Interaction of Lipopolysaccharides of Helicobacter pylori with

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1 NFETON AND MMUNTY, Sept. 1994, p /94/$4.+ opyright 1994, Amerian Soiety for Mirobiology Vol. 62, No. 9 nteration of Lipopolysaharides of Heliobater pylori with Basement Membrane Protein Laminin KAJA H. VALKONEN,1 TORKEL WADSTROM,' AND ANTHONY P. MORAN2* Department of Medial Mirobiology, University of Lund, S Lund, Sweden, 1 and Department of Mirobiology, University ollege, Galway, reland2 Reeived 18 Otober 1993/Returned for modifiation 2 January 1994/Aepted 2 June 1994 The ability of hemagglutinating and poorly hemagglutinating strains of the gastroduodenal pathogen Heliobater pylori to bind 125-radiolabelled laminin was quantitated in a liquid phase assay. Although all strains bound laminin, some hemagglutinating strains were good binders of laminin (maximum of 31% binding), whereas poorly hemagglutinating strains bound intermediate to small amounts of laminin (minimum of 6% binding). Sine a hydrophobi omponent of the baterium has been reported to be involved in binding of laminin (T. J. Trust, P. Doig, L. Emody, Z. Kienle, T. Wadstrom, and P. O'Toole, nfet. mmun. 59: , 1991), we investigated the role of lipopolysaharide (LPS) in the interation of both types of strains with laminin. Although the extent of inhibition varied among strains, laminin binding to hemagglutinating and poorly hemagglutinating strains was inhibited with homologous and heterologous smooth-form LPS. The ability of heterologous rough-form LPS to produe inhibition omparable to that shown by smooth-form LPS indiated that the side hain of H. pylorn LPS was not involved in the interation. Further inhibition experiments with dephosphorylated LPS, isolated ore oligosaharide, and free lipid A suggested that a phosphorylated struture in the ore oligosaharide mediates the interation of a hemagglutinating strain of H. pylori with laminin, whereas a onserved nonphosphorylated struture in the ore oligosaharide mediates the interation of a poorly hemagglutinating strain. Furthermore, we showed that the interation of H. pylori LPS with 1251-radiolabelled laminin in a solid phase assay was saturable, speifi, and inhibitable with unlabelled laminin. t was postulated that the initial reognition and binding of laminin by H. pyloni may our through LPS and that subsequently a more speifi interation with a letin-like adhesin on the baterial surfae ours. Heliobater pyloni is the ausal agent of ative hroni gastritis in humans, is assoiated with the development of duodenal ulers in patients, and may be assoiated with gastri aner (27, 33, 34, 42). The baterium is thus an important human pathogen and has been shown to olonize gastri muus and to adhere to muosal ells, espeially at interellular juntions (18, 41). Various binding speifiities and putative tissue adhesins of H. pylori have been desribed, inluding hemagglutination ativities with different speifiities (9, 21, 49), an N-aetylneuraminyllatose-binding fibrillar hemagglutinin (11), binding to GM3 ganglioside and sulfatides (37, 4), and binding to a speies of phosphatidylethanolamine in the gastri muosa (22), but these interations alone may not explain the tissue tropism of H. pyloni. H. pyloni produes ytotoxins and enzymes whih may degrade the epithelium, ausing ellular destrution (33, 41, 42) and exposure of the underlying basement membrane (16). Ultimately, in suh ulerated tissue the various onnetive tissue proteins and extraellular basement membrane strutures ould beome exposed and olonized by H. pyloni. Laminin is a omplex, nonollagenous glyoprotein whih is important for the struture of the basement membrane by its formation of networks with type V ollagen, entatin/nidogen, and heparan sulfate proteoglyans (53). Laminin bears reeptors involved in ell adhesion by normal ells, metastating tumor ells, and ertain pathogeni bateria (15, 23, 43, 44). Surfae-exposed proteins of bateria that bind laminin have * orresponding author. Mailing address: Department of Mirobiology, University ollege, University Road, Galway, reland. Phone: Fax: been shown to exist on Streptoous pyogenes (45) and uropathogeni Esherihia oli (5), and laminin-binding proteins have been identified on Staphyloous aureus (24) and on tissue-invasive E. oli (47). Similarly, strains of H. pylori have