Characterization of outer membrane and secreted proteins of Leptospira interrogans serovar pomona

Microbial Pathogenesis 1991 ; 10 : 311-322

Characterization of outer membrane and secreted proteins of Leptospira interrogans serovar pomona Richard L . Zuerner,' William Knudtson, 2 .3 Carole A . Bolin,' and Gabriel Trueba" 'Leptospirosis and Mycobacteriosis Research Unit, National Animal Disease Center, U .S . Department of Agriculture, Agricultural Research Service, P.O. Box 70, Ames, Iowa 50010, U.S.A ., 2National Veterinary Services Laboratory, U .S . Department of Agriculture, Animal Plant Health Inspection Service, P .O . Box 844, Ames, Iowa 50010, U .S .A ., 3 Sanofi Animal Health Inc ., 12300 Santa Fe, Lenexa, Kansas 66215, U .S.A . and 'Department of Veterinary Microbiology, Iowa State University, Ames, Iowa 50011, U .S .A .

(Received November 23, 1990 ; accepted in revised form January 11, 1991)

Zuerner, R . L . (Leptospirosis and Mycobacteriosis Research Unit, National Animal Disease Center, U .S . Dept of Agriculture, Agricultural Research Service, P .O . Box 70, Ames, Iowa 50010, U .S .A .), W . Knudtson, C . A . Bolin and G . Trueba . Characterization of outer membrane and secreted proteins of Leptospira interrogans serovar pomona . Microbial Pathogenesis 1991 ; 10 :311-322 .

Outer membrane and secreted proteins were isolated from Leptospira interrogans serovar pomona and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblot and radioimmunoprecipitation techniques . The L . interrogans outer membranes were extracted with Triton X-1 14 and contained several proteins . The major cellular protein with a molecular mass of 31 kDa was associated exclusively with the L. interrogans outer membrane . Using a whole cell immunoprecipitation method, five hydrophobic, Triton X-114 extractable proteins (22, 26, 31, 36 and 42 kDa) were exposed on the surface of L . interrogans. The 31 kDa protein was heat labile and was a potent antigen in animals experimentally infected with L . interrogans serovar pomona . Several proteins were secreted by L . interrogans including a 60 kDa protein tentatively identified as the L . interrogans hemolysin . Key words: Leptospira interrogans ; outer membrane; secreted proteins ; antigens .

Introduction Leptospira interrogans is the etiological agent of leptospirosis, a widespread zoonosis

in humans and an important disease in animals . Leptospira interrogans infections in humans can range in severity from subclinical infections to death as a result of renal or hepatic failure .' One of the most common signs of leptospirosis in animals is reproductive failure (abortions, stillbirths and infertility) . 2-5 Leptospirosis in animals is controlled by vaccination . Current L . interrogans vaccines composed of heat-killed or chemically killed bacteria can protect animals against many clinical signs of leptospirosis .' However renal infections (resulting in leptospiuria) or reproductive failure may still occur in vaccinated animals .' •$ Since urinary shedding of viable L . interrogans is an important source of infection for humans and non-vaccinated animals,' improvement of existing vaccines is needed . Additionally, safe and effective vaccines for humans are not available . 0882-4010/91/040311 +12 $03 .00/0

