~'2,terinary Microbiology, 28 ( 1991 ) 7 5 - 9 2 Elsevier Science Publishers B.V., A m s t e r d a m

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Relationship between the iron regulated outer membrane proteins and the outer membrane proteins of in vivo grown Pasteurella multocida Keumhwa Choi-Kim a, Samuel K. Maheswaran a'*, Lawrence J. Felice b and T h o m a s W. M o l i t o r c aDepartments of Veterinary Pathobiology, bDiagnostic Investigation, and CLargeAnimal Clinical Science, College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota 55108 USA (Accepted 21 November 1990)

ABSTRACT Keumhwa, C.-K., Maheswaran, S.K., Felice, L.J. and Molitor, T.W., 1991. Relationship between the iron regulated outer membrane proteins and the outer membrane proteins of in vivo grown Pasleurella multocida. Vet. Microbiol., 28: 75-92. The SDS-PAGE patterns of the outer membrane protein ( O M P ) extracts of Pasteurella multocida strain P1059, grown under iron-restricted, iron-replete and in vivo conditions, were examined. The results showed that the iron-regulated outer membrane proteins (IROMPs) with molecular masses of 76 kDa~ 84 kDa, and 94 kDa were expressed by bacteria grown in iron-restricted media. They were also expressed by in vivo grown P. multocida. Convalescent-phase sera, obtained from turkeys which had survived pasteurellosis, contained antibodies that reacted intensly with the three IROMPs. This indicated that these proteins were expressed in vivo. Bacteria expressing the IROMPs showed greater binding to Congo Red when compared to cells not expressing IROMPs. Cells expressing the IROMPs or its OMP extracts grown in iron-restricted media also showed greater binding to 59Fe-pasteurella siderophore (multocidin) when compared to bacteria or its extracts not expressing IROMPs. Convalescent-phase sera, which contained antibodies against the IROMPs, blocked this specific 59Fe-multocidin binding to IROMPs. Autoradiography was used to determine which of these IROMPs functioned as a receptor for the iron-multocidin complex. The results suggested that these three IROMPs have specific epitopes for binding to the iron multocidin complex.

INTRODUCTION

Pasteurella multocida is the etiological agent of avian pasteurellosis. It is a highly contagious, often fatal, septicemic disease of poultry (Rhoades and Rimler, 1984). In domesticated poultry, strains belonging to capsular type A and different somatic types ( 16 in all) ofP. multocida (Brogden et al., 1978 ) are recognized as the primary cause of disease. Of these types, the pathogen *Author to w h o m correspondence should be addressed.

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© 1991 - - Elsevier Science Publishers B.V.

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most often isolated belong to capsular type A, somatic type 3 (Carter, 1972; Heddleston, 1962; Hofacre and Glisson, 1986; Namioka and Bruner, 1963 ). It is designated as serotype A:3 (Carter and Chengappa, 1981 ). To date, the virulence factors ofP. multocida serotype A:3 such as the capsule (Carter and Annau, 1953; Heddleston et al., 1964; Maheswaran et al., 1973; Snipes and Hirsh, 1986 ), endotoxin (Ganfield et al., 1976; Heddleston et al., 1966; Rebers and Rimler, 1984), and an outer membrane protein (Truscott and Hirsch, 1988) have been examined with organisms grown in the laboratory. Studies using other bacterial pathogens, such as Escherichia coli and Vibrio cholerae, have established that when they multiplied in vivo during infection, they were phenotypically quite different from bacteria grown in vitro because they expressed novel surface antigens (Griffiths et al., 1983; Jonson et al., 1989). There was evidence that in vivo grown avian strains of P. multocida expressed novel surface antigens which were not expressed by in vitro grown bacteria (Choi et al., 1989). These novel antigens, when used as immunogens in turkeys, induced protective immunity against pasteurellosis caused by both homologous and heterologous serotypes of P. multocida (Rimler and Rhoades, 1981 ). The putative antigens responsible for cross immunity were termed cross-protection factor (CPF) immunogens (Brogden and Rimler, 1983 ). We have been particularly interested in the possibility that the CPF immunogens expressed by in vivo grown P. multocida may be modulated by iron because the level of freely available iron in the healthy avian host was severely restricted. Many other bacterial pathogens, such as E. coli, Pseudomonas aeruginosa, Haemophilus influenza, and Actinobacillus pleuropneumoniae expressed iron-regulated outer membrane proteins (IROMPs) when growing in an iron-restricted environment (Deneer and Potter, 1989; Griffiths, 1987; Griffiths et al., 1985; Sokol and Woods, 1983 ) and antibodies specific to the IROMPs were protective in some studies (Bolin and Jensen, 1987; Sokol and Hirsh, 1986). Previous studies from our laboratory have shown that P. mulIocida serotype A:3, grown under iron-restricted conditions, produced a siderophore termed multocidin (Hu et al., 1986 ). Ikeda and Hirsh ( 1988 ) have shown that the same organism also expressed several high molecular weight IROMPs when grown under iron-limiting conditions. They also observed that all serotypes, with the exception of serotype A: 12, expressed several antigenically related high molecular weight IROMPs. However, which of these IROMPs functioned as receptors for iron-multocidin complex was not determined. In this study we demonstrate that P. rnultocida serotype A:3 grown under iron-restricted conditions expressed IROMPs and present evidence that they are identical to the novel OMPs expressed by in vivo grown bacteria. Furthermore, we report the identification of the IROMPs which bind to 59F-multocidin complex.