been reported to bind to laminin and also to type V ollagen in a speifi and saturable manner (3, 46, 48). The interation of H. pyloni with laminin involves a baterial adhesin reognizing ertain sialylated oligosaharides of the glyoprotein (48). Sine the arbohydrate moiety of laminin is believed to play a role in euaryoti ellular adhesion proesses (3, 6), suh an interation of H. pylori with laminin ould further disorganize the gastri epithelium. Supporting this, H. pyloni was shown to interfere with the interation between laminin and a laminin reeptor in gastri epithelial membranes (39). n partiular, lipopolysaharide (LPS) of H. pyloni an inhibit the interation between laminin and its reeptor on epithelial ells (39), but LPS has reeived only limited attention as a potential tissue adhesin (3). hemially, LPSs are omposed of a poly- or oligosaharide moiety and a lipid omponent, termed lipid A, whih anhors LPS in the outer membrane and endows the moleule with its toxi properties (2, 35). High-moleular-weight smooth-form (S-) LPS onsists of an side hain (whih is a polymer of repeating oligosaharide units), a ore oligosaharide, and lipid A, whereas low-moleular-weight rough-form (R-) LPS laks the side hain (2). Strains produing LPS laking the side hain, beause of defets in biosynthesis of LPS, have proven useful in biologial and strutural studies on the ore oligosaharide and lipid A of those bateria whih normally produe S-LPS (2, 35, 36). The present study was performed to evaluate the possible 364

2 VOL. 62, 1994 role of LPS in the binding of H. pylon to laminin. Sine purified S-LPS was shown to inhibit binding of a number of strains, R-LPS of H. pylon, and derivatives therefrom, were employed to identify the moleular basis within the LPS moleule for binding of laminin. The evidene presented suggests that two different mehanisms mediate the interation of LPS of hemagglutinating and poorly hemagglutinating strains of H. pylon with laminin. MATERALS AND METHODS Baterial strains and growth onditions. H. pylon strains for investigation were seleted from a olletion of hemagglutinating and poorly hemagglutinating strains with differing abilities to bind laminin (48). Four hemagglutinating strains (UG 17874, UG 1916, 1139, and 12225) and two poorly hemagglutinating strains (UG and 33) were used in these binding studies. H. pylon UG and UG (same as NT and NT 11638, respetively [National olletion of Type ultures, London, England]) and UG 1916 (strain Pylo 1 [F. Megraud, entral Regional Hospital, Bordeaux, Frane]) were obtained from the ulture olletion of the University of Goteborg, Goteborg, Sweden. The other strains were isolates from endosopi biopsies obtained at the University Hospital, Lund, Sweden. An additional strain, S-24 (provided by D. Danielsson, Medial enter Hospital, Orebro, Sweden), besides UG 17874, was used for LPS prodution. Stok ultures were maintained at -7 in 15% (vol/vol) glyerol-tryptiase soy broth (BBL Mirobiology Systems, okeysville, Md.). H. pylori strains were grown routinely on blood agar under miroaerobi onditions at 37 for 48 h (29). To obtain H. pylon expressing S-LPS, strains were grown in a broth of brain heart infusion (Oxoid Ltd., London, England) ontaining 2% (vol/vol) fetal alf serum (Oxoid) as desribed by Moran and Walsh (31). solation of LPS. After pretreatment of bateria (about 2 g [dry weight]) with pronase (albiohem, Los Angeles, alif.) (4), LPS was extrated from UG and S-24 by the hot -phenol-water tehnique (51). The LPS preparations were purified by treatment with RNase (Sigma hemial o., St. Louis, Mo.), DNase (Sigma), and proteinase K (Sigma) and by ultraentrifugation as desribed previously (29). Dephosphorylation of LPS. Preparative dephosphorylation of UG R-LPS was performed by treating LPS (2 mg [dry weight]) with 48% aqueous hydrofluori aid (Merk, Darmstadt, Germany) at 4 with onstant stirring for 48 h. The resulting suspension was dialyzed against water, and the retentate (dephosphorylated LPS) was freeze-dried. Preparation of free lipid A and ore oligosaharide. R-LPS of UG was treated with 1% aeti aid at 1 for 1.5 h (29). Free lipid A was preipitated after entrifugation (3, x g, 4, 3 min), washed with water, freeze-dried, and subsequently solubilized by soniation after the addition of triethylamine to ph 8. The supernatant of the hydrolysate was