n 1991 Academic Press Limited



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The primary targets for immunoglobulin-mediated killing of L . interrogans are situated in the outer membrane . 10 The immune response to one of the outer membrane components, lipopolysaccharide (LPS), has received considerable attention . Monoclonal antibodies against leptospiral LPS are opsonic 11 and passive immunization with anti-LPS monoclonal antibodies protects hamsters and guinea-pigs against lethal challenge 12 (W . Knudtson, unpublished data) . Immunization with an outer membrane glycolipid, presumably LPS, protects hamsters against homologous challenge ." These studies show that antibody against LPS is an important component of the protective immune response in some animal models . However, cattle with high antibody titers to LPS are not protected against experimental challenge .' $ These results indicate that antibody to other components of L . interrogans outer membranes, perhaps protein, may be needed to develop protective immunity . Little is known about the outer membrane proteins of L . interrogans. Few leptospiral outer membrane proteins (OMPs) have been identified ."' The presence of major cellular proteins in the outer membrane of L . interrogans has not been demonstrated . The purpose of this study was to characterize OMPs from L . interrogans serovar pomona type kennewicki, a common pathogen of livestock . The results of this study show that the outer membrane of L . interrogans serovar pomona contains a major cellular protein which is a potent antigen in infected animals . In addition, we show that L . interrogans serovar pomona secretes several proteins including a previously described hemolysin . Results Extraction of L . interrogans outer membranes Triton X-114 has been used previously to isolate and characterize outer membranes from several organisms including two other spirochetes : Treponema pa/lidum 18 • 1 9 and Borrelia burgdorferi. 2021 In the present study the ability of this detergent to solubilize the outer membrane of L . interrogans serovar pomona was tested . Leptospira interrogans cells were treated with 2% Triton X-1 14 and the soluble material separated by centrifugation . Electron microscopic analysis of detergent insoluble material showed that Triton X-1 14 removed the leptospiral outer membrane but cells retained periplasmic flagella and helical shape (data not shown) . Based on these results, it was concluded that the Triton X-114 soluble fraction contained outer membrane and perhaps periplasmic material . Since L . interrogans are grown in complex media containing serum proteins, bacterial proteins were labeled by growth in the presence of [ 35S]-methionine and analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography . A sample of cells taken before detergent solubilization contained many radiolabeled proteins [Fig . 1 (a)] . Many of these proteins remained associated with the protoplasmic cylinder after detergent treatment [Fig . 1 (a)] . The 31 and 42 kDa proteins were the most prominent proteins in the Triton X-114 solubilized fraction [Fig . 1 (a)] . No differences were seen in protein profiles of Triton X-1 14 extracts using lower (0 .1-1 %) detergent concentrations (data not shown) . Two proteins (45 and 46 kDa) apparent in whole cells were destroyed during detergent solubilization of the outer membrane . Hydrophobic proteins (e .g . integral membrane proteins) can be separated from hydrophilic proteins by phase partitioning using Triton X-114 . 22 Phase partitioned Triton X-1 14-solubilized outer membrane material from L . interrogans showed that several proteins (including the 42 kDa protein) appeared to be amphiphilic, being extracted into both aqueous and detergent phases [Fig . 1 (a)] . The results do not



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Fig . 1 . Characterization of L . interrogans proteins. (a) Radiolabeled L . interrogans proteins were characterized by SDS-PAGE and fluorography . Lanes : 1, whole cells ; 2, protoplasmic cylinder ; 3, Triton X114 extract ; 4, Triton X-1 14 extract heated at 37'C for 30 min ; 5, aqueous phase; 6, detergent phase; 7, aqueous phase prepared with 20 mm CaCl 2 ; 8, detergent phase prepared with 20 mm CaCl2 ; 9-11, surface exposed proteins radioimmunoprecipitated after adsorption of whole cells with buffer (lane 9), normal rabbit sera (lane 10), or hyperimmune rabbit sera (lane 11) . Approximately equal amounts of radioactivity were loaded onto lanes 1-8, and equal volumes of immunoprecipitated proteins onto lanes 9-11 . (b) Leptospira interrogans fractions separated by SDS-PAGE and stained with silver . Lanes : 1, whole cells ; 2, protoplasmic cylinder ; 3, Triton X-1 14 extract. Migration of molecular mass markers (kDa) is indicated to the left .

preclude the possibility that these 'amphiphilic' proteins are actually distinct protein species which comigrate in SDS-PAGE . Some proteins were found exclusively in the detergent phase [Fig . 1 (a)] as was the leptospiral LPS (data not shown) . Some proteins present in the detergent phase (lanes 6 and 8) were not seen in samples of whole cells or outer membranes, except when the film was overexposed . Since these proteins represent a small fraction of the overall radioactive protein in samples of whole cells and outer membranes, their prominence in the detergent phase indicates that they are concentrated by phase partitioning . The 31 kDa protein was degraded during the incubation step used to effect phase partitioning [Fig . 1 (a)] . Loss of the 31 kDa protein coincided with the appearance of a 12 kDa protein which was not seen in extracts before incubation [Fig . 1 (a)] . Western blot analysis suggests that the 12 and 31 kDa proteins are immunologically related (data not shown), thus the 12 kDa protein is thought to comprise part of the degraded 31 kDa protein . Calcium chloride (20 mm) stabilized the 31 kDa protein (see below) and was added to Triton X-1 14 extracts before phase partitioning . In the presence of CaCI 2 , the 31 kDa protein partitioned exclusively into the detergent phase [Fig . 1 (a)] . Since proteins exposed on the surface of L . interrogans may be important targets of