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MATERIALS AND METHODS

Chemicals and glassware To minimize iron contamination, all glassware was soaked in 0.5% ethylenediamine tetraacetic acid (EDTA, Sigma Chemical Co., St. Louis, MO) overnight and rinsed 10 times in ion-free/organic-free water purified by the Milli Q system (Millipore Corp., Bedford, MA.). Radioactive iron was purchased from Amersham Corp., (Arlington Heights, IL.) as 59FeC13 in 0.1 N HC1 (specific activity, 3.7 m C i / m g iron) and 2,2'-dipyridyl was purchased from Sigma. All media and solutions were made in Milli Q water autoclaved under slow exhaust for 90 min to get rid of endotoxin contamination.

Bacteria and growth conditions Pasteurella multocida strain P 1059, serotype A:3, which is highly virulent to domestic poultry, was used in all studies. For each experiment, one lyophilized culture was rehydrated in sterile brain heart infusion broth (BHI, Difco Laboratories, Detroit, MI) and propagated overnight on blood agar at 37 ° C. Several colonies were inoculated into 5 ml of BHI or iron-restricted chemically defined m e d i u m ( C D M ) and allowed to grow for 6 h on a shaker. The composition and procedure used for deferration of CDM was reported earlier (Hu et al., 1986). Aliquots of BHI-propagated P. multocida was then transferred to large volumes of BHI and growth from CDM to BHI ÷ 100/~M 2,2'dipyridyl ( B H I + D P ) , or C D M + 50/~M FeC13 ( C D M + F e ) , or C D M + 100 /~M 2,2'-dipyridyl (CDM + DP ), or CDM. The BHI and CDM ÷ Fe were considered as iron-replete media, while CDM, CDM + DP, and BHI ÷ DP were considered to be iron-restricted media. The iron-restricted media contained less than 0.02/~g/ml of iron as determined by the Ferrozine assay (Sigma) (Holzberg and Artis, 1983). In vivo grown bacteria were harvested from the blood of adult turkeys in the terminal stage of experimental pasteurellosis as described by Brogden and Rimler ( 1983 ). Purity, plus confirmation that the in vivo grown bacteria were indeed P. multocida was shown by Gram staining and biochemical testing. The in vivo grown bacteria were stored at - 7 0 ° C until use.

Preparation of pasteurella siderophore (multocidin) Large scale multocidin production was accomplished using the procedure described earlier from our laboratory (Hu et al., 1986). The culture filtrates obtained from spent media were filter sterilized, and concentrated by freezedrying. The presence of multocidin in the media was estimated using a previously described assay reported by Hu et al. ( 1986 ).