freeze-dried, redissolved in water, and subjeted to gel hromatography on Bio-Gel P-6 (Bio-Rad, Herules, alif.; olumn, 1.2 by 1 m) to obtain purified ore oligosaharide. haraterization of LPS and derivatives. The purities of LPS, free lipid A, ore oligosaharide, and dephosphorylated LPS were verified by hemial analyses. Preparations were examined by previously desribed (29) analytial tehniques and proedures for determining phosphate, 3-deoxy-D-manno- 2-otulosoni aid, neutral sugar, amino sugar, and fatty aids. Radioiodination of laminin. Laminin, purified from Engelbrett-Holm-Swarm transplantable mouse tumor (AMS Biotehnology, Stokholm, Sweden), was labelled with (sodium LAMNN BNDNG BY H. PYLOR 3641 salt; speifi ativity, 14.5 mi/,ug; Amersham nternational, Amersham, England) aording to a modified hloramine-t method using odobeads (26). The speifi ativity of the labelled protein was about 2 x 16 pm/jig. The protein onentration was determined aording to the method of Lowry et al. (25). Binding assays. (i) Liquid phase assays. Binding assays were performed as desribed previously (48). Briefly, baterial growth was harvested from ultures, washed one with phosphate-buffered saline (PBS), and finally adjusted to an A595 of 1. (18 ells per ml). The baterial suspension (1,ul, 17 ells) was mixed with.55 pmol of 1251-labelled laminin in.1 M PBS ontaining.1% (wt/vol) bovine serum albumin (PBS- BSA) and inubated at 2 for 6 min in air or under miroaerobi onditions. Subsequently, bateria were pelleted by entrifugation (2, x g, 4, 15 min) and washed with PBS. The total radioativity and the radioativity of the pellet were determined by onventional sintillation ounting in a gamma ounter (Walla, Turku, Finland). Assays were performed in tripliate, unless stated otherwise, and the radioativity bound in the pellet is expressed as a perentage of the total radioativity added to the sample. To assess the role of LPS in laminin binding by H. pylon strains, inhibition of baterial binding to laminin was investigated with purified S- and R-LPS, dephosphorylated R-LPS, ore oligosaharide and lipid A of H. pyloni UG 17874, and R-LPS of H. pyloni S-24. nhibition assays were performed by inubation of.55 pmol of 125-laminin in PBS-BSA with different amounts of the inhibitors (5 to 3 jig) at 2 for 1 h, followed by addition of 1 jil of baterial suspension (17 ells) and inubation for a further 1-h period. The extent of baterial binding to '25-laminin was determined as desribed above. Also, to determine whether LPS-mediated binding to laminin was restrited to H. pylon, E. oli 26:B6 LPS (Difo Laboratories, Detroit, Mih.) was used as ontrol material in idential inhibition experiments. The effet of an antiuler drug on baterial binding to laminin was investigated with niteapone [3-(3,4-dihydroxy-5- benzylidiene)-2,4-pentadione], whih was kindly provided by P. Pohto, Orion Pharmaeutia, Espoo, Finland. The drug was stored in the dark and before use was suspended in PBS as desribed by Slomiany et al. (39). nhibition experiments were performed in three ways. First, baterial suspension (1 jl, 17 ells) was inubated with different onentrations of niteapone (5 to 5 jig) at 2 for 2 h in the dark and then inubated with.55 pmol of 1251-laminin for 1 h. Seond, under the same inubation onditions,.55 pmol of 125-laminin was inubated with different onentrations of niteapone and subsequently with baterial suspension. Third, S-LPS of UG (1 jig) was inubated with niteapone, and the treated LPS was used to inhibit the interation between 125-laminin and baterial suspension. The resulting ell suspensions from these three inhibition experiments were entrifuged (2, x g, 15 min), the pellets were washed with PBS, and the amount of bound radioativity was determined. To determine the roles of both LPS and protein adhesins in laminin binding by H. pylon, baterial suspensions (1 jil, 17 ells) were heat treated (8, 1 min) or inubated with pronase (5 jig/ml, 37, 1 h) and then inubated with.55 pmol of 1251-laminin for 1 h. Alternatively, the treated ell suspensions were inubated with niteapone (2 jig) before inubation with 125-laminin. The extent of baterial binding to 125-laminin was determined as desribed above. (ii) Solid phase assays. Mirotiter plates (ostar, ambridge, Mass.) were oated with LPS aording to a modifiation of the method of Freudenberg et al. (17). Briefly, an LPS

