(~) INSTITUTPASTEUR/ELsEVIER Paris 1990

Res. Microbiol. 1990, 141, 1077-1094

ISOLATION, PURIFICATION AND PARTIAL ANALYSIS OF THE LIPOPOLYSACCHARIDE ANTIGENIC DETERMINANT RECOGNIZED BY A MONOCLONAL ANTIBODY TO L E G I O N E L L A P N E U M O P H I L A SEROGROUP 1

F. Petitjean (l), E. Dournon (2), A.D. Strosberg (l) and J. Hoebeke (1) (t) Laboratoire d'Immunopharmacologie Moldculaire, Institut Cochin de Gdndtique Moldculaire, 22 rue Mechain, 75014 Paris, and (2) Unitd de Pathologie Infectieuse, H6pital Raymond Poincard, 92380 Garches (France)

SUMMARY Monoclonal antibody I I-6-18 recognizes a serogroup-l-specific Legionella pneumophila antigenic determinant which has been shown to be virulenceassociated. We previously reported the physicochemical characterization by means of a quantitative fluorometric assay of monoclonal antibody II-6-18 binding to L. pneumophila, and its implications concerning the nature of the antigen. We describe here the isolation and the purification of the antigen by chemical and immunological methods, followed by its partial chemical analysis. The results demonstrate that the epitope - - an immunodominant carbohydrate which includes a fucosamine-like residue - - is part of the cell wall lipopolysaccharide (LPS). It is localized in the polysaccharide moiety of the LPS which contains KDO, rhamnose, mannose, glucosamine and an unidentified aminodideoxyhexose XI, but no heptose. The aminodideoxyhexose X1 could be fucosamine and is probably the immunodominant residue in the epitope, localized, at least partially, at the end of the polysaccharide chain. KEY-WORDS" Legionella pneumophila, LPS, Epitope; Serogroup 1, Virulence, Fucosamine, Immunodominance.

Submitted May 29, 1990, accepted July 17, 1990.

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INTRODUCTION The preparation of monoclonal antibodies (mAb) to Legionella, and their use for diagnostic and epidemiological purposes have been reported by several authors. Their potential use in the study nf Legionella antigens has been s~,ggested. However, only a few of these mAb ha,,e been used until now to characterize these antigens at the molecular level (Gosting et al., 1984; Barthe et al., 1988). In a previous paper (Petitjean et al., 1987), we described the physicoch~:mical characterization of a mAb binding to a serogroup-l-specific L. p n e u m o p h i la Philadelphia 1 antigenic determinant by means of a quantitative fluorometric assay. The specificity of our mAb II-6-18 (Guillet et al., 1983) was shown to be the same (Petitjean et al., 1987) as that of CDC mAb2 (McKinney et al., 1983), which was demonstrated by Dournon et al. (1988) to be specific for a virulence-associated L. p n e u m o p h i l a serogroup 1 (Lpl) antigenic determinant. In our previous study, the results obtained using indirect investigation methods indicated that the epitope recognized by mAb II-6-18 was an immunodominant carbohydrate including a fucosylamine-like residue, and suggested that it was most probably localized on the lipopolysaccharide (LPS) of the bacterial cell wall. This would be in agreement with the data reported by several authors. Indeed, the LPS was described as the serogroup-specific antigen in Lp (Wong and Feeley, 1984; Gabay and Horwitz, 1985 ; Cieselski et al., 1986; Conlan and Ashworth, 1986; Nolte et aL, 1986; Otten et al., 1986), while protein antigens were shown to be broadly cross-reactive (Gosting et al., 1984; Ehret and Ruckdeschel, 1985; Wong et al., 1979). However, Barthe et al. (1988) demonstrated that a common epitope recognized by an mAb - - most probably a minor epitope - - was also present on the LPS of Lp serogroups 1 to 8. When compared to the classical LPS associated with Gram-negative bacteria, Legionella LPS was shown to be rather unusual. Its SDS-PAGE migration pattern was of the same type as that of smooth LPS but it differed, in particular, in the size distribution of the molecules: the average size was intermediate between that of smooth and rough Escherichia coil LPS, and the band spacing was tighter (Gabay and Horwitz, 1985; Cieselski et al., 1986; Conlan and Ashworth, 1986; Nolte et al., 1986; Otten et al., 1986). Its unique fatty acid composition was characterized by high amounts of branched-chain fatty acids and by the absence of hydroxy fatty acids generally associated with

ELISA GLC HVE IC w lg KDO Lp

= = = = = = =

enzyme-linked i m m u n o s o r b e n t assay. gas-liquid c h r o m a t o g r a p h y . high voltage electrophoresis. 50 070 inhibitory c o n c e n t r a t i o n . immunoglobulin. 2-keto-3-deoxyoct o n a t e . Legionella pneumophila.

Lpl LPS mAb PBS SDS TLC

= = = = = =

L. p n e u m o p h i l a s e r o g r o u p I. lipopolysaccharide. monoclonal antibody. p h o s p h a t e - b u f f e r e d saline. s o d i u m dodecyl sulphate. thir~ layer c h r o m a t o g r a p h y .

LPS A N T I G E N I C D E T E R M I N A N T OF LEGIONELLA

1079

lipid A in classical endotoxins (Otten et ai., 1986; Moss et al., 1977; Finnerty et al., 1979). In addition, the endotoxicity in Legionella differed from the classical endotoxicity of Gram-negative bacteria in that it had very low biological activity (weak pyrogenic response in rabbits and low toxicity for mice) and a relatively high activity in gelation of the limulus amoebocyte lysate (Cieselski et al., 1986; Wong et al., 1979; Para et aL, 1985). It was therefore hypothesized that Legionella LPS may b c a novel type of bacterial LPS (Wong and Feeley, 1984; Gabay and Horwitz, 1985; Otten et al., 1986). The aim of the present study was to confirm that the epitope recognized by mAb II-6-I8 was part of the cell wall LPS, using direct m~thods, and to further characterize the antigenic determinant. Consequently, we proceeded to isolate, purify and partially analyse the antigen by means of chemical and immunochemical methods.

MATERIALS AND METHODS Organisms and culture conditions.