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Preparation of outer membrane protein-enriched extracts The outer membrane proteins (OMPs) were extracted by the method of Barenkemp et al. ( 1981 ). Briefly, cells were harvested, washed twice and resuspended in 10 mM HEPES buffer (pH 7.4) and sonicated as described by Choi et al. (1989). Unbroken cells and debris were removed by centrifugation at 1700 g for 20 min. The supernatant was collected and centrifuged at 100 000 g for 60 min at 4 ° C. The pellet, which contained the total membrane, was resuspended in 2 ml of 2% sodium lauryl sarcosinate detergent in 10 mM HEPES buffer (pH 7.4) and incubated at 22°C for 60 rain. The detergent insoluble OMP-enriched extracts were obtained by centrifugation at 100 000 g for 60 min at 4°C. They were then washed 2 times in distilled water (DW), dialyzed against DW for 48 h, lyophilized, and stored at - 2 0 °C. Sera Reference negative sera were obtained from 10 turkeys which did not contain any antibodies against P. multocida serotype A:3 as had been determined by ELISA (Marshall et al., 1981 ). Post infection immune sera (convalescent-phase sera) were obtained from 3 turkeys one month after their survival of experimental pasteurellosis. Specific antisera (OMP antisera) against the OMPs extracted from in vitro-grown (BHI broth) P. multocida P 1059 were obtained from immunized turkeys. Five hundred micrograms of OMP extracts (Choiet al., 1989) were mixed with Freund's incomplete adjuvant (Difco) and 0.5 ml of the mixture was injected intramuscularly into several turkeys. The animals were injected two times at 2-week intervals. Two weeks after the second injection, blood was collected and the antisera were pooled. All sera were filter-sterilized and stored in aliquots at - 80 ° C until used. SDS-PAGE The OMP-enriched extracts from P. multocida P 1059 grown in BHI broth, BHI + DP, CDM, CDM + DP, CDM + Fe and in vivo grown bacteria were subjected to discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 7.5% or 10% separation gel according to the method of Laemmli (1970). Protein ( 15-20 #g) in 50/tl of sample buffer (Laemmli, 1970) was heated at 100°C for 4 min, and loaded into each well. Staining was done with 0.2% Coomassie Blue R-250 (Bio-Rad Laboratories, Richmond, CA) in 45% methanol and 10% acetic acid in DW. Molecular weight of the OMP was determined, using standard proteins of known molecular weight. Western blots Electrophoretic transfer of proteins to 0.45 j~m nitrocellulose paper (BioRad) and western blotting (immunoblotting) was performed essentially as

IRON REGULATED AND NORMAL OUTER MEMBRANE PROTEINS OF P. MULTO(TDA

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described by Towbin et al. (1979). Efficiency of transfer was determined by staining the nitrocellulose paper with 0.5% Ponceau S (Helena Lab., Beaumont, TX) for 10 min and washing with DW for 10 rain at room temperature. Blots were then soaked in 3% BSA in 50 mM Tris-200 mM sodium chloride buffer (TBS, pH 7.2) at 37°C for 1 h to block the nonspecific binding sites. This was followed by washing, and incubation at 37°C for 1 h with either a 1 : 100 dilution of convalescent-phase sera, a 1 : 100 dilution of OMP antisera or a 1:20 dilution of reference negative sera diluted in TBS (pH 7.2 ) containing 1% BSA and 0.05% Tween-20. A 1 : 1000 dilution in the same buffer of peroxidase-labelled goat anti-turkey IgG ( H ÷ L , Kirkegaard and Perry Laboratories, Gaithersburg, MD) was used as the secondary antibody by incubating at 37 °C for 1 h. The membranes were washed extensively with three changes of TBS (pH 7.4) containing 0.1% BSA and 0.05% Tween-20. The blots were tlaen immersed in 80 ml of TBS containing 20 ml of methanol, 60 mg of 4-chloro-l-naphthol (Sigma) and 50/tl of 30% hydrogen peroxide for 15 rain, and the reaction was stopped by washing with distilled water.

Congo Red binding assay Comparison of Congo Red binding to P. multocida cells grown under different conditions was measured using the procedure described by Deneer and Potter (1989). Briefly, P. multocida P1059 grown in BHI, B H I + D P , CDM, C D M + D P , C D M + F e and in vivo were harvested, washed in phosphate buffered saline (PBS, pH 7.2) and suspended at a density of 1.5 measured at OO62o. Congo Red was added to a final concentration of 30/~g/ml. A 1-ml sample was removed immediately, centrifuged in an Eppendorfcentrifuge for 30 s to pellet the cells and the supernatant was used to measure the density of the Congo Red spectrophotometrically at OD480 using a Beckman DU-50 spectrophotometer (Beckman Instruments, Inc. Fullerton, CA). The remaining cells were further incubated on a rotary shaker at 37°C and assayed for residual Congo Red concentration every 30 rain by the method previously mentioned. The experiment was repeated four times and the mean value and standard error were calculated. Iron-m ultocidin uptake assay Twenty microliters of 59FeC13 (specific activity of 3.7 mci/mg; Amersham) was dissolved in 200/A of 100/~M nitriloacetic acid (pH 7.2) and added to 100/~g of multocidin and incubated at 22 ° C for 10 min. The resulting mixture contained 59Fe-multocidin complex. Bacteria at a concentration of 1 × 107 C F U / m l grown in an iron-restricted or iron-replete medium were incubated with either ~9Fe-multocidin or free ~gFe at 37 ° C. Samples (0.5 ml ) were removed at 5-rain intervals, filtered through Metricel membrane filters (0.45/~m, 13 mm, Gelman Instrument Co., Ann Arbor, MI ) and washed with 20 ml of saline (pH 7.2 ). The ~9Fe retained by the filters was counted using a