3 3642 VALKONEN ET AL. NFEr. MMUN. 4 T 3+ )._ 2+ -m n 1± FG. 1. Effets of S-LPS of H. pylon UG on binding of 125-laminin to H. pylon strains. Binding of laminin without preinubation with LPS (_) and after inubation with 1 pug of LPS (FZ) is shown. Data represent the means of three determinations, and error bars indiate standard deviations. oating solution (1 jig/ml) was prepared by mixing 1,ul of LPS stok solution (1 mg/ml) with 9,ul of a hloroformethanol mixture (1:9, vol/vol). This solution was added to wells and evaporated overnight. Thereafter, wells were washed three times with PBS ontaining.1% Tween 2 (PBS-Tween), further inubated with PBS-BSA at 2 for 2 h, and finally washed three times with PBS-Tween. Binding of laminin to LPS-oated plates was estimated by inubation of 125-laminin in oated wells at 2 for 2 h in air or under miroaerobi onditions, followed by three washes with PBS and determination of the amount of bound radioativity by onventional sintillation ounting with a gamma ounter. The speifiity of 1251-laminin binding to immobilized LPS was investigated in two ways. First, nonspeifi binding of 1251-laminin to wells oated with 1% (wt/vol) BSA was determined and used as a ontrol. Seond, inhibition of 1251-laminin binding to LPS-oated wells was determined in the presene of exess unlabelled laminin. Statistial analysis. Student's t test was used to assess the signifiane of differenes between means in binding assays. RESULTS Quantitation of laminin binding to H. pylori strains. Liquid phase assays were used sine they allowed diret assessment of laminin-baterial interation and the formulation of inhibition experiments. dential results were obtained when the assays were performed in air or under miroaerobi onditions. Binding of 1251-laminin by the seleted hemagglutinating and poorly hemagglutinating H. pylon strains varied between 6 and 31% (Fig. 1). Three groups of binding affinities were observed. Some hemagglutinating H. pylori strains bound large to intermediate amounts of laminin (UG and 1139); others bound small amounts (UG 1916 and 12225). Poorly hemagglutinating strains (UG and 33) that were reported previously to bind large amounts of laminin (48) bound small to intermediate amounts of laminin during the more extensive testing of this study. nhibition of laminin binding by H. pyloni with LPS and LPS derivatives. S-LPS of UG 17874, in addition to inhibiting the binding of 125-laminin to the same strain, inhibited binding 6 T 4 %._O._ z O- / T O 2/ S nhibitor (ug) FG. 2. Effets of onentration of H. pylon UG S-LPS on inhibition of binding of 125-laminin to UG (@) and UG () in the liquid phase assay. Data points represent the means of three determinations, and error bars indiate standard deviations. of the protein to other hemagglutinating and poorly hemagglutinating strains (Fig. 1). The extent of inhibition varied among strains. Hemagglutinating strains ould be divided into two groups: those whose laminin binding was signifiantly different after inhibition by LPS (UG [P <.1] and UG 1916 [P <.1]) and those whose inhibition was not statistially signifiant (1139 [P >.1] and [P >.1]). The greatest inhibition of laminin binding (at most 5%), whih was statistially signifiant (P <.1), was observed with poorly hemagglutinating strains (UG and 33). Quantitation of the inhibition of laminin binding to H. pylori as a funtion of inreasing S-LPS onentration showed that the inhibition was saturable for both hemagglutinating and poorly hemagglutinating strains (Fig. 2), and indiated that the interation between LPS and laminin involved a limited number of sites. S-LPS of UG did not inhibit laminin binding to UG 17874, although the onverse ourred (Fig. 1 and 2). Furthermore, LPS of E. oli 26:B26 inhibited binding of laminin to UG in a dose-dependent saturable manner, with maximal inhibition of 2% at 1,ug (Fig. 3) omparable to that obtained with S-LPS of UG (19% at 1 jig [Fig. 2]; not signifiantly different [P >.1]). Laminin binding to UG 17875, however, was not inhibited by LPS of E. oli 26:B26 (Fig. 3). - ~o_ 3 2! 1 o*ee-e---- (D nhibilor (ug) FG. 3. nhibition of binding of '25N-laminin to H. pylori UG () and UG () with E. oli 26:B26 LPS in the liquid phase assay. Data points represent the means of three determinations, and error bars indiate standard deviations.