Lpl Philadelphia strain l (ATCC 33 i52), Lpl 2-passaged CB 81-13 virulent strain and Lp3 (ATCC 33155) were cultured and harvested as described previously (Petitjean et al., 1987). Lpl CB 81-13 strain was isolated from the lung of a patient who had died of legionnaires' disease in a Paris hospital. The bacteria were killed by adding 1 070 formaldehyde for ELISA. The suspension was titrated by measuring absorbance at 600 nm. Enzyme immunoassay (EI,ISA).

All incubations were performed at 37°C for 1 h in phosphate-buffered saline (PBS) containing 0.25 070 gelatin (Sigma) and 0.1 070 Tween-20 (Sigma). Indirect immunoassay. The indirect ELISA was performed by coating the bacteria on a 96-well microtitration plate (Immulon, Dynatech Corp.), incubating them with mAb II-6-18 ascitic fluids and then with peroxidase-labelled rabbit anti-mouse immunoglobulins (Ig) (Miles Laboratories), as described previously (Petitjean et al., 1986). For the coating of purified bacterial antigen, we used "Titertek" polyvinyl 96-well microtitration plates. An aliquot of 50 ~d/well of a 50 ~tg/ml antigen solution in coating buffer (0.5 M NaHCO3 pH 9.6) was incubated at 37°C overnight until dessiccated. Indirect sandwich immunoassay. The indirect sandwich ELISA was performed using 96owell microtitration plates (Immulon, Dynatech Corp.). The purified monoclonal IgG were coated by incubating 50 ~d/well of a 5 ptg monoclonal IgG solution per ml PBS. After saturation of the wells with PBS + gelatin + Tween, the bacterial antigen was incubated at 4°C overnight. Dilutions (1/1,000) of polyclonal anti-Lpl rabbit antiserum and peroxidase-

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ET AL.

labelled goat anti-rabbit IgG (Miles Laboratories) were added successively and revealed with ABTS-H202 substrate. Purification of mAb and preparation of an immunoadsorbant. The purification of mAb II-6-18 was described previously (Petitjean et al., 1987). Purified monoclonal IgG (30 mg) were coupled to 6 ml activated "Ultrogel AcA22" (IBF) according to the process recommended by the producer. The affinity gel was stored in PBS containing 0.02 °70 sodium azide at 4°C in the dark. Purification of the antigen by immunoaffinity. The II-6-18-AcA22 affinity gel was used (1) to purify the antigen from the saline supernatant of the bacteria and (2) as an additional purification step to separate the antigenic fraction from the polysaccharide part of Lp LPS. PBS was used as loading and washing buffer. The elution buffer was 0.2 M glycine-HCl pH 2.7. The antigen was incubated overnight at room temperature with constant recirculation in the gel. The immunoadsorbant was washed successively with 10 volumes PBS, 3 ml PBS containing 1 M NaCl, 10 volumes PBS, 3 ml glycine-HCl and 10 volumes PBS. The serological activity of the pass-through and the different fractions was checked by indirect sandwich ELISA. Isolation and purification of the antigen by chemical methods. The hot phenol/water LPS extraction was performed according to Westphal and Jann (1965). Tris-EDTA extraction of LPS was performed as described by Darveau and Hancock (1983), with an increase in pH at certain steps because of the relative insolubility of the material. PAGE. SDS-PAGE was performed as described by Laemli and Favre 0973) using a 4 070 stacking gel and a l0 or 12.5 070 separating gel. Samples were denatured by boiling 5 min in 2 070SDS 5 070~-mercaptoethanol sample buffer. Migration was performed in Tris-glycine-SDS buffer. The gels were stained using either Coomassie blue or the periodic acid/silver staining described by Tsai and Frash (1982). A commercial E. coli LPS preparation (Malinckrodt Inc.) was loaded as the control. Chemical analysis of the antigen. Mild acid hydrolysis o f LPS. - - LPS was hydrolysed with I °70 acetic acid at 100°C for 1 h and 30 min. The sediment and supernatant were separated by centrifugation at 6,700 g (SS34, Sorvall) for 15 min and then lyophilized. KDO, hexose and rhamnose determinations. - - KDO was determined using the microassay described by Karkhanis et al. (1978), hexose by the anthrone test according to Shields and Burnetts (1960), using glucose or rhamnose as standards. Rhamnose was determined using the cysteine-H2SO4 assay (Kabat and Mayer, 1961). Quantitative determination o f the neutral sugars by gas-liquid chromatography (GLC). - - The polysaccharide fraction of LPS was hydrolysed with 0.1 N HC! at

100°C for 48 h. After hydrolysis, a defined quantity of galactose was added as internal standard. The sample was neutralized and the alditol acetates were prepared from

LPS ANTIGENIC

DETERMINANT

O F LEGIONELLA

1081

the aldoses according to Sawardeker et ah (1965). A chloroform solution of alditol acetates was injected into the gas chromatograph. The analyses were performed using a gas chromatograph equiped with a flame ionization detector and a capillary glass column filled with 1.5 % "Silar 7 CP". The carrier gas was nitrogen. The chromatograph was run at 1900C. Quantitative determination o f the amino sugars by GLC. - - The polysaccharide fraction of LPS was hydrolysed overnight at 100°C with 4 N HCI. The amino sugars were N-acetylated and the samples were analysed by GLC as above. Thin layer chromatography (TLC). ~ Polysaccharides were hydrolysed with 0.1 N HCI at 100°C for 48 h. TLC was performed using precoated 20x 20 cm silica gel 60 thin-layer plates (Merck). The solvant used was water-saturated phenol with 1% NH4OH. The chromatograms were stained with ninhydrin (Condsen et al., 1944), anilin phtalate (Partridge, 1949) or silver. High voltage electrophoresis ( H V E ) on paper. - - Electrophoreses were run on "Schleicher and Schul12043a paper" under 150 mA for 60 to 90 rain. The electrolyte was composed of pyridin, acetic acid and water (10/4/86). The temperature was maintained between 10 and 200C. The electrophoregrams were stained with alcaline silver nitrate (Trevelyan et al., 1950) or Elson-Morgan reagent (Okhuma, 1963). Periode oxidation. - - Four mg of the polysaccharide fraction were dissolved in 1 ml PBS, and sodium periodate was added to a final concentration of 0.05 M. After 4 da'ys at 4°C, the reaction was stopped by adding 0.4 ml ethylene glycol. The mixture was fractionated on a prepacked "Sephadex G25" column and the polysaccharide fraction was collected and lyophilized. A m i n o acid analysis. ~ Amino-acid analyses were performed using a "Kontron analyser". Nucleic acid determination. - - Nucleic acid contamination was determined by measuring the absorbance at 260 nm of a 100 i~g/ml sample solution in 0.01 N NaOH and referring to an RNA standard curve.