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a protective immune response, 10 surface-exposed proteins were identified by a wholecell immunoprecipitation technique . 14 Rabbit serum prepared against L . interrogans serovar pomona type kennewicki was adsorbed to intact radiolabeled bacteria, the outer membranes solubilized, and antibody-bound proteins precipitated with staphylococcal protein A . 14 Using this method the most prominent surface-exposed proteins were the 22, 26, 31, 36, 42 and 63 kDa proteins [Fig . 1 (a)] . Control experiments showed that none of the proteins identified as surface-exposed by this method corresponded to periplasmic flagella (not shown) . This result suggests that the bacteria were intact and only surface proteins were immunoprecipitated . To identify major cellular proteins, samples of whole L . interrogans serovar pomona type kennewicki cells, a detergent insoluble protoplasmic cylinder, and detergent soluble outer membrane fraction were separated by SDS-PAGE and stained with silver . The most prominent stained protein in samples of whole cells was the 31 kDa protein [Fig . 1 (b)] . This protein was associated exclusively with the outer membrane fraction after Triton X-114 extraction . Heat sensitivity of the 31 kDa protein The heat sensitivity of the 31 kDa protein was examined in greater detail . The lability of this protein in situ was determined by incubating radiolabeled cells at either 30°C (the normal in vitro growth temperature), 37 ° C, or on ice . Cells were harvested by centrifugation, the outer membranes extracted with Triton X-114, and then analysed by SDS-PAGE . The outer membranes of cells incubated at 37°C underwent several changes as compared with control samples . After incubation at 37°C, the outer membranes contained proportionately more of the 63 kDa protein and less of the 31, 42, 55 and 78 kDa proteins (Fig . 2) . The amounts of the 42, 55 and 78 kDa proteins present in the outer membrane were reduced through the process of protein secretion (see below, Fig . 4), rather than degradation . In contrast, the 31 kDa protein did not appear to be secreted by these cells and was the only protein degraded at 37°C (see Fig . 3) . Protein profiles of outer membranes extracted from cells incubated on ice or at 30°C were similar to a control sample obtained before incubation (Fig . 2) . Stabilization of the 31 kDa protein Initial experiments showed that the 31 kDa protein was the only heat-sensitive outer membrane and that CaC1 2 stabilized this protein during incubation at 37°C [Fig . 3(a)] . The effects of other cations and the ion chelator ethylene diamine tetraacetic acid (EDTA), each at 20 mm concentration, on the stability of the 31 kDa protein were determined . In addition to CaCI 2 , both CuC1 2 and ZnC1 2 stabilized the 31 kDa protein, whereas MgC1 2 , SrCI 2 , MnC1 2 , CoC1 2, NiC1 2 , KCI, RbCI, CsCl and EDTA had no effect (data not shown) . The concentrations of CaC1 2 , CuC1 2 and ZnC1 2 were titrated to determine the minimal concentration of each compound which stabilized the 31 kDa protein . The results show that the 31 kDa protein was stable in the presence of 0 .5 mm CaCI 2 , 2 mm ZnC1 2 , or 20 mm CuCI 2 [Fig . 3(b)] . In reactions containing 0 .05 mm to 5 mm ZnCI 2 , three proteins with molecular weights of 17 kDa, 24 kDa and 28 kDa were seen which were not present in unheated samples (data not shown) and are thought to represent partial digestion products of the 31 kDa protein . This result suggested that the 31 kDa protein was degraded by an endopeptidase . Several protease inhibitors were tested for the ability to stabilize the 31 kDa protein . These included antipapain dihydrochloride, [(4-amidinophenyl)-methanesulfonyl fluoride], aprotinin, bestatin, chymostatin, E-64, leupeptin, pepstatin, phenylmethanesulfonyl fluoride and phosphoramidon . Of these, only E-64, an inhibitor of cysteine