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gamma counter (Biogamma II, Beckman Instruments). The results were expressed as counts per minute (CPM) of bound 59Fe. Iron binding to OMPs was measured using OMP extracts from P. rnultocida P 1059 grown either in iron-restricted, iron-replete media or in vivo and incubating for 10 min at 22°C with 59Fe-multocidin or free59Fe. Samples (0.5 ml) were removed, filtered through 0.2/zm Metricel membrane filters (Gelman Instruments) and counted as described above.

Inhibition o f iron-rnultocidin binding to IROMPs To test the blocking activity of iron-multocidin binding by antibodies to IROMPs, 100/~1 amounts of convalescent-phase sera, OMP antisera or reference negative sera were preincubated at 37°C for 1 h with 100 #g of OMP extracts from P. multocida grown in iron-restricted media such as CDM or CDM + DP. 59Fe-multocidin was then added to the reaction mixtures, incubated at 22°C for 20 min, and filtered through 0.2 ~tm Metricel membrane filters (Gelman Instruments). This was followed by washing with 20 ml of saline (pH 7.2). The 59Fe retained on the filters was counted in a gamma counter and the results expressed as described in previous sections. To test for potential inhibition of iron binding to intact bacteria by antibodies, bacteria were grown under different conditions and then exposed to different sera. Overnight cultures of P. multocida grown in CDM and C D M + DP were centrifuged at 7000 g for 40 min, and the pellets were adjusted to 50 Klett Units with sterile PBS (pH 7.2). A 1.5 ml sample of the suspended bacterial cells was centrifuged again in Eppendorf tubes at 15 000 rpm for 15 min, and the supernatant was discarded. The bacterial pellets were preincubated with 1 ml of 1 : 100 convalescent-phase sera, 1 : 100 dilution of OMP antisera, or 1:20 dilution of reference negative sera at 37°C for 1 h. Iron-multocidin complex was then added, followed by incubation at 22°C for 20 rain. Samples of 0.5 ml were filtered through 0.45/~m Metricel membrane filters (Gelman), followed by washing with 20 ml of saline (pH 7.2). The radioactivity of 59Fe was counted in a gamma counter. Autoradiography Visualization of the binding of ~gFe to OMPs was demonstrated by autoradiography. One hundred micrograms of OMP extract from P. rnultocida grown in either iron-restricted media, iron-replete media or in vivo were dissolved in a denaturing sample buffer containing 0.06 M Tris (pH 6.8 ), 1.2% SDS, 0.5% 2-mercaptoethanol, and 11.9% glycerol, and subjected to SDSPAGE containing 7.5% acrylamide. The gels were electrically transferred to nitrocellulose membranes. The membranes were either not renatured or renatured by soaking in PBS buffer (pH 7.2) containing Tween 20 and M I~glucose for 1-2 days at 37°C. The renaturing step has been used previously (Birk and Koepsell, 1987) to achieve optimal renaturation of the binding

IRON REGULATED AND NORMAL OUTER MEMBRANE PROTEINS OF P. MULTOCIDA

81

sites of the electroblotted proteins. The membranes were then exposed to 59Femultocidin for 20 min at 37 °C and washed exhaustively with TBS (pH 7.2). The 59Fe binding was visualized by autoradiography for 2-3 days at - 7 0 °C on R P / R 2 film (Eastman, Kodak).

Statistical method Statistical significance of differences between groups was determined by the multiple comparison analysis (Devore and Peck, 1986). For all analysis, P~< 0.05 was chosen as the level of significance. RESULTS