RESULTS

Isolation and purification of the LPS by chemical means. The extraction of L e g i o n e l l a LPS using hot phenol/water (Westphal and Jann, 1965) yielded, in the water phase, 0.3 ~g material per mg cell dry weight for Lpl CB 81-13 strain, 1.2 ~g for Lpl Philadelphia strain 1 and 1.0 ~tg for Lp3. We did not attempt to isolate LPS from the phenol phase. Extracts of serogroup 1 bacteria gave a positive reaction when tested against mAb II-6-18 in indirect sandwich-type ELISA. Lp3 LPS did not react with mAb II-6-18 in the same assay. Tris-EDTA extraction accompanied by several enzymatic digestions and followed by purification steps according to Darveau and Hancock (1983) yielded 32 ~g material per mg cell dry weight for Lpl CB 81-13 strain. The extract was recognized by mAb II-6-18 in direct and indirect sandwich ELISA. This method was used to prepare the material necessary for chemical analysis of the antigen. A f t e r migration in SDS-PAGE and periodic acid/silver staining (Tsai and Frash, 1982), L e g i o n e l l a LPS showed a typical smooth-type LPS ladder-like

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F. P E T I T J E A N E T A L .

A

B

C

D

92"* 66--

45"*

31"*

21.*

14.*

FIG. 1. -- SDS-PAGE analysis of Lpl LPS. The migration was performed in a 12.5 % acrylamide gel. Lanes A, B and D were revealed by periodic acid/silver staining; lane C was stained using Coomassie blue. Lane A = Lpl Philadelphia strain l LPS purified by immunoaffinity. Lanes B and C=Tris-EDTAextracted Lpl CB81-13 strain LPS preparation. Lane D = commercial E. coli LPS preparation (Malinckrodt Inc.).

pattern (fig. 1). The molecular weight range o f the bands was between 20 and 30 kDa as calculated using protein standards. T h e profile o f Legionella L P S differed markedly f r o m that obtained with a commercial E. coil L P S preparation, especially with regard to molecular weight (fig. 1). T h e L p l L P S pattern showed that the polysaccharide moiety o f the molecule was a small polysaccharide, whose O side-chain can be composed o f between 1 to 10 or 12 repeating units, according to the n u m b e r o f distinct bands seen on the gel. Protein staining with Coomassie blue o f a c o m p a n i o n gel showed a single band. T h e molecular weight o f this protein was 29 kDa.

LPS ANTIGENIC

O F LEGIONELLA

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1083

Purification of the antigen by immunoaffinity. Purification of the antigen on the mAb II-6-18-AcA22 affinity gel was performed as a control from the saline supernatant of the Lpl Philadelphia strain 1 bacteria. After overnight incubation of the supernatant in the gel and collection of the pass-through, the column was washed with PBS and with a few ml of a 1-M NaCI solution in order to disrupt non-specific interactions. The gel was washed again in PBS and submitted to low pH (0.2 M glycine/HCl pH 2.7) to elute specifically bound antigen. The pass-through and different fractions collected were checked for their serological activity using mAb II-6-18 in an indirect sandwich-type ELISA. Part of the serological activity was recovered in the pass-through and the 1-M-NaCl-eluted material, but most of it was found in the glycine/HCl-eluted fraction. As in the case of the phenol/water- and Tris-EDTA-extracted LPS, this antigenic fraction migrated in SDS-PAGE to yield a pattern composed of regularly spaced bands between 20 and 30 kDa (fig. 1) after periodic acid/silver staining.

I;t

•

8"/

/

/

Y/ 3

2

o

I - Ioo

rc'l

FIG. 2. -- Serological activity of the whole Lpl LPS molecule (Q) and the polysaccharide (0) and lipid (A) fractions obtained after mild acid hydrolysis, tested in indirect ELISA using mAb 11-6-18.

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F. P E T I T J E A N E T A L .

C h e m i c a l analysis o f the T r i s - E D T A - e x t r a c t e d a n t i g e n .

Mild acid hydrolysis usually cleaves LPS at the acid-labile KDO linkage to release the polysaccharide (core and O side-chain) which is soluble, and the lipid part (lipid A) which precipitates. As expected, mild acid hydrolysis of the antigen yielded a soluble and an insoluble fraction (supernatant and sediment, respectively). After separation by centrifugation, lyophilization and weighing, the supernatant represented 51 °70 (wt/wt) of the material obtained and the sediment 49 o70 (mean value of 5 experiments). Both fractions were tested against mAb II-6-1 8 by indirect and inhibition ELISA. Serological activity was found only in the supernatant (fig. 2) which was positive in both types of ELISA. The results of the chemical analysis of the antigen are summarized in table I. Amino acid analysis of both fractions showed that the sediment contained most of the protein (1 1.3 070 wt/wt) and few amino sugars (0.2 %), while the supernatant had a lower protein content (1.4 070) but more amino sugars (3.7 07o). KDO determination showed that the whole preparation contained 1.7 070 KDO (wt/wt) and the polysaccharide fraction, 2.4 070.Anthrone tests using glucose as a standard demonstrated that the supernatant contained twice as much hexose (5.9 070 wt/wt) than the whole antigen ( 3 . 1 % ) , confirming that this fraction contained the polysaccharide part of the molecule. The amount of total hexose in the polysaccharide fraction determined by the anthrone test was however significantly higher (8.3 070 wt/wt) when rhamnose was used as the standard. Analysis of the sugar and amino sugar content of the polysaccharide fraction was performed by TLC, GLC, HVE and amino

TABLEI. - - Chemical composition of Lpl CB 81-13 strain LPS. Whole LPS

Polysaccharide fraction

Lipid fraction

(a) (b) (c) (d) (d) (e) (f)

31 ND ND ND ND 17 12

59 83 37 7 12 24 24

ND ND ND ND ND ND 2

(f) (f) (g)

6 53 ND

13 14 50

0 157 ND

Compounds Hexose Rhamnose Mannose KDO Glucosamine Aminodideoxyhexose XI Protein Nucleic acid

The results are expressed as ~g/mgsample.ND= not determined.The letterin bracketsrefersto the analyticalmethodused:(a)=anthronetest withglucoseas standard;(b)=anthronetest withrhamnose as standard;(c)= cysteine-H2SO4assayfor rhamnose;(d)= GLC;(e)= colorimetricr,~icroa~sayfor KDO; (f)= aminoacid analysis;(g)= spectrophotometricdetermination.