4 VOL. 62, 1994 LAMNN BNDNG BY H. PYLOR %-O._1-2 /j 1 1 / 1 /l i',..' * nhibitor (ug) FG. 4. Effets of onentration of H. pylori S-24 R-LPS on inhibition of binding of "M-laminin to UG () and UG () in the liquid phase assay. Data points represent the means of three determinations, and error bars indiate standard deviations. The ability of UG S-LPS to ross-inhibit binding of laminin to heterologous strains (Fig. 1 and 2) suggested that strutures ommon to the LPS of a number of H. pyloni strains ould interat with laminin. R-LPS of another H. pylori strain, S-24, was used in inhibition experiments to test this hypothesis, sine R-LPS lak the side hain and sine the ore oligosaharide and lipid A moieties are the regions of LPS more onserved in struture in strains of a given baterial speies (2, 35, 36). The R-LPS of S-24 inhibited binding of laminin to both hemagglutinating and poorly hemagglutinating H. pylori strains (Fig. 4), and the inhibition was omparable to that indued by S-LPS of UG For example, 4% inhibition of binding of UG was observed with 1,ug of S-LPS of UG (Fig. 2), and 43% inhibition of binding was observed with 1,ug of R-LPS of S-24 (Fig. 4) (not signifiantly different [P >.1]). LPS of H. pyloni strains, inluding UG and S-24, are phosphorylated (5, 28), and thus the potential role of phosphorylated strutures in the LPS-laminin interation was investigated. Dephosphorylated R-LPS of UG inhibited binding to both hemagglutinating (UG 17874) and poorly hemagglutinating (UG 17875) strains (Fig. 5) in a saturable manner, similar to data obtained with S-LPS and R-LPS (Fig. 2 and 4). Nevertheless, for UG the maximal inhibition of laminin binding observed with dephosphorylated LPS was 6% (Fig. 5), whih was signifiantly different (P <.1) from results obtained with native LPS (19% inhibition with 1,ug [Fig. 2]), indiating that phosphorylated strutures were involved in the interation of LPS of this strain with laminin. Although hydrofluori aid treatment of H. pylor LPS an, in addition to dephosphorylation, liberate fuose, it is unlikely that this sugar is involved in the interation with laminin sine fuose is present in small, nonstoihiometri amounts in R-LPS (29), in ontrast to that in S-LPS, and no appreiable differenes between inhibition of binding by S-LPS and R-LPS were observed (Fig. 2 and 4). For UG 17875, however, the maximal inhibition of laminin binding observed with dephosphorylated R-LPS of UG (49% inhibition with 5,g, [Fig. 5]) was omparable to, and not signifiantly different from (P >.5), that obtained with native LPS (4% [Fig. 2]). The latter result showed that some ommon struture in the R-LPS, but not a phosphorylated struture, was involved in the LPS-laminin interation with this poorly hemagglutinating strain nhibitor (ug) FG. 5. nhibition of binding of "N-laminin to H. pylori UG () and UG () with dephosphorylated R-LPS of UG in the liquid phase assay. Data points represent the means of three determinations, and error bars indiate standard deviations. omparative inhibition assays were also performed with isolated ore oligosaharide and free lipid A to determine the portion of H. pylori R-LPS involved in the interation with laminin. Both ore oligosaharide and free lipid A of UG inhibited binding of laminin to the homologous strain in a dose-dependent, saturable manner (Fig. 6), refleting the presene of phosphorylated strutures in the ore oligosaharide and lipid A (29). n ontrast, ore oligosaharide (Fig. 6), but not free lipid A, of the same strain inhibited binding of laminin to UG 17875, whih showed that a onserved, nonphosphorylated struture in the ore oligosaharide was involved in the LPS-laminin interation of the latter, poorly hemagglutinating strain. Efet of niteapone on LPS-mediated binding of laminin. A series of inhibition experiments whih investigated the effets of the antiuler drug niteapone on laminin binding by H. pylon UG and on the laminin-lps interation were ol nhibitor (ug) FG. 6. nhibition of binding of "M-laminin to H. pylon strains with ore oligosaharide and free lipid A in the liquid phase assay. Shown are the inhibition of binding to UG after preinubation of 125-laminin with ore oligosaharide () and free lipid A (V) from R-LPS of the same strain and the inhibition of binding to UG after preinubation of '21-laminin with ore oligosaharide () and free lipid A (V) from R-LPS of UG Data points represent the means of three determinations, and error bars indiate standard deviations.