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Fig . 2. Heat lability of 31 kDa protein in situ. Outer membranes were prepared from cells before (lane 1 ) or after incubation on ice (lanes 2 and 3), at 30°C (lanes 4 and 5), or 37°C (lanes 6 and 7) for 1 h (lanes 2, 4 and 6), or 2 h (lanes 3, 5 and 7) . Migration of molecular mass markers (kDa) is indicated to the left .

proteases23 protected the 31 kDa protein (data not shown) . Attempts to detect protease activity using other protein substrates were unsuccessful . Protein secretion by L . interrogans We tested the possibility that L . interrogans secretes protein by obtaining cleared supernatants from cells incubated at either 37°C or on ice for 2 h in buffer . Analysis of cell-free supernates by SDS-PAGE showed that although the cells appeared intact after heating at 37°C, several proteins were released from these cells [Fig . 4(a)] . Included among these were the 42 kDa and 78 kDa proteins which were lost from the outer membrane of cells incubated at 37°C (Fig . 2) . Several proteins were also secreted into the culture medium during growth of L . interrogans at 30°C [Fig . 4(a)] . In this sample, protein migration between 50 and 70 kDa was distorted as a result of the presence of bovine serum albumin from the culture medium . The antigenicity of radiolabeled leptospiral proteins secreted into the culture medium during growth of L . interrogans was determined . Several secreted proteins were immunoprecipitated with sera from rabbits immunized with viable L . interrogans serovar pomona including two prominent proteins [52 and 60 kDa ; Fig . 4(a)] . Similarly, several secreted proteins including the 52 and 60 kDa proteins were precipitated with serum collected from pigs 35 days after infection with L . interrogans serovar pomona [Fig . 4(a)] .



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Fig . 3 . Stabilization of the 31 kDa protein . (a) Outer membrane samples were solubilized by boiling in SDS-PAGE sample buffer before (lane 1) or after incubation at 37°C for 30 min (lanes 2 and 3) . Incubated samples lacked (lane 2) or contained 20 mm CaCl 2 (lane 3) . Migration of molecular mass markers (kDa) is indicated to the left . (b) Titration curve for stabilizing the 31 kDa protein using CaCl 2 (Ca), ZnCl 2 (Zn), and CuC1 2 (Cu) . Samples of outer membrane were heated at 37°C for 30 min in the absence (lane 1) or presence of the appropriate cation (lanes 2 to 10) at the following concentrations, lanes : 2, 0.05 mm ; 3, 0.1 mm ; 4, 0.2 mm ; 5, 0 .5 mm ; 6, 1 mm ; 7, 2 mm ; 8, 5 mm ; 9, 10 mm ; 10, 20 mm . Only the relevant portion of the gel is shown, with the 31 kDa protein near the bottom of each panel and the 42 kDa protein, for comparison, near the top .

Leptospira interrogans secretes a hemolysin which lyses sheep erythrocytes .24 Since L . interrogans secretes several proteins [Fig . 4(a)], the possibility that one or more of these proteins were associated with hemolytic activity was tested . Since detergents present in the culture medium for L . interrogans are toxic and lyse sheep erythrocytes, the samples used for analysis of hemolytic activity were cell-free supernates of washed cells incubated at 37°C in buffer . The results of this experiment show that hemolysin was secreted by L . interrogans, and that secretion of both protein (measured in a parallel experiment using radiolabeled cells) and hemolysin coincided and were temperature dependent [Fig . 4(b)] . Immunological analysis of L . interrogans OMPs Serum samples from pigs infected with L . interrogans were used to identify outer membrane antigens which were recognized by the host animal during infection . Western blot analysis of Triton X-1 14 extracted outer membranes was used to examine both IgM [Fig . 5(a)] and IgG [Fig . 5(b)] responses to L . interrogans infection . Both pig IgM and IgG reacted predominantly with the 31 kDa protein, although reactions with some additional proteins were seen [Fig . 5(a) and (b)] . Antibodies to LPS were