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acid analysis. The polysaccharide was shown to be composed of two sugars (rhamnose-and mannose) and two amino sugars (glucosamine and an unidentified amino sugar). No heptose could be detected. An additional fast-migrating spot was sometimes observed after TLC or HVE analysis of the hydrolysed polysaccharide fraction. This component was not identified but may be an additional unidentified amino sugar. The relative amount of glucosamine and fucosamine was 2/1 as shown by amino acid analysis. The relative amount of rhamnose and mannose in GLC was approximatively 3/5. However, there was an important discordance between the amount of rhamnose in the polysaccharide fraction determined by the cysteine-H2SO4 assay (3.7 °70 wt/wt) or GLC (0.7 070). Periodate oxidation of the polysaccharide decreased the serological activity in ELISA, Gas chromatographic analysis of the material after periodate oxidation showed that rhamnose was unchanged but mannose was destroyed. Amino acid analysis showed that glucosamine almost completely disappeared while the unidentified amino sugar was only partially destroyed. The amino sugars of the polysaccharide part of Lpl LPS analysed by TLC (table lI) resolved in 2 spots, one whose migration rate (0.26) was close to glucosamine and galactosamine (0.26 and 0.27, respectively), and the other whose migration rate (0.42) was close to fucosylamine and fucosamine (0.43 and 0.41, respectively). When the aminosugars of Lpl LPS and two polysaccharities of E. coli (O4-specific LPS and K87-specific capsular polysaccharide) known to contain both glucosamine and N-acetyl-L-fucosamine (Jann et al., 1971 ; Schmidt et al., 1983) were co-analysed by TLC or HVE, similar migration patterns were observed (table II). In amino acid analysis, the retention time of the unidentified amino sugar relative to glucosamine differed slightly from that of fucosamine (1.18) but was the same as quinovosamine (1.12).

TABLEII. -- TLC and HVE analysis of the amino sugars present in the polysaccharide part of Lpl LPS and in O4-specific LPS and K87-specific capsular polysaccbaride of E. cog, both known to contaia glucosamine and N-acetyI-L-fucosamiue. Polysaccharide or amino sugar Lpl PS 04 LPS K87 PS Glucosamine Galactosamine Fucosylamine Fucosamine

Migration rates in TLC Spot l Spot 2 0.26 -0.25 0.26 0.27 ---

0.42 -0.38 --0.43 0.41

Migration rates in HEV Spot l Spot 2 0.62 0.62 0.61 0.62 --m

0.65 0.66 0.65 -m ---

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lmmunochemical analysis of the antigen. As mentioned above, the epitope recognized by mAb II-6-18 was shown to be part of the polysaccharide moiety of the LPS molecule (fig. 2), and the serological activity decreased after periodate oxidation, which resulted in the destruction of mannose and glucosamine residues and the partial cleavage of the unidentified amino sugar, without any change in the rhamnose content. Different sugars, substituted sugars and amino sugars were tested in inhibition of mAb binding to the antigen in ELISA. No inhibition was found with D-mannose, c~-Me-D-mannoside, L-fucose and L-rhamnose. Among the different amino sugars tested (glucosamine, N-acetyl-glucosamine, galactosamine, L-fucosylamine and N-acetyl-D-fucosamine), only L-fncosylamine and N-acetyl-D-fucosaminewere inhibitory with an IC50 of 28.6_+ 6.1 x 10-3 and 20.1 _+9.8 x 10- 3 M, respectively. The O4-specific LPS and the K87-specific capsular polysaccharide of E. coli, which both contain N-acetyl-L-fucosamine (Jann et al., 1971 ; Schmidt et al., 1983) did not inhibit the ELISA.

DISCUSSION In a previous paper (Petitjean et al., 1987), we reported data suggesting that the epitope recognized by mAb II-6-18 was part of the polysaccharide moiety of Lpl LPS and included a fucosylamine-likeresidue. In order to confirm this hypothesis based on the results obtained by means of a fluorometric binding assay, we attempted to isolate, purify and analyse this molecule. The demonstration (1) that the antigenic determinants recognized by mAb 11-6-18 and CDC mAb2 were identical (Petitjean et aL, 1987), and (2) that the presence of the mAb2-specific epitope on Lpl could be considered as a virulence marker (Dournon et al., 1988) provided the impetus for further study and characterization of this antigenic determinant. After control by means of inhibition ELISA of the identity between the antigenic determinant present on both the Lpl Philadelphia strain 1 and the Lpl CB 81-13 virulent strain, we chose to use the latter for further characterization of the epitope. Indeed, in order to perform experiments on a pathogenic microorganism, we preferred to use a virulent clinical strain (such as the 2-passaged CB 81-13 strain), rather than a multipassaged laboratorymodified non-virulent strain (such as the Philadelphia strain 1).