5 3644 VALKONEN ET AL. NFET. MMUN. -o r " m ' m A redued laminin binding (e.g., 34% inhibition with 2,ug [Fig B]) (statistially signifiant [P <.1]). Furthermore, no redution of the extent of laminin binding by UG was observed when niteapone-treated S-LPS of UG was 27-1 used to inhibit laminin binding to bateria (Fig. 7) (not * signifiantly different [P >.1]). olletively, these results * 1 J indiate that niteapone interferes with the binding of laminin /l mediated by LPS. Further experiments using niteapone were undertaken to T investigate the role of protein adhesins, in addition to LPS, in 9 1 laminin binding by H. pyloni. Binding of laminin to UG and UG was redued by 85 and 65%, respetively, when strains were heat treated (8, 1 min). Treatment of baterial ells with 1 pronase redued laminin 3 binding o loo by 8 and 6%, respetively. Both results suggest the involve- Niteopone (ug) ment of protein adhesins in binding of laminin. n addition, when heat-treated or enzyme-treated bateria were inubated B with niteapone (2,ug), omplete inhibition of laminin 36-- ( '% Xr 36 binding was observed, indiating the involvement of both LPS and protein adhesins in binding of laminin. Solid phase analysis of laminin binding. The moleular 27 interation of LPS with laminin was investigated further to onfirm the speifiity of the interation and to determine its binding onstant. Sine it was not possible to formulate an 181 assay system with immobilized laminin to give sensitive dete _ tion of bound LPS, an assay using LPS-oated wells of miro T/Xl l - l titer plates with detetion of bound 125-laminin was utilized. 9{1 j Binding of 125-laminin to immobilized S-LPS of UG /showed saturation (Fig. 8A). Sathard plot analysis (38) gave a straight line indiative of a single lass of binding interation / with a binding onstant of 2.5 nm (Fig. 8B). The speifiity of the interation was further demonstrated by inhibition of 1251-laminin binding to LPS by unlabelled laminin in a onen- Niteapone (ug) tration-dependent manner (Fig. 9). dential results were obtained when assays were performed in air or under miroaerobi onditions DSUSSON The preise role that attahment plays in the pathogenesis of H. pylori has not, to date, been unequivoally established, but /l l J the baterium shows a strong ability to adhere in various in vitro test systems (1, 13, 14, 32) and an adhere to gastri muosal ells in vivo (18). Furthermore, the adhesive proess is apparently omplex, sine the organism produes a variety of different adhesins (9, 11, 21, 22, 3, 37, 4, 49). t has been suggested that this multipliity of adhesins may reflet that H. pyloni adherene is a multistep proess involving different adhesion and reognition interations at different stages of the proess (7) and that H. pyloni expresses several adhesins mediating adherene to various targets in gastri tissue (42). Niteopone (ug) The latter possibility is supported by the finding that in vivo not FG. 7. Effets of niteapone on binding of 1251-laminin to H. pylon all H. pyloni ells are in diret ontat with the gastri muosal UG in the liquid phase assay. (A) 12"-Laminin was pre- epithelium, but rather some are found within the muus gel treated with niteapone before inubation with bateria. (B) Baterial overlying the epithelium, whereas others an be observed in ells w,ere pretreated with niteapone before inubation with 1- lose proximity to interellular juntions (18, 42). laminin. () S-LPS of UG was pretreated with niteapone The apaity of H. pylori strains to bind to basement before attempted inhibition of 251-laminin binding to baterial ells, membrane laminin is unlikely to be involved in the initial Datum points represent the means of three determinations, and error bars in( dliate standard deviations. olonization of gastri muosal ells (46), sine speifi primary adhesins reognize reeptors in the muus layer and on the epithelial ell surfae (11, 22, 37, 4, 41). Nevertheless, this binding property would assume major importane one the underttaken. Laminin pretreated with niteapone bound to basement membrane beame exposed. This exposure may bateria in a saturable manner and to the same extent as result from the mirobial ativity of H. pyloi (33, 39, 41, 42) or untreated laminin (Fig. 7A) (not signifiantly different [P > feeding (52). Eletron mirosopy studies have shown that H..1]).. Pretreatment of bateria with niteapone, however, pylori an disrupt the epithelial ells after olonization of the