Effects of iron restriction or in vivo growth on the OMP composition of P. multocida P 1059 Outer membrane proteins (OMPs) from P. multocida P 1059 grown in ironrestricted media (BHI + DP, CDM, and CDM + D P ) and in vivo were compared by SDS-PAGE with OMPs from bacterial cells grown in iron-replete media (BHI, and CDM + Fe ). The maj or OMPs with molecular masses of 29 kDa, 34.5 kDa and 45 kDa were expressed by bacteria grown in iron-restricted media, iron-replete media and in vivo. However, iron restriction induced the expression of several additional OMPs with the molecular masses 76 kDa, 85 kDa, and 94 kDa (Fig. 1 ). These novel OMPs were also expressed by in vivo grown cells (Fig. 1 ). In contrast, bacteria grown in iron-replete media did not express these novel OMPs. Bacteria grown in CDM containing 20-400/~M of DP also expressed the novel OMPs and the major OMPs, although quantitative variations were detected (data not shown). These three novel OMPs with molecular masses 76 kDa, 84 kDa, and 94 kDa were designated as iron regulated outer membrane proteins (IROMPs) because they were expressed only by bacteria grown in iron-restricted media and in vivo. Western blot analysis of lROMPs Results, as shown in Fig. 2, revealed that convalescent-phase sera contained antibodies that showed intense reactivity with the three IROMPs which suggest that these proteins were expressed in vivo. In contrast, the OMP antisera and reference negative sera failed to react with the IROMPs. Congo Red binding by P. multocida Experiments using the Congo red binding assay provided evidence that the IROMPs ofP. multocida played a role in iron acquisition. A marked increase in binding to Congo Red was observed with bacteria grown in BHI + DP, CDM, C D M + D P , and in vivo which expressed IROMPs (Fig. 3). In con-

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~i~ii~iiiii~ii~iiiJiiiiiii!~iiiiiiii Fig. 1. Comparison of SDS-PAGE profiles of OMP extracts from P. multocida P1059 grown in vivo (Lane B), in BHI (Lane C), BHI plus 100/~M dipyridyl (Lane D), CDM plus 50 #M FeCI~ (Lane E), CDM (Lane F) and CDM plus 100 j~M dipyridyl (Lane G). Samples of 15/~g protein from each extract was analyzed in 7.5% SDS-polyacrylamide gel and stained with Coomassie Blue. Molecular masses (in kDa) arc listed in Lane A of the Figure. Arrow heads on Lanes B, D, F, and G denote the presence of 76 kDa, 84 kDa and 94 kDa IROMPs expressed by organisms grown in vivo and under iron restricted conditions.

trast, bacteria grown in either BHI, or C D M + F e that do not express the IROMPs, bound modestly to Congo red.

Iron-multocidin uptake The ability of I R O M P s to specifically bind iron-multocidin complex as opposed to iron alone, was demonstrated by measuring the uptake of either free 59Fe or 59Fe-multocidin by P. multocida grown in iron-restricted or iron-replete media. Only bacteria grown in iron-restricted media (BH! + DP, CDM,

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Fig. 2. Western blots of OMP extracts from P. tnultocida P1059 grown under different conditions reacted with convalescent-phase sera (Panel [), OMP antisera (Panel I I ), or reference negative sera (Panel III). OMP extracts from P. multocida grown in BHI (Lane A), in vivo (Lane B), BHI plus 100 ~tM dipyridyl (I-ane C), C DM plus 50/~M FeC13 (Lane D), CDM (Eane E), and CDM plus 100 ~M dipyridyl (Eane F). Note that the convalescent-phase sera (Panel I) contained antibodies that reacted with the 76 kDa, 84 kDa, and 94 kDa IROMPs from P. multocida grown in vivo (Lane B) or under iron-restricted conditions (I~anes C, E, and F). However, OM P antisera (Panel II) did not contain any antibodies against these IROMPs. The reference negative sera only recognized some of the major OMPs (Panel IIl). Arrow heads indicate the IROMPs.

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Fig. 4. (a) Binding of ~gFe-multocidin by P. multocida P1059 cells. Cultures of P. rnultocida grown in BHI ((3), BHI plus 100/~M dipyridyl ( × ), CDM ( ~ ) , CDM plus 50 ~M FeCI3 ( • ) ov CDM plus 100 #M dipyridyl ( I ) were incubated with 59Fe-multocidin. Samples were removed at various times, filtered and the 59Fe retained by the filter was counted in a gamma counter as described in Materials and Methods. Binding is expressed as counts per minute (CPM) × 1000. Each point represents the mean counts per minute of two experiments. (b) Binding of ~gFe-multocidin to outer membrane proteins. Outer membrane protein extracts isolated from P. multocida P1059 grown in BHI, BHI plus 100 ~M dipyridyl, CDM plus 50 l~M FeC13, CDM, CDM plus 100/tM dipyridyl, and in vivo were incubated with free ~9Fe ( I ) or ~gFe-multocidin ( [ ] ) . Samples were removed, counted and binding expressed as described in Fig. 4(a). Each bar represents the mean counts per minute +_standard error of four experiments.