Isolation and purification of the antigen. We tried at first to isolate LPS by the classical hot phenol/water procedure (Westphal and Jann, 1965), but only a very little amount of material (0.3, 1.2 and 1.0 Izg per mg cell dry weight for Lpl CB 81-13, Lpl Philadelphia 1 and Lp3, respectively) was recovered in the water phase. We did not

LP~ ANTIGENIC

DETERMINANT

O F LEGIONELLA

1087

attempt to isolate LPS from the phenol phase. These results are in agreement with those obtained by several authors using the same method who found little or no LPS detectable in the water phase (Gabay and Horwitz, 1985; Cieselski et al., 1986; Conlan and Ashworth, 1986; Nolte et al., 1986; Otten et al., 1986) but in some cases were able to isolate the molecule from the phenol phase (Conlan and Ashworth, 1986; Otten et al., 1986). The isolates from Lpl CB 81-13 and Philadelphia 1 were recognized by mAb II-6-18 in indirect sandwich ELISA, but the isolates from Lp3 were not. The second procedure we used was the Tris-EDTA extraction method described by Darveau and Hancock (1983). The extract from Lpl CB 81-13 strain obtained by this method was recognized by mAb II-6-18 in direct and indirect sandwich ELISA, This technique yielded more material than the first, with 32 ~tg per mg cell dry weight for Lpl CB 81-13 strain, i.e. 0.54 ~tg KDO per mg cell dry weight (mean value of 6 experiments). The yield was in the same range as those obtained by Cieselski et al. (0.008 to 0.726 izg KDO per mg cell dry weight) (Cieselski et al., 1986), but was lower than that obtained by Gabay and Horwitz (2.28 ~tg per mg cell dry weight) who used the same procedure with some modifications (Gabay and Horwitz, 1985). As a control, the antigen was purified from the saline supernatant of Lpl bacteria using an affinity gel prepared with mAb II-6-18. Indeed, we previously reported the presence of soluble antigen in the saline supernatant of Lp (Petitjean et al., 1987). The serological activity of the fractions eluted from the affinity column was controlled again in ELISA and most of it was recovered in the glycine/HCl-eluted fraction. $DS-PAGE analysis of the antigen. Each of the LPS preparations, obtained either by the hot phenol/water procedure, Tris-EDTA or immunoaffinity gave the same SDS-PAGE pattern after periodic acid/siver staining. This ladder-like pattern (fig. 1) is known to be the typical smooth-type LPS migration profile and is considered to be due to the heterogeneity of LPS preparations, the different bands corresponding to LPS molecules with different numbers of repeating units in their O sidechain (Mayer, 1985). Several authors observed a similar migration profile for Lp LPS (Petitjean et al., 1984; Gabay and Horwitz, 1985; Nolte et al., 1986; Otten et al., 1986; Petitjean et al., 1987). The molecular weight range of the different bands (fi~,. 1) was between 20 and 30 kDa (approximation according to protein standards). About 10 to 12 bands were seen, indicating that the Lpl LPS O si~.e-chain ought to be a small polysaccharide containing between 1 to 10 or 12 repeating units. Figure 1 also shows that our Lpl LPS preparation was contaminated (5.3 °70wt/wt) by a single 29-kDa protein species as reveale,: by Coomassie blue staining. Gabay and Horwitz (1985) also found a single protein contamination (2 070wt/wt) in their preparation whose molecular weight was 28 kDa. They demonstrated that this 28-kDa protein was the major outer membrane protein and also the most abundant protein in Lp,

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F. P E T I T J E A N E T A L .

and that it was exposed at the surface of the cell and functioned as a porin (Gabay and Horwitz, 1985; Gabay et al., 1985). Barthe et aL (1988) also reported that a 29-kDa protein was tightly bound to Legionella LPS. Like the data obtained by other authors (Barthe et aL, 1988; Gabay and Horwitz, 1985; Cieselski et al., 1986; Colan and Ashworth, 1986; Nolte et al., 1986; Otten et aL, 1986), our results showed that Lp LPS was smooth in type according to its SDS-PAGE profile but nevertheless behaved as a hydrophobic molecule. In addition, variation in the physicochemical parameters during binding, enabled us to characterize the hydrophobic interaction between mAb II-6-18 and Lpl (Petitjean et al., 1987). The hydrophobicity of Lp LPS may be due to the presence of deoxysugars and deoxyamino sugars in the polysaccharide chain (Conlan and Ashworth, 1986; Nolte et aL, 1986), the high lipid content, and to its unusual fatty acid composition characterized by the predominance of branched-chain fatty acids and the absence of the hydroxy fatty acids generally associated with lipid A in classical endotoxins (Otten et al., 1986; Moss et aL, 1977; Finnerty et aL, 1979).

Chemical analysis of the LPS polysaccharide moiety. Mild acid hydrolysis of the antigen followed by chemical analysis of the two fractions obtained (supernatant and pellet) showed that the pellet which contained the fatty acids and protein corresponded to the lipid part of the molecule, while the supernatant which included the sugars and amino sugars contained the polysaccharide moiety. The presence of the characteristic component KDO in the molecule (1.7 °70 wt/wt in the whole preparation) is a confirmation of the lipopolysaccharide nature of the antigen. Since serological tests by indirect and inhibition ELISA showed that the epitope was part of the polysaccharide moiety of the molecule, our interest during chemical analysis focussed mainly on this fraction of the molecule. The results obtained using TLC, GLC, HVE and amino acid analysis allowed us to determine the sugar and amino sugar composition (rhamnose, mannose, glucosamine and an unidentified amino sugar) of the polysaccharide part of the molecule. No heptose was detected in our preparation. The unidentified amino sugar most likely corresponds to the unusual 2-aminodideoxyhexose X1 of unknown structure found in Lp (Otten et al., 1986; Walla et al., 1984; Fox et al., 1984). Our results are compatible with the carbohydrate composition of whole Lp cells (Walla et al., 1984; Fox et al., 1984a, b) and with the sugar composition of Lp LPS as determined by Otten et al. (1986). However, the latter found an additional unidentified sugar X2 in the molecule. According to previous data (Petitjean et al., 1987) concerning the antigenic determinant recognized by mAb II-6-18, aminodideoxyhexose X1 is a fucosylamine-like residue which is the immunodominant residue in the epitope. However, the relationship between the immunodominant residue and fucosylamine is only a structural analogy because the latter cannot be integrated into a polysaccharide chain. In amino acid analysis, aminodideoxyhexose X1