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Fig . 4 . Protein secretion by L . interrogans. (a) Cell-free supernatants obtained before (lane 1) or after L . interrogans cells were incubated for 2 h on ice (lane 2), or at 37'C (lane 3) . Radiolabeled proteins released during growth of L . interrogans in the presence of [ 35 S]-methionine (lane 4) . Lanes 5-10 are radioimmunoprecipitated secreted proteins . Lanes: 5, no sera ; 6, normal rabbit sera ; 7, hyperimmune rabbit sera ; 8, serum from a germ free pig; 9, infected pig no. 14, 35 days p .i . ; 10, infected pig no . 15, 35 days p .i . Migration of molecular mass standards is indicated to the left. (b) Comparison of radioactivity released from intact cells with hemolytic activity . The amount of radioactivity released from cells is compared with hemolytic activity of cell-free supernates measured in parallel experiments .

not detected in pig sera by this method . However, control immunoblots using serum from a rabbit immunized with L . interrogans serovar pomona [Fig . 5(c)] and a mouse monoclonal antibody against L . interrogans serovar pomona LPS (data not shown) confirmed the presence of LPS on these blots (M r approximately 28 kDa) . In addition to LPS, hyperimmune rabbit antiserum also reacted with several outer membrane antigens including the 31 kDa protein . Since sera from infected pigs agglutinated L . interrogans serovar pomona but did not appear to react with LPS, the agglutinating antibodies may be directed against one or more surface proteins . Discussion The results of this study show that Triton X-1 14 efficiently removed the L . interrogans outer membrane, but left the protoplasmic cylinder intact. Thus the Triton X-114 extracted material is thought to contain outer membrane and perhaps some periplasmic but not cytoplasmic constituents . The results reported here are consistent with the results of similar experiments performed with T. pa//idum 18 • 1 9 and B . burgdorferi 20,2'



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Fig . 5. Immunoblot analysis of L . interrogans outer membrane antigens . Western blots containing serovar pomona outer membranes were reacted with sera from a pig infected with L . interrogans (a and b) . Lanes : 1, 1 day p .i . ; 2, 10 days p .i . ; 3, 17 days p .i . ; 4, 24 days p .i . ; 5, 30 days p .i . ; 6, 35 days p .i . Blots were reacted with goat anti-pig IgM (a) or anti-pig IgG (b) horseradish peroxidase conjugated secondary antibody . (c) Control immunoblot reacted with normal (lane 1) or hyperimmune (lane 2) rabbit sera followed by reaction with horseradish peroxidase conjugated secondary antibody directed against rabbit IgG . Migration of molecular mass markers is indicated to the left .

using Triton X-114 . Combined, these studies show that Triton X-114 is a detergent well suited for analysis of spirochete outer membrane components . Based on the surface exposure and hydrophobicity of several Triton X-1 14 extracted proteins (22, 26, 31, 36 and 42 kDa), we conclude that these proteins are integrated in the outer membrane . 22 The major cellular protein in L . interrogans serovar pomona is a 31 kDa hydrophobic protein located in the outer membrane and is exposed on the surface of the cell . The presence of a major OMP in L . interrogans has not been shown previously . Antiserum from infected animals showed that the 31 kDa protein is the predominant leptospiral immunogen in the outer membrane . This finding is consistent with similar experiments done with T. pa//idem' $ and B . burgorferi 21 which show that integral OMPs are highly immunogenic . Since the 31 kDa protein was digested in situ by an endogenous bacterial protease at 37°C, infected animals probably mount an immune response to a digestion product of this protein, and not the native 31 kDa protein . The role of the 31 kDa protein in virulence of L . interrogans is unknown . Since the 31 kDa protein is the only heat labile OMP, it may be analogous to a thermolabile Vi antigen described in L . interrogans serovars icterohaemorrhagiae 25 and pomona . 26 The L . interrogans serovar pomona Vi antigen protects the bacteria from antibodycomplement mediated killing . 26 Although a virulence associated 31 kDa OMP was identified in L . interrogans serovar copenhageni 15 the stability of this protein at 100 ° C led those investigators to assume that the protein was heat stable . The results of the present study showed that although the 31 kDa protein is stable at 100°C, it is unstable