IRON REGULATEDAND NORMALOUTER MEMBRANE PROTEINS OF P. MULTOCIDA

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CDM + DP ) bound to the 59Fe-multocidin complex (Fig. 4a). The same bacteria grown under the same conditions did not bind to free 59Fe (data not shown). Pasteurella multocida not expressing IROMPs (grown in BHI, or C D M + F e ) did not bind to ~9Fe-multocidin or to free 59Fe (Fig. 4a). The same effect was observed when iron binding to IROMPs were measured, using OMP extracts from bacteria was incubated with either 59Fe-multocidin or free ~9Fe. Only OMP extracts containing IROMPs from bacteria grown under iron-restricted conditions (BHI + DP, CDM, CDM + DP and in vivo) bound significantly to the 59Fe-multocidin complex, but not to free ~9Fe (Fig. 4b). Binding of either 59Fe-multocidin complex or free 59Fe to bovine serum albumin and multocidin was negligible (data not shown ).

Effect of antibody on iron-multocidin binding to IROMPs Results, as shown in Fig. 5, revealed that bacterial cells induced to express IROMPs (grown in CDM, or CDM ÷ DP) when preincubated with convalescent-phase sera significantly decreased the amount of bound 59Fe-multocidin ( P < 0.05 ). To demonstrate that this antibody inhibition of 59Fe-multocidin binding was specific, the effects of the OMP antisera on 59Fe-multocidin binding were examined. Intact bacteria or OMP extracts containing IROMPs preincubated with OMP antisera bound 59Fe-multocidin slightly less than was observed when they were preincubated with normal turkey sera. This decrease was not statistically significant. To further demonstrate that this inhibition of iron binding to the bacteria or its OMP extracts was due to antibody 15

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Fig. 5. Effects of antibodies on binding o f i r o n - m u l t o c i d i n to P. multocida P 1059 or to IROMPs. Convalescent-phase sera ( L a n e A), O M P antisera (Lane B), reference negative sera ( L a n e C) or no sera ( L a n e D ) was p r e i n c u b a t e d with P. multocida grown in C D M ( [ ] ) , C D M plus 100 p M dipyridyl, ( . ) , or O M P extracts isolated from P. multocida grown in C D M ( [] ), a n d C D M plus 1 0 0 / t M dipyridyl ( [ ] ) . 59Fe--multocidin was a d d e d to the reaction mixtures a n d incubated as described in Materials a n d Methods. Samples were removed, counted a n d binding expressed as described in Fig. 4 (a). Each bar represents the m e a n counts per m i n u t e __+s t a n d a r d error of five experiments.

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C.-K. KEUMHWA ET AL,

directed against the iron-multocidin binding IROMP, the experiment was repeated with OMPs extracted from bacteria grown in iron-replete media. Results showed that when P. multocida P 1059 cells grown in iron-replete media or its OMP extracts containing OMPs were preincubated with convalescent-phase sera, the amount of bound 59Fe-multocidin was not altered when compared to the controls which had not been preincubated with the sera (data not shown.

Autoradiography To further substantiate the binding of the 59Fe-multocidin to the IROMPs, we employed autoradiography. Renatured OMP extracts from bacteria grown in iron-restricted media ( B H I + D P , CDM, and C D M + D P ) and in vivo inA

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Fig, 6. Autoradiogram of OMP extracts from P. multocida P1059 grown under iron-replete, iron-restricted or in vivo conditions electrophoretically transferred to nitrocellulose. The membranes were renatured as described in Materials and Methods and then incubated with 59Femultocidin. Lane A, OMP extracts from bacteria grown in vivo; Lane B, grown in BHI; Lane C, grown in BHI plus 100 I~M dipyridyl; Lane D, grown in CDM plus 50/~M FeC13; Lane E, grown in CDM; Lane F, grown in CDM plus 100/~M dipyridyl. The positions of the 76 kDa, 84 kDa, and 94 kDa IROMPs bound to 59Fe-multocidin are indicated by arrow heads.

IRON REGULATED AND NORMAL OUTER MEMBRANE PROTEINS OF P. MULTOCIDA

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cubated with 59Fe-multocidin, showed three OMPs with molecular masses 76 kDa, 84 kDa, and 94 kDa bound to the 59Fe-multocidin (Fig. 6, lanes A, C, E, and F). Our earlier results had shown that these three OMP extracts were indeed the IROMPs. In contrast, OMP extracts from bacteria grown in iron-replete media (BHI, and CDM + Fe) did not bind to 59Fe-multocidin (Fig. 6, lanes B and D). Deletion of multocidin eliminated the binding of SgFe to any of the OMPs. The same OMPs electroblotted to nitrocellulose membrane which was not renatured, when reacted with ~9Fe-multocidin complex showed non-specific binding of the complex to several OMPs which were not IROMPs (data not shown). DISCUSSION