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and fucosamine had retention times relative to glucosamine which were slightly different (1.12 and 1.18, respectively). When the amino sugar composition of Lpl LPS polysaccharide was analysed by TLC (table II), 2 distinct spots were seen: one had a migration rate (0.26) close to glucosamine and galactosamine (0.26 and 0.27, respectively), the other had a migration rate close to fucosamine (0.42 vs. 0.41). In addition, when the amino sugar composition of Lp 1 polysaccharide and two E. coil polysaccharides (O4-specific LPS and K87-specific capsular polysaccharide) containing both glucosamine and N-acetyl-L-fucosamine (Jann et al., 1971; Schmidt et al., 1983) were coanalysed using either TLC or HVE, similar migration patterns were obtained (table II). The relative amounts of the 2 amino sugars present in the poly~accharide fraction as calculated from the amino acid analysis spectrum was two glucosamine for one aminodideoxyhexose X1 residue. The relative amounts of rhamnose and mannose determined by GLC was 3/5, but the true ratio could also be 1/1 because rhamnose can be partially lost by evaporation during the procedure used to prepare alditol acetates. Indeed, the rhamnose content as determined using the cysteine-H2SO4 assay was much higher (table I). Otten et al. (1986) found approximately the same amount of rhamnose and mannose in Lpl LPS.

lmmunochemical analysis of the antigen. The serological tests (direct and inhibition ELISA) showed that the epitope recognized by mAb II-6-18 was localized in the polysaccharide part of the LPS. The serological activity of the polysaccharide in ELISA was significantly lower after periodate oxidation (data not shown). This result is in agreement with our previous observation (Petitjean et al., 1987) that the serological activity of the soluble antigen in the saline supernatant of the bacteria decreased after periodate oxidation. Periodate oxidation resulted in the destruction of mannose and glucosamine, but the rhamnose content was unchanged. Otten et al. (1986) also reported that mannose residues in Lp LPS were completely destroyed after periodate oxidation. Aminodideoxyhexose XI was only partially sensitive to periodate oxidation, which suggests that XI residues could be present in at least two different sites in the molecule, one inside the chain and the other at the end of the chain. In order to define which sugar and/or amino sugar residues were directly implicated in recognition, inhibition of mAb binding to LPS was performed using different sugars (D-mannose, ct-Me-D-mannoside, L-fucose and L-rhamnose) and amino sugars (glucosamine, galactosamine, N-acetylglucosamine, L-fucosylamine and N-acetyl-D-fucosamine), and with other microbial polysaccharides (O4-specific LPS and K87-specific capsular polysaccharide of E. coh) both containing a N-acetyl-L-fucosamineresidue in their chain (Jann et al., 1971 ; Schmidt et al., 1983). The fact that none of the sugars present in the molecule was able to inhibit the binding does not neces-

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sadly mean that they do not contribute to the serological activity. It could also be due to the fact that a monosaccharide is not sufficient to obtain detectable inhibition. Disaccharides may give better results, but specific disaccharides are not usually available. Among the amino sugars tested, only L-fucosylamine and N-acetyl-D-fucosamine inhibited binding. Their ICs0 were 28.6_+6.1 × 10 -3 and 20.1 _+9.8x 10 -3 M, respectively. The IC50 for fucosylamine determined here by ELISA was slightly higher than the value obtained previously (1.7 _+0.5 × 10- 2 M) using a fluorometric assay (Petitjean et ai., 1987). This difference may reflect the greater sensitivity of the fluorometric assay compared to ELISA. The fact that a single residue (fucosylamine or N-acetyl fucosamine) was able to inhibit the binding means that this residue is probably localized at the end of the polysaccharide chain (main or side chain), and could be considered to be the immunodominant residue. Thus, the immunodominant residue in the epitope is probably fucosamine-like (fucosylamine cannot be integrated into a polysaccharide chain). Both E. coli polysaccharides tested in inhibition, each containing N-acetyl-L-fucosamine inside the chain (not at the end), had no effect on binding. This result is in agreement with the conclusion that the immunodominant residue should be localized at the end of the chain. To summarize, the results of chemical and immunochemical analysis of Lpl LPS (1) confirm that the antigenic determinant recognized by mAb II-6-18 is localized in the polysaccharide moiety of LPS, (2) show that the latter contains rhamnose, mannose, glucosamine, an unidentified aminodideoxyhexose XI and KDO, but no heptose, (3) suggest that the immunodominant residue in the epitope is aminodideoxyhexose X1, localized, at least partially, at the end of the polysaccharide chain, and (4) allow us to hypothesize that aminodideoxyhexose X I may be fucosamine or a structurally similar residue. In addition, the present work confirms the unusual character of Legionella LPS which is smooth in type but nevertheless hydrophobic. More work is needed in order to identify the structure of aminodideoxyhexose XI, to define the other sugar components of Lp LPS from among those which are part of the epitope, and to determine the whole structure of the antigenic determinant. Further characterization at the molecular level of Legionella LPS, its antigenic determinants and the relationships between these structural features and the biological properties, is of interest especially since LPS has been shown to play a major role in antibody response both in patients (Otten et al., 1986) and in animals (Friedman et al., 1987), and since some of the clinical manifestations of legionnaires' disease may be endotoxinmediated. The LPS and, in particular, the epitope described here could be implicated in the virulence of the organism as suggested by epidemiological and laboratory data (Dournon et al., 1988; Plouffe et al., 1983, 1985 ; Bollin et al., 1986; Molina et al., 1987; Dournon and Rajagopalan, 1987).