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at 37 and 56°C (not shown) . The temperatures at which we show the 31 kDa protein to be unstable are consistent with the temperatures used to establish the presence of a heat-labile antigen .", " The stability of the 31 kDa protein at 100°C is also consistent with our conclusion that this protein is degraded by a cysteine protease, which would be inactivated by boiling . The presence of this protease was shown by the stabilization of the 31 kDa protein by E-64, a specific inhibitor of cysteine proteases . 23 The 31 kDa protein was also stabilized by CaC1 2 , ZnC1 2 and CuCl 2 . Although common in eukaryotic organisms, cysteine proteases are rarely found in bacteria . 27 Since the 31 kDa protein was the only OMP degraded by this protease, and no alternative protease substrates were identified, the proteolytic activity described here appears to be specific for targets within the 31 kDa protein, and may be related to processing this OMP . It should be noted that a cysteine protease identified in group A staphylococci may also be involved with processing bacterial surface proteins . 27 Several proteins are secreted by L . interrogans serovar pomona including a previously recognized hemolysin . 24 Nucleotide sequence analysis of cloned L . interrogans serovars hardjo and pomona hemolysin genes predicts synthesis of a 63 268 dalton precursor protein containing a 3175 dalton signal peptide . 25 These predictions are in excellent agreement with a 60 kDa secreted protein and a 63 kDa cell associated protein identified in this study as the mature and precursor forms of the serovar pomona hemolysin, respectively . Although most of the 63 kDa protein is associated with the protoplasmic cylinder after Triton X-1 14 extraction, some of the 63 kDa protein is detergent soluble and is exposed on the surface of L . interrogans cells . Also, outer membranes extracted from cells incubated at 37°C contain proportionately more 63 kDa protein than control samples . Since the sizes of the 63 kDa proteins in outer membranes and protoplasmic cylinders are identical, it appears that this protein is not processed during migration to the outer membrane as often occurs during protein secretion in Escherichia co/i. 29 Instead, the signal peptide is removed during or after release of this protein from the outer membrane . Immunoprecipitation experiments showed that both a 52 kDa protein and the 60 kDa protein were expressed during infection . Hence these proteins may be important in virulence of the bacteria . Leptospira interrogans-infected pigs failed to develop detectable antibodies to leptospiral LPS . This result is surprising since leptospiral LPS is often a potent immunogen ." We are currently attempting to determine if the apparent lack of antibodies to LPS in infected pigs is due to the route of challenge exposure or to an inability of the pigs to mount an immune response to serovar pomona LPS . The results of this study establish a foundation for further analysis on the immunobiology and pathogenesis of L . interrogans infections .

Materials and methods

Bacterial strains . Leptospira interrogans serovar pomona type kennewicki isolate RM211 30 was colony-purified by plating on Ellinghausen-McCullough-Johnson-Harris medium 31 supplemented with 1 % rabbit sera and solidified with 1 % agar . Leptospira interrogans serovar pomona type kennewicki isolate 11000-45A was used for infection of piglets . Leptospira interrogans was propagated in bovine serum albumin-Tween 80 medium at 30°C . 31 Outer membrane extraction . Leptospira interrogans proteins were labeled by growth in media containing 10pCi ml -1 [ 35 S]-methionine (Tran 35 S-label, ICN Pharmaceuticals, Irvine, California, 1611 Ci mmol - ') for three to four generations." Cells were harvested by centrifugation (16000xg for 15 min) and washed twice with 50 mm Tris-HCI, pH 7 .2, 150 mm NaCl (TBS) . The cell pellet (approximately 3x 10 9 cells) was suspended in 1 .5 ml TBS containing 2% Triton X-1 14 (Sigma Chemical, St Louis, Missouri) and held on ice for 20 min . Detergent-soluble outer membrane material was separated from a detergent-insoluble protoplasmic cylinder by