A number of earlier studies have affirmed some exciting findings in the area of experimental immunogens for the prevention of avian pasteurellosis in domestic poultry. Heddleston and Rebers ( 1972 ), and Rimler et al. ( 1979 ) stimulated great interest when they demonstrated that, in turkeys, a bacterin prepared from in vivo grown P. multocida strain P1059 induced cross-immunity against heterologous serotypes of the organism. Furthermore, they demonstrated (Heddleston et al., 1970) that turkeys which survived natural or experimental infection (convalescent turkeys) were cross-protected against heterologous serotypes of the organism. In contrast, a bacterin prepared from bacteria grown artificially in c o m m o n laboratory media induced only a serotype-specific immunity against the homologous serotype (Heddleston et al., 1970; Rebers and Heddleston, 1977 ). Turkeys vaccinated with an attenuated live vaccine also showed this cross-immunity (Bierer and Derieux, 1972a; Bierer and Derieux, 1972b). Humoral immunity was responsible for this protection against both homologous and heterologous serotypes of P. multocida (Rebers and Heddleston, 1977) in that antibodies from turkeys immunized with live attenuated vaccine or bacterin from in vivo grown bacteria passively cross-protected turkeys against challenge by heterologous serotype organisms (Rebers et al., 1975; Rimler, 1987). Collectively, these results clearly indicate that in vivo grown P. multocida expressed unique immunogens (which were not expressed by in vitro grown bacteria) which were responsible for cross protection. The putative immunogens were named cross-protection factor (CPF) immunogens and found to be associated with the outer membrane vesicles of the bacteria and found to be proteinaceous (Brogden and Rimler, 1983). A more recent study (Lu et al., 1988) established that an OMP was the immunogen responsible for protective immunity against rabbit pasteurellosis. However, the relationship between this OMP and CPF immunogen was not known. Studies generated from our laboratory have recently shown (Choi et al., 1989 ) that in vivo grown P. multocida P 1059 expressed several novel OMPs

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which were not expressed by in vitro grown bacteria. In a series of experiments presented here, we have shown that P. multocida grown both in vitro under iron-restricted conditions and in vivo, expressed several OMPs. Three large molecular weight OMPs, with molecular masses 76 kDa, 84 kDa, and 94 kDa, were expressed by in vivo grown and those grown in iron-restricted media. These three OMPs were designated as the iron-regulated OMPs ( IROMPs ). Studies with other bacterial pathogens have established that they produced high-affinity iron-uptake systems when grown in iron-restricted environments, both in vitro and in vivo, during infections (Griffiths, 1987; Ichihara and Mizushima, 1977 ). An essential feature of these systems was the production of siderophores, and concomitant expression of IROMPs, some of which functioned as receptors of ferric-siderophore during iron uptake of the bacteria. There has been increasing evidence presented that indicate that the iron-uptake systems behave as virulence determinants. Immunoblot analysis was used to determine whether the IROMPs were expressed when the bacteria grows in vivo. We found that only convalescentphase sera from turkeys recovered from experimental pasteurellosis contained antibodies that reacted with IROMPs in immunoblots. In contrast, antisera against OMPs from in vitro grown bacteria (OMP antisera) did not contain antibodies against the IROMPs. We therefore have concluded that the IROMPs were expressed in vivo. Earlier studies (Rimler, 1987) have shown that convalescent-phase sera contained antibodies against the CPF immunogens which passively protected turkeys against challenge with various serotypes of P. multocida. These results, in conjunction with our findings, have tempted others (Ikeda and Hirsh, 1988 ) as well as ourselves to speculate that the IROMPs and the CPF immunogens may be the same molecules. If the 76 kDa, 84 kDa, and 94 kDa OMPs are indeed the IROMPs, then they should be involved in the acquisition of iron by P. multocida. Earlier studies have shown with other bacterial species (Daskaleros and Payne, 1987; Deneer and Potter, 1989) that the Congo Red dye binds only to IROMPs. Our studies revealed that only P. multocida expressing the IROMPs showed modest binding with the dye (Fig. 3). In some species of bacteria the ability to bind to Congo Red has been shown to be an in vitro correlate with virulence (Daskaleros and Payne, 1987; Payne and Finkelstein, 1977; Surgalla and Beesley, 1969). It has not been shown however, whether or not this is true with P. multocida. In other bacterial systems, some of the IROMPs have been shown to function as receptors for the iron-siderophore complex in iron transport (Ichihara and Mizushima, 1977; Sokol and Woods, 1983 ). Previously, we reported that P. multocida produced a unique siderophore called multocidin when grown under iron-restricted conditions (Hu et al., 1986). In a series of experiments presented here, we examined which of these IROMPs were capable of multocidin-mediated iron binding. We found that bacteria grown in iron-re-

