimmunogenic for experimental animals. MAbs against neitherthe enterobacterial common antigen (29) nor the genus-specific chlamydial exoglycolipid (36) reacted with the antigen
1 2 3 4 1 2 3 4 (data not shown). S-LPS was detected in yolk sac- and tissueculture-grown chlamydiae; however, in case of the latter, onlysome bacteria made S-LPS which could be detected by immu-nofluorescence but was below the detection limits of Westernblots.When we detected S-LPS for the first time in chlamydiae
grown in embryonated eggs, we were immediately aware of thepossibility of contamination with another bacterium. Althoughthe harvested yolk sacs were negative for aerobic and anaero-
"IiJ-n bic bacteria (blood and chocolate agar and thioglycolate brothwere used), these media did not exclude the presence of a
1 2 3 4 1 2 3 4 fastidiously growing microorganism of known or unknownnature or the presence of an obligatory intracellular bacterium..6. SDS-PAGE and Western blot of S-LPS and R-LPS from Specific attention was paid to exclude contamination withc-grown C trachomatis LI (A and B) and C. psittaci PK5082 (C .cxieatbention whc is ao grown inoaborator in). Chlamydiae were partially purified by differential centrifuga- . 'nd extracted with phenol-water. Shown are the water phases Bratislava. At the beginning, SDS-PAGE followed by silver1 and 3) and phenol phases (lanes 2 and 4) of the low-speed staining was the only tool to detect S-LPS. This methodI and 2) and high-speed (lanes 3 and 4) sediments after required 25 ,ug of EBs for a positive signal. Considering an LPSon with proteinase K. In lanes 1 to 4, 100, 1,000, 50, and 100 ,ug, content of 1 to 5% of the dry bacterial mass and a detectiontively, were separated on a 15% gel, blotted, and developed with limit of 100 ng of LPS in silver-stained gels, 10% contamina-iologous polyclonal rabbit serum (A and C) or MAb S25-23 tion with another bacterium would be enough to attribute thethe genus-specific epitope of chlamydial R-LPS (B and D). For appearance of S-LPS to a contamination. After we had pre-
r details, see Materials and Methods. pared antisera and MAbs against the S-LPS, we performedWestern blots in which 200 ng of EBs (corresponding to 2 to 10ng of LPS) still gave a positive result. This result showed that
hydrochloric acid hydrolysis, and that this reactivity could the S-LPS could not be derived from a minor contamination.wve arisen from R-LPS. No lipid A reactivity was detected Only a pure culture of a microorganism other than a memberacetic acid hydrolysis, indicating that the Kdo-to-lipid A of the genus Chlamydia could explain this result. That ourre was more stabile in S-LPS than in R-LPS. This is not an preparation was indeed of the genus Chlamydia was proven byal finding, since it has been shown that the Kdo-to-lipid (i) electron microscopy showing only the typical morphology of-age is even more stabile in rough mutants carrying other chlamydial EBs, (ii) the protein profiles as detected by Coom-in addition to Kdo in their core oligosaccharide (5). assie blue-stained gels displaying bands at 62, 40, and 12 kDa,
:ection of S-LPS in yolk sac-grown chlamydiae other than which is in accordance with published data (13), and (iii) PCRttaci 6BC. Polyclonal antisera were raised in rabbits with with primers for the gene gseA encoding the Kdo transferase of'ac-grown EBs of C. psittaci PK5082 and tissue culture- C. psittaci 6BC (23).C. trachomatis L2. With these antisera, S-LPS was In other preparations of EBs from C. psittaci 6BC, we found
Led in phenol-water extracts by Western blots. The results that the S-LPS bands observed in Western blots differed byiown in Fig. 6. As in the case of C. psittaci 6BC, the S-LPS their molecular weights and their antigenic properties.redominantly present in the water phases and the R-LPS Whereas all five preparations reacted with the polyclonalredominantly present in the phenol phases. antiserum, only two of them bound the MAbs. Although this is
not the usual situation encountered with S-LPS, one canDISCUSSION explain this observation on the basis of our knowledge of the
immunochemistry of 0 antigens, e.g., in Salmonella spp. (20).has been known from the beginnings of chlamydial Many of the Salmonella spp. have a repeating unit with a basicrch that these bacteria possess a genus-specific antigen trisaccharide backbone composed of galactose, mannose, andit which antibodies are made by the infected host. During rhamnose which may be substituted by additional sugars asst decade, it has been shown that this antigen is an LPS side chain substituents. Such additional sugars, such as thee rough phenotype, and its molecular and antigenic family of 3,6-dideoxyhexoses, are often highly immunogenic,ure has been elucidated (3, 4, 8, 18, 26, 27). However, the and in some cases, even the presence of a single 0-acetyl groupYical significance of the LPS, except for its being an can change the immunoreactivity as in the case of factor 5 (20).;n, has remained obscure. In particular, the fact that Regarding this as the basis to discuss the phenomenon de-)dies against LPS failed to neutralize infectivity, although scribed here, chlamydiae make a basic oligosaccharide presentvere directed against surface-exposed epitopes on extra- in all five batches of C. psittaci 6BC and against which theitracellular chlamydial forms, was in contrast to what is polyclonal antiserum is directed. This backbone oligosaccha-n of the biological effects of LPS antibodies in general ride carries different sugars (varying from one batch to anoth-
er), which causes the differences in molecular weight withouthindering the polyclonal antibodies from binding to theirepitopes. However, the MAbs recognize an epitope onlypresent or only accessible in two of the five preparations. Suchvariations in 0 side chain biosynthesis have been reported tooccur in E. coli and in Klebsiella pneumoniae (11, 37).