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centrifugation (16000xg for 10 min at 4°C) . Phase partitioning of detergent soluble material was achieved by incubating extracts at 37°C for 30 min followed by centrifugation (16000xg for 10 min at 22°C) . Each phase was washed three times with buffer (detergent phase) or detergent (aqueous phase) .' $ All samples were stored at -20°C until analysis . Batch preparations of outer membranes were prepared for Western blot analysis from cells grown to mid-logarithmic phase in 500-1000 ml cultures and by scaling up the detergent solubilization . Heat lability of the 31 kDa protein. Outer membrane preparations were incubated at 37°C for 30 min prior to analysis by SDS-PAGE . The effects of various compounds on the 31 kDa protein were determined by adding the compound before incubation . Protease inhibitors used to stabilize the 31 kDa protein were obtained from Boehringer-Mannheim Biochemicals (Indianapolis, Indiana), and were used at the concentrations recommended by the supplier . Protein secretion . Leptospira interrogans cells were labeled with [ 35 S]-methionine, harvested by centrifugation, and washed twice with TBS as described above . Cells were suspended in TBS and incubated at 37°C, or on ice . Samples were removed after 2 h, the cells pelleted by centrifugation (16000xg for 10 min), and the supernatant fluid centrifuged again . Cell-free supernatants were prepared for assay of hemolytic activity by the same technique, using cells harvested from a 500 ml culture . Hemolytic activity of cell-free supernates was determined using sheep erythrocytes .32 Antisera . Rabbit antiserum was prepared against L . interrogans serovar pomona type kennewicki isolate RM211 as described previously ." Caesarean-derived colostrum-deprived pigs (48 days old) were infected with 0 .2 ml (105-106 cells ml - ') logarithmic phase cultures of L . interrogans serovar pomona type kennewicki isolate 11 000-45A by conjunctival instillation .' Pigs were kept in isolation and sera collected at 1, 10, 17, 24, 30 and 35 days post-infection (P-0Immunoprecipitation . Leptospira interrogans cells were labeled with [ 35 S]-methionine, harvested by centrifugation, and washed twice with TBS as described above . The cells were resuspended in TBS at an approximate concentration of 1 )<10 9 cells ml - ' . Equal amounts of the cell suspension were mixed with either 25 pl TBS, serum from a control rabbit, or serum from a rabbit immunized with L . interrogans serovar pomona, and incubated for 90 min at 4°C . Immunoreactive surface exposed proteins were precipitated from washed and solubilized cells . 14 Culture medium from cells grown with [ 35 S]-methionine used as a source of radiolabeled secreted proteins was cleared by two successive centrifugations at 30 000 xg . Proteins contained in 1 ml cleared culture medium were reacted with 100 fd serum then immunoprecipitated ." Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis . Cell fractions were boiled 5 min in sample buffer (100 mm Tris-HCI, pH 6 .8, 2% SDS, 1 .2% glycerol, 0 .25 M 2-mercaptoethanol, 0 .01% bromophenyl blue) . Protein samples were fractionated by discontinuous Tris-Tricine SDS-PAGE in 10% gels ." Radiolabeled proteins were visualized by fluorography . Proteins were stained with silver using a commercial kit (Bio-Rad Laboratories, Richmond, California) . 14 C-labeled molecular mass standards were purchased from Amersham (Arlington Heights, Illinois) . Western blots were prepared by transferring SDS-PAGE separated proteins to Immobilon-P PVDF membranes (Millipore, Bedford, Massachusetts) in 25 mm Tris, 192 mm glycine at 0 .1 A for 16 h followed by 2 h at 0 .5 A using a Transphor electrophoretic transfer unit (Pharmacia LKB Biotechnology, Piscataway, New Jersey) at 4°C . Membranes were blocked for 1 h at 22°C with 0 .05% Tween 20-PBS (TPBS) . Membrane bound antigens were incubated overnight at 4°C with pig serum diluted 1 :200 or rabbit sera diluted 1 :500 in TPBS . Membranes were washed with TPBS then incubated for 1 h at 22°C with a 1 :200 dilution of secondary antibody (goat anti-pig IgM, goat anti-pig IgG or goat anti-rabbit IgG antisera conjugated with horseradish peroxidase ; Kirkegaard and Perry Laboratories, Gaithersburg, Maryland) . Membranes were washed three times with TPBS, then three times with PBS . Membranes were incubated in PBS containing 0 .6 mg 4-chloro-1-naphthol ml - ' and 1 .8% H 2 0 2 for approximately 10 min . Enzyme reactions were terminated by washing membranes with distilled H 2 0 .

We wish to thank T . Casey and E . Dean for helpful suggestions, and A . Handsaker and J . Foley



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for technical assistance . We thank E . Dean, S . Halling and J . Ridpath for reviewing the manuscript .