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RI~SUMI~ ISOLEMENT,PURIFICATIONET ANALYSEPARTIELLE DU DETERMINANTANTIGi~NIQUELIPOPOLYOSIDIQUE RECONNUPAR UN ANTICORPSMONOCLONAL ANTI-LEGIONELLAPNEUMOPHILASI~ROGROUPE1 L'anticorps monoclonal (AcM) II-6-18 reconnait un d6terminant antig6nique de

Legionellapneumophila sp6cifique du s6rogroupe 1, dont i'association avecla virulence a 6t6 d6montr6e. Dans une publication ant6rieure, nous avons rapport6 ia caract6risation physico-chimique par fluorim6trie quantitative de ia liaison de I'AcM 11-6-18 L. pneumophila et ses implications concernant la nature de l'antig6ne; nous d6crivons ici l'isolement et la purification de l'antig6ne, par des m6thodes chimiques et immunochimiques, suivis de son analyse chimique partiell¢. Les r6sultats obtenus montrent que l'6pitope - - un hydrate de carbone immunodominant comprenant un r6sidu analogue A la fucosamine - - est port6 par le lipopolyoside (LPS) de la bact6rie. II est localis6 dans la partie polyosidique de cette mol6cule comprenant du KDO, du rhamnose, du mannos¢, de la glucosamine et un aminodid~soxyhexose XI de structure non identifi6e, mais ne contenant pas d'heptose. Le r~sidu X 1, qui pourrait ~tre un r6sidu fucosamine, est vraisemblablement le r6sidu immunodominant dans l'6pitope; il est localis6 au moins partiellement/t l'extr6mit6 de la chaine polyosidique. MOTS-CL~S: Legionella pneumophila, Epitope, LPS; Virulence, Fucosamine, Immunodominance.

ACKNOWLEDGEMENTS The authors thank Dr. P. Rajagopalan for Legionella cultures, Dr. B. Jann and Dr. K. Jann for their help in the chemical characterization of Legionella LPS and for reviewing the manuscript, Dr. H. Mayer and Dr. N. Sharon for providing fucosamine and N-acetylfucosamine, Dr. B. Vray for polyclonal rabbit antiserum to L. pneumophila serogroup l, and Dr. C. Galanos for helpful discussion. Mr. M. Wiesner is thanked for having performed the amino acid analyses. This work was supported by grants from : CNRS (ATP Hybridomas), INSERM (Contrat Recherche Externe), Minist6re de la Recherche et de la Technologic, Comit6 National contre les Maladies Respiratoires et la Tuberculose, European Molecular Biology Organization and Max-Planck Geselschaft.

REFERENCES BARTHE, C., JOLY, J.R., RAMSAY,D., BOISSINOT,M. & BENHAMOU,N. (1988), Common epitope on the lipopolysaccharide of Legionella pneumophila recognized by a monoclonal antibody. J. clin. Microbiol., 26, 1016-1023. BOLLIN, G.E., PLOUFFE, J.F., PARA, M.F. & PRIOR, R.B. (1985), Difference in virulence of environmental isolates of Legionella pneumophila. J. clin. Microbiol., 21, 674-676. CIESELSKI, C.A., BLASER, M.J. & WANG, W.L.L. (1986), Serogroup specificity of Legionella pneumophila is related to lipopolysaccharide characteristics. Infect. Immun., 51, 397-404. CONDSEN, R., GORDEN, A.H. & MARTIN, A.J.P. (1944), Qualitative analysis of proteins: partition chromatographic method using paper. Biochem. J., 38, 224.

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CONI.AN, J.W. & ASHWORTH,L.A.E. (1986), The relationship between the serogroup antigen and iipopolysaccharide of Legionellapneumophila. J. Hyg. (Camb.), 96, 39-48. DARVEAU, R.P. & HANCOCK,E.W. (1983), Procedure for isolation of bacterial lipopolysaccharides from both smooth and rough Pseudomonas aeruginosa and Salmonella tiphymurium strains. J. Bact., 155, 831-838. DOURNON, R., BlaB, W.F., RAJAGOPALAN,P., DESPLACES,N. • McKINNEY, R.M. (1988), Monoclonal antibody reactivity as a virulence marker for Legionella pneumophila serogroup 1 strains. J. infect. Dis., 157, 496-501. DOURNON,E. & RAJAGOPALAN,P. (1987), Correlation between monoclonal antibody pattern, serum sensitivity and intracellular growth of Legioneila pneumophila serogroup 1 strains, in "Abstracts of the 87th Annual Meeting of the American Society for Microbiology". American Society for Microbiology, Washington, D.C. EHRET, W. & RUCKDESCHEL,G. (1985), Membrane proteins of Legionellaceae I. m Membrane proteins of different strains and serogroups of Legionella pneumophila. Zbl. Bakt. Hyg., A 259, 433-445. FINNERTY,W.R., MAKULA,R.A. & FEELEY,J.C. (1979), Cellular lipids of the legionnaires' disease bacterium. Ann. Int. Med., 90, 631-634. Fox, A., LAU, P.Y., BROWN, A., MORGAN, S.L., ZHU, Z.T., LEMA, M. & WALl.A, M.D. (1984), Capillary gas chromatographic analysis of carbohydrate of Legionella pneumophila and other members of the family Legionellaceae. J. clin. Microbiol., 19, 326-332. Fox, A., LAU,P.Y., BROWN,A., MORGAN,S.L., ZHU, Z.T., LEMA,M. & WALLA,M. (1984), Carbohydrate profiling of some Legionellaceae by capillary gas chromatography-mass spectrometry, in "'Legionella, Proc. 2nd Int. Symp." (C. Thornsberry, A. Balows, J.C. Feeley & W. Jakubowski) (pp. 71-73). American Society for Microbiology, Washington D.C. FRIEDMAN, H., NEWTON, C., BLANCHARD, D.K., KLEIN, T.W., WIDEN, R. & WONG, K.H. (1987), lmmunogenicity and adjuvanticity of lipopolysaccharide from Legionella pneumophila (424666). Proc. Soc. exp. Biol. (N.Y.), 184, 191-196. GABAY, J.E. & HORWlTZ, M.A. (1985a), Isolation and characterization of the cytoplasmic and outer membranes of the legionnaires' disease bacterium (Legionella pneumophila). J. exp. Med., 161,409-422. GABAY, J.E., BLAKE,M., NILES, W.D. & HORWITZ,M.A. (1985b), Purification of Legionella pneumophila outer membrane protein and demonstration that it is a porin. J. Bact., 162, 85-91. GOSTING, L.H., CABRIAN,K., STURGE,C. & GOLDSTEIN,L.C. (1984), Identification of a species specific antigen in Legionella pneumophila by a monoclonal antibody. J. clin. Microbiol., 20, 1031-1035. GUILLET, J.G., HOEBEKE,J., TRAM, C., MARULLO,S. & STROSBERG,A.D. (1983), Characterization, serological specificity and diagnostic possibilities of monoclonal antibodies against Legionella pneumophila. J. clin. Microbiol., 18, 793-797. JANN, B., JANN, K. & SCHMIDT,G. (1971), Comparative immunochemical studies of the surface antigens of Escherichia coli strains O8:K87(B?):HI9 and (O32):K87(B?):H45. Europ. J. Biochem., 23, 515-522. KABAT,E.A. & MAYER,M.M. (1961), Methylpentose determination, in "Experimental Immunochemistry" (538-541). C.C. Thomas, Springfield, IL. KARKHANIS,Y.D., ZELTNER,J.Y., JACKSON,J.J. & CARLO,D.J. (1978), A new improved microassay to determine 2-keto-3-deoxyoctonate in lipopolysaccharides of Gram-negative bacteria. Analyt. Biochem., 85, 595-601. LAEMtl, U.K. & FAVRE, M. (1973), Maturation of the head of bacteriophage T4. J. Mol. Biol., 80, 575-599. MAYER,H. (1985), Analysis of lipopolysaccharides of Gram-negative bacteria. Meth. Microbiol., 18, 156-207.