Certainly, many colleagues will ask why this phenomenonhas been overlooked so far. Two facts are, in our opinion,contributing to this failure. First, the major activities in basicChlamydia research are focused more on understanding host-parasite relationships than on studying the physiology of thebacterium, and the tools required for these investigationspredominantly use the approaches of molecular genetics orcellular immunology. Therefore, the embryonated egg is usedonly in a few laboratories to grow chlamydiae. Second, thekilling of chlamydiae with phenol instead of formalin seems tobe crucial in our purification protocol. In earlier preparations(inactivated with formalin), we were also unable to detectS-LPS; obviously, the formalin-fixed S form EBs went duringthe purification protocol into a fraction which has not beenanalyzed.To facilitate the following discussion, we shall assume that
the biosynthesis of chlamydial S-LPS is organized geneticallythe same as in other gram-negative bacteria, e.g., in membersof the families Enterobacteriaceae (1, 7, 17, 21, 22), Vibrion-aceae (35), and Pseudomonadaceae (9), in which the genes forthe assembly of the 0 side chains are clustered in the rfb locus.It has been reported that a cloned rjb locus can express 0chains that are different in the recipient from those in thedonor (12, 35). Our data show that the synthesis of 0 sidechains in Chlamydia spp. is influenced by currently unknownsignals provided by the host environment. One known param-eter influencing 0 side chain biosynthesis is the availableenergy pool, as shown for Salmonella typhimurium (25).
Until we understand the molecular basis for this host-parasite interaction, we can only speculate on its possiblebiological significance. In other gram-negative bacteria, the 0polysaccharide protects the microorganism against host de-fense mechanisms, on the one hand, such as killing by com-plement or phagocytosis; on the other hand, it activates thehost's immune response and becomes a target for antibodieswith bactericidal or opsonic activities. Thus, the ability toswitch back and forth between the synthesis of R-LPS andS-LPS or between different forms of the latter would be highlyadvantageous for a bacterium in order to survive in a host. Foran obligatory intracellular parasite such as a Chlamydia organ-ism, this switching mechanism may be particularly useful; oncethe EBs have entered into the safe environment of thephagosome, there is no need for the protecting 0 antigen andthe continued synthesis of the polysaccharide would reduce thesubstrate and energy pool of the host. This may be the reasonwhy facultative intracellular mucosal pathogens such as Neis-seria gonorrhoeae lack an 0 chain (10); however, they have alsoevolved mechanisms to escape the host attack by modifyingtheir LPS, i.e., by sialylation to mimic host structures (24, 28).
Finally, the fact that 0 side chain expression has not beenrecognized in this bacterial genus so far may allow the hypoth-esis that the mechanism of LPS phase variation also takesplace in other pathogenic bacteria which have been reported tolack an 0 side chain in their LPS, such as Haemophilusinfluenzae, Bordetella pertussis, N. gonorrhoeae, Neisseria men-ingitidis, Acinetobacter calcoaceticus, and Bacteroides fragilis(15).
ACKNOWLEDGMENTS
We thank D. Bitter-Suermann and A. B. MacDonald for providingMAbs against the enterobacterial common antigen and the genus-specific chlamydial exoglycolipid, respectively; S. Kusomoto for syn-thetic lipid A; and U. Albert, V. Susott. and G. Benkovicova for experttechnical assistance.We thank the Deutsche Forschungsgemeinschaft for financial sup-
port (grants Br731/9-l and SFB 367/B1).
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