6 VOL. 62, 1994 LAMNN BNDNG BY H. PYLOR 3645 E -o._. E -J A een 4-_ / B LA. a.4- z m m.2 T were low- to intermediate-level binders of laminin, suggesting a multipliity of baterial-glyoprotein interations. A hydrophobi surfae omponent of H. pylon is apparently involved in the interation with laminin (46). nitially, binding of H. pylon to laminin was attributed to an afimbrial 19.6-kDa T protein adhesin (7), but further haraterization of the protein o - T * revealed that it was a ytosoli iron-binding protein whose /ltl adherene to laminin was due to nonspeifi hydrophobi T l interations (8). Nevertheless, sine LPS is relatively hydrophobi and surfae exposed, it is a potential andidate for involvement in the interation. The observed ability ofh. pylon LPS to inhibit binding of hemagglutinating and poorly hemagglutinating strains to laminin supports this hypothesis, al- though the extent of inhibition of binding with LPS also supports the involvement of another moleular interation in Laminin added (fmol) the adhesion proess. n addition, denaturation experiments indiated the involvement of both protein adhesins and LPS in the interation with laminin. Although previously we observed that an LPS preparation from NT (same as UG 17874) ompletely inhibited laminin binding by the same strain (3), further analysis has revealed that the LPS preparation was ontaminated by protein and hene potentially by the other adhesin involved in laminin binding. On the basis of the results obtained in the earlier parts of this study, two strains, one hemagglutinating and a high-level binder of laminin (UG 17874) and the other poorly hemagglutinating and an intermediate-level laminin binder (UG 17875), were seleted for more detailed studies of LPS-laminin interations. t is interesting that inhibition by UG LPS, even though the LPS was from a heterologous strain, was greater in the poorly hemagglutinating strain than in the hemagglutinating one, emphasizing differenes Laminin Bound (fmol) between the strains in baterial-laminin interation. Support- FG. 8. Binding of ' 1251-laminin to immobilized S-LPS of H. pylo ing this onlusion, LPS of UG did not inhibit UG in the solid phase assay. (A) The total amount of binding of laminin to the heterologous hemagglutinating '25-laminin bound to immobilized LPS () was determined, and strain. The ability of E. oli LPS to inhibit binding of the subsequently, after subttration of nonspeifi binding of 1N-laminin to hemagglutinating strain, but not the poorly hemagglutinating BSA-oated surfaes, the amount of laminin bound speifially to LPS strain, onfirmed that different modes of binding of laminin by (@) was estimated. DaLta points represent the means of three deter- LPS existed in the two H. pylori strains. minations, and error bears indiate standard deviations. (B) Sathard Nevertheless, even though two separate modes of interadata. plot of speifi binding gastri muosa, leaving the underlying basement membrane bare (16). Alternatively, a high rate of ell turnover in the gastri epithelium may expose laminin and other extraellular matrix omponents for the baterium to interat with (29, 46). Tissue damage may be further enhaned by bakflow of gastri seretions into these mirolesions, and thus laminin binding may enable the pathogen to survive in hronially inflamed tissue. n addition to binding to gastri muosal ells in vivo, different H. pylon strains exhibit different hemagglutination speifiities in vitro (49), further emphasizing a multipliity of adhesin prodution. However, the relevane of hemagglutinins to pathogenesis of H. pylon in vivo remains unlear, and the adherene mehanisms of poorly hemagglutinating strains were, to date, unknown. n ontrast to the diversity of hemagglutination speifiities, onserved binding of laminin and ollagen type V by H. pylon strains has been reported (46), but the onserved nature of this binding between strains has been disputed (3). Upon examination of laminin binding to various H. pylon strains in the present study, we found that hemagglutinating strains ould be divided into high- and low-level binders of laminin, whereas poorly hemagglutinating strains tion of LPS with laminin were apparent in the hemagglutinating and poorly hemagglutinating strains, it was possible to ross-inhibit binding of both types of strains with heterologous Unlabelled laminin added (pmol) FG. 9. nhibition of binding of '25-laminin to immobilized S-LPS of H. pylon UG with unlabelled laminin in the solid phase assay. Data points represent the means of three determinations, and error bars indiate standard deviations.