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McKINNEY, R.M., THACKER, L., WELLS, D.E., WONGS, M.C., JONES, W.J. & BIBB, W.F. (1983), Monoclonal antibodies to LegioneUa pneumophila serogroup 1 : possible applications in diagnostic tests and epidemiolog~cal studies. Zbl. Bakt. Hyg., I Abt. Orig. A 255, 91-95. MOLINA,J.M., RAJAOOPALAN,P. & DOURNON,E. (1987), Monoclonal antibody pattern of Legionella pneumophila serogroup 1 associated with virulence in guinea-pigs, in "Abstracts of the 87th Annual Meeting of the American Society for Microbiology". American Society for Microbiology, Washington, D.C. Moss, C.W., WEAVER,R.E., DEES, S.B. & CHERRY,W.B. (1977), Cellular fatty acid composition of isolates from legionnaires' disease. J. clin. Microbiol., 6, 140-143. NOLTE, F.S., CONUN, C.A. & MOTLEY,W.A. (1986), Electrophoretic and serological characterization of the lipopolysaccharides of Legionella pneumophi!a. Infect. Immun., 52, 676-681. OKHUMA,S. (1963), Spray methods for the detection of 2-amino-2-deoxyhexosesand their N-acety, derivatives on paper chromatograms. Proc. Jap. Acad., 39, 400-405. OTTEN, S., IYER,S., JOHNSON,W. & MONTGOMERY,R. (1986), Serospecific antigens of Legionella pneumophila. J. Bact., 167, 893-904. PARA, M.F., PLOUFFE,J.F. & KRAViTZ,G.R. (1985), Legionella endotoxin: relationship of altered lipid A structure to biological activity. Abstracts of the 24th ICAAC, 150. PARTRIDGE,S.M. (1949), Aniline hydrogen phtalate as a spraying reagent for chromatography of sugars. Nature (Lond.), 164, 443. PETITJEAN, F., GUILLET,J.G., VRAY, B., TRAM, C., HOEBEKE,J. & STROSBERG, A.D. (1984), Standardization for diagnostic purposes of a monoclonal antibody against Legionella pneumophila. Develop. Biol. Standard., 57, 99-105. PETITJEAN, F., GUILLET,J.G., VRAY, B., HOEBEKE,J. & STROSBERG,A.D. (1986), Legionellapneumophila, in "Methods of enzymatic analysis" (H.U. Bergmeyer) Xi (pp. 173-88). V.C.H., Weinheim. PETITJEAN,F., GUILLET,J.G., VRAV,B., STROSBERG,A.D. & HOEBEKE,J. (1987), Partial characterization of a Legionella pneumophila serogroup 1 immunodominant antigenic determinant recognized by a monoclonal antibody. Comparative ImmunoL, Microbiol. Infect. Dis., 10, 9-23. PLOUFFE, J.F., PARA, M.F., MAHER,W.E., HACKMAN,B. & WEBSTER,L. (1983), Subtypes of Legionellapneumophila serogroup 1 associated with different attack rates. Lancet, II, 649-650. PLOUFFE, J.F., PARA, M.F. & FULLER,K.A. (1985), Serum bactericidal activity against Legionella pneumophila. J. clin. Microbioi., 22, 863-864. SAWARDEKER,J.S., SLONEKER,J.H. & JEANES,A. (1965), Quantitative determination of monosaccharides as their alditol acetates by gas chromatography. Analyt. Chem., 37, 1602-1604. SCHMIOT,M.A., JANN, B. & JANN, K. (1983), Cell-wall lipopolysaccharide of the urinary tract infective Escherichia coli O4:KI2:H-. Structure of the polysaccharide chain. Europ. d. Biochem., 137, 163-171. SHIELDS, R. & BURNETT,W. (1960), Determination of protein-bound carbohydrate in serum by a modified Anthrone method. Analyt. Chem., 32, 885-886. TSAI, C.M. & FRASH,C.E. (1982), A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Analyt. Biochem., 119, 115-119. TREVELYAN,W.E., PROCTER,D.P. & HARRISON,J.S. (1950), Detection of sugars on paper chromatograms. Nature (Lond.), 166, 444-445. WALLA,M.D., LAU, P.Y., MORGAN,S.L., Fox, A. & BROWN,A. (1984), Capillary gas chromatography-mass spectrometry of carbohydrate components of Legioneila and other bacteria. J. Chromat., 288, 399-413.

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F. P E T I T J E A N E T A L .

WESTPHAL, O. ~,r JANN, K. (1965), Bacterial lipopolysaccharides: extraction with phenol-water and further applications of the procedure. Meth. Carbohydr. Chem., $, 83-91. WONG, K.H., SHALLA,W.O., ARKO,R.J., BULLARD,J.C. & FEELEY,J.C. (1979), Immunochemical, serologic and immunologic properties of major antigens isolated from the legionnaires' disease bacterium. Ann. Intern. Med., 90, 634-638. WONG, K.H. & FEELEY,J.C. (1984), Lipopolysaccharide of Legionella as adjuvant for intrinsic and extrinsic antigens. Proc. Soc. exp. Biol. (N.Y.), 177, 475-481.