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stricted media (BHI + DP, CDM, and CDM + D P ) and in vivo bound only to 59Fe-multocidin complex and not to free 59Fe (Fig. 4a). Bacteria grown in iron-replete media which did not express IROMPs showed minimal binding. Furthermore, OMP extracts prepared from P. multocida grown in iron-restricted media and in vivo also showed high binding of 59Fe-multocidin, while those derived from bacteria grown in iron-replete media showed minimal binding (Fig. 4b ). These findings suggested that the increased binding of 59Femultocidin was directly related to the presence of IROMPs. An additional immunological approach was used to demonstrate the involvement of IROMPs in iron acquisition. Preincubation of bacteria or its membrane extracts expressing IROMPs with convalescent-phase sera blocked significantly the binding of the ~gFe-multocidin. This blocking activity of antibodies present in convalescent-phase sera was found to be specific. Our results also showed that bacterial cells or OMPs extracts containing IROMPs when preincubated with OMP antisera inhibited the binding of ~gFe-multocidin slightly (Fig. 5, lane B), when compared to preincubation with reference negative sera or no sera (Fig. 5, lanes C and D). Most likely, this may be caused by nonspecific binding between IROMPs and antibodies present in the OMP antisera. Finally, autoradiographic analysis was used to determine which of these OMPs, the 76 kDa, 84 kDa, or the 94 kDa IROMPs, functioned as receptor(s) for the iron-multocidin complex. Results derived from these studies showed that when OMP extracts exposed to 2-mercaptoethanol and heat separated in SDS-PAGE were transferred to the nitrocellulose membranes, renatured, and then exposed to radioactively labeled iron-multocidin, all three IROMPs, 76 kDa, 84 kDa, and 94 kDa bound to the complex (Fig. 6). However, when OMPs electroblotted to nitrocellulose membranes were not renatured and then reacted with radioactively labeled iron-multocidin, several OMPs which were not IROMPs showed non-specific binding to the complex (data not shown ). In all likelihood, the renaturation step may have quenched this non-specific binding of the iron-multocidin complex to all blotted proteins. A recent study by Birk and Koepsell (1987) demonstrated that non-specific binding of antibodies to plasma membrane proteins was increased by 2mercaptoethanol or heat denaturation of the antibodies. Furthermore, they found that specific antibody binding was drastically reduced by denaturation of the plasma membrane proteins caused by SDS. They demonstrated that this denaturation by SDS, 2-mercaptoethanol and heat was reversed considerably by exposing the blotted proteins in a renaturing buffer (Birk and Koepsell, 1987 ). Whether these results were compatible with ours will require further clarification. Although these three IROMPs bound specifically with 59Femultocidin complex, it was difficult to conclude that all three IROMPs were indeed iron binding receptors because the denaturing gel system has been

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known to cause the denaturation a n d / o r dissociation of proteins. Sokol and Woods ( 1983 ) reported that ferripyochelin complex bound to a 14 kDa OMP in the P. aeruginosa iron transport system. They also found that when the outer membranes were treated with 2-mercaptoethanol and heat, it abolished the binding of the ferripyochelin to the OMP. They speculated that reduction of the sulfhydral groups in the pyochelin molecule or in the OMP, destroyed the ability to bind the complex. This may be the case with our system. It is quite possible that the 76 kDa, 84 kDa, and 94 kDa IROMPs may be subunits of a larger protein which is dissociated by SDS and still contained the specific binding epitopes for iron-multocidin complex. Additional studies are however necessary for further confirmation. To our knowledge, this study represented the first time that a comparison had been made between the novel OMPs expressed by in vivo grown P. multocida and the IROMPs expressed by bacteria grown under iron-restricted conditions. The data presented, clearly demonstrated that the three IROMPs with molecular masses 76 kDa, 84 kDa, and 94 kDa were also expressed by in vivo grown bacteria. However, the data presented did not provide enough evidence to prove our original hypothesis that IROMPs and CPF immunogens were the same molecules. Clearly, additional experimentation such as vaccination challenge studies using domesticated poultry will provide further insights into the identity of CPF immunogens and IROMPs. ACKNOWLEDGMENTS

Contribution No. 17947 of the Minnesota Agricultural Experiment Station based on research supported by AES grant MN 63-053.

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