7 3646 VALKONEN ET AL. S- and R-LPS. Thus, a relatively onserved region (ore oligosaharide or lipid A), but not a strain-speifi region (O side hain), in LPS is involved in the interation. omparative inhibition experiments with dephosphorylated R-LPS showed that phosphorylated strutures in the ore of H. pylon LPS were involved in the interation of the hemagglutinating strain, but not the poorly hemagglutinating strain, with laminin. Further, the presene of phosphate, phosphorylethanolamine, and pyrophosphorylethanolamine as substituents in the ore oligosaharide and lipid A of LPS of members of the family Enterobateriaeae (2, 35, 36) explains the ability of E. oli LPS to ross-inhibit the interation of the hemagglutinating strain with laminin. Although both isolated ore oligosaharide and free lipid A inhibited binding of laminin by the hemagglutinating strain, it is unlikely that phosphorylated strutures in lipid A mediate the interation between baterial ells and laminin, sine lipid A is embedded in the outer membrane of gram-negative bateria and is overed by the sugars of the inner ore oligosaharide (2). Only ore oligosaharide inhibited binding of the poorly hemagglutinating strain. These results, therefore, suggest that a phosphorylated struture in the ore oligosaharide mediates the interation of LPS of the hemagglutinating strain with laminin, whereas in ontrast, a onserved nonphosphorylated struture in the ore oligosaharide mediates the interation of the poorly hemagglutinating strain. Of partiular interest is that although the LPS-laminin interations of the hemagglutinating and the poorly hemagglutinating strains were mediated by different mehanisms, LPS of the hemagglutinating strain was able to ross-inhibit the interation of the poorly hemagglutinating strain with laminin. t would therefore appear that despite the LPS of the hemagglutinating strain possessing the responsible struture in its ore oligosaharide, it is masked on the surfae of that baterial strain and annot mediate the interation of the hemagglutinating strain with laminin. n addition to a hydrophobi omponent (46), a letin-like baterial adhesin (48) has been impliated in the binding of laminin by H. pylori. The inomplete inhibition of laminin binding to H. pylori by LPS is therefore in agreement with the existene of a seond type of interation between laminin and the baterial surfae in the adhesion proess. Thus, initial reognition and binding of H. pyloni to laminin may our through LPS, and subsequently a more speifi interation with a letin-like protein adhesin on the baterial surfae may our. Likewise, MSweegan and Walker (28) showed that both LPS and another surfae struture, possibly flagella, mediated adhesion of ampylobater jejuni to epithelial ells. A similar adhesion mehanism involving LPS and protein reeptors on epithelial ells has been desribed for Shigella flexneri (19), and LPS of Atinobaillus pleuropneumoniae has been identified as the moleule mediating adhesion of the pathogen to porine trahea (1). However, the interation of H. pylon LPS with laminin is the first haraterization of suh an interation by a spiral, gram-negative, muus-olonizing baterium. Slomiany et al. (39) demonstrated that H. pyloni ells, and in partiular H. pylon LPS, inhibit the interation between a laminin reeptor in gastri epithelial ells and laminin. The subsequent disruption of epithelial ell-basement membrane interation by LPS would explain the development and ontinuane of lost muosal integrity during H. pyloni infetion (3). The antiuler drug niteapone, however, abolishes the inhibitory effet of LPS on the epithelial ell-laminin interation (39). Our observation that niteapone-treated LPS no longer inhibited binding of laminin to H. pylori indiates that NFET. MMUN. the drug also interferes with the adhesion mediated by LPS. Furthermore, the inomplete inhibition of baterial adherene by the drug is onsistent with a seond moleular interation in the adhesion proess. Binding of laminin to H. pylori ells has been shown to be saturable and of high affinity (3, 46, 48). Likewise, we demonstrated that the interation of laminin with LPS in the solid phase assay was saturable, speifi, and inhibitable with unlabelled protein. The binding onstant of 2.5 nm for the LPS-laminin interation lies within the range of 8.5 pm to 7.9 nm that has been alulated for the interation of laminin with H. pylon (46, 48). Also, the LPS-laminin binding onstant is slightly higher than the 2.9 nm laminin binding onstant reported for S. aureus (24) but signifiantly higher than the values of 4 to 8 nm reported for streptooi (44, 45). However, the protein adhesin with letin-like properties involved in the interation between laminin and H. pylon remains to be identified. Of interest is that the H. pylori letin-like adhesin reognizes N-aetylneuraminyllatose oligosaharides of laminin (48), and binding to ell surfaes with suh oligosaharides has been demonstrated in vitro (1). Furthermore, an N-aetylneuraminyllatose-binding hemagglutinin of H. pylori has been desribed and loned by Evans et al. (11, 12); this hemagglutinin thus represents a potential andidate for the other adhesin involved in laminin binding. n summary, the interation of H. pylori LPS with laminin is, to our knowledge, the only binding of laminin by a nonproteinaeous baterial surfae omponent identified to date. Furthermore, the interation of LPS with extraellular matrix proteins may represent a previously unreognized group of surfae interations produed by signifiant gastrointestinal pathogens of humans. The in vitro laminin adherene assay should prove useful in further haraterizing H. pylori adhesinreeptor interations and for identifying potentially therapeuti drugs against H. pylori that inhibit adherene of this human pathogen. AKNOWLEDGMENTS This study was supported by grants from the rish Heliobaterpylori Study Group to A.P.M., from the Swedish Medial Researh ounil to T.W. (16X-4723) and to KV. (16V ), and from the Nordisk Forskeruddannelseakademie to K.V. REFERENES 1. Belanger, M., D. Dubreuil, J. 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