References 1 . Feigin RD, Anderson DC . Human leptospirosis . CRC Crit Rev Clin Lab Sci 1975 ; 11 : 413-67 . 2 . Ellis WA, O'Brien JJ, Bryson DG, Mackie DP . Bovine leptospirosis : some clinical features of serovar hardjo infection . Vet Rec 1985; 117 : 101-4 . 3 . Ellis WA, O'Brien JJ, Neill SD, Bryson DG . Bovine leptospirosis : experimental serovar hardjo infection . Vet Microbiol 1986 ; 11 : 293-9 . 4 . Ellis WA, O'Brien JJ, Neill SD, Hanna J, Bryson DG . The isolation of a leptospire from an aborted bovine fetus . Vet Rec 1976 ; 99 : 458-9 . 5 . Thiermann AB . Experimental leptospiral infections in pregnant cattle with organisms of the hebdomadis serogroup . Am J Vet Res 1982 ; 43 : 780-4 . 6 . Tripathy DN, Hanson LE, Mansfield ME . Evaluation of the immune response of cattle to leptospiral bacterins . Am J Vet Res 1976 ; 37 : 51-5 . 7 . Bolin CA, Thiermann AB, Handsaker AL, Foley J . Effect of vaccination with a pentavalent leptospiral vaccine on Leptospira interrogans serovar hardjo type hardjo-bovis infection of pregnant cattle . Am J Vet Res 1989 ; 50: 161-5 . 8 . Bolin CA, Zuerner RL, Trueba G . Effect of vaccination with a pentavalent leptospiral vaccine containing Leptospira interrogans serovar hardjo type hardjo-bovis on type hardjo-bovis infection of cattle . Am J Vet Res 1989 ; 50 : 2004-8 . 9 . Michna SW . Leptospiroris . Vet Rec 1970 ; 86 : 484-96 . 10 . Anderson DL, Johnson RC . Electron microscopy of immune disruption of leptospires : action of complement and lysozyme . J Bacteriol 1968 ; 95 : 2293-309 . 11 . Farrelly HE, Adler B, Faine S . Opsonic monoclonal antibodies against lipopolysaccharide antigens of Leptospira interrogans serovar hardjo. J Med Microbiol 1987 ; 23 : 1-7 . 12 . Schoone GJ, Everard COR, Korver H et al. An immunoprotective monoclonal antibody directed against Leptospira interrogans serovar copenhageni. J Gen Microbiol 1989; 135 : 73-8 . 13 . Masuzawa T, Nakamura R, Shimizu T, Iwamoto Y, Morita T, Yanagihara Y . Immunological characteristics of the glycolipid antigen of Leptospira interrogans serovar lai. Infect Immun 1989; 57 : 2502-6 . 14 . Nunes-Edwards PL, Thiermann AB, Bassford PJ Jr, Stamm LV . Identification and characterization of the protein antigens of Leptospira interrogans serovar hardjo . Infect Immun 1985 ; 48 : 492-7 . 15 . Niikura M, Ono E, Yanagawa R . Molecular comparison of antigens and proteins of virulent and avirulent clones of Leptospira interrogans serovar copenhageni, strain Shibura . Zbl Bakt Hyg A 1987 ; 266 : 45362 . 16 . Chapman AJ, Adler B, Faine S . Antigens recognized by the human immune response to infection with Leptospira interrogans serovar hardjo . J Med Microbiol 1988; 25 : 269-78 . 17 . Hata K, Ono E, Yanagawa R . Comparative studies of strains Ictero No . I and RGA as the type strain of Leptospira interrogans : agglutinin absorption test, protein and antigen profiles, and enzyme activities . Microbiol Immunol 1988 ; 32 : 817-32 . 18 . Radolf JD, Chamberlain NR, Clausell A, Norgard MV . Identification and localization of integral membrane proteins of virulent Treponema pallidum subs . pallidum by phase partitioning with the nonionic detergent Triton X-1 14 . Infect Immun 1988 ; 56 : 490-8 . 19 . Radolf JD, Norgard MV . Pathogen specificity of Treponema pallidum subsp pallidum m integral membrane proteins identified by phase partitioning with Triton X-114 . Infect Immun 1988; 56 : 1825-8 . 20 . Brandt ME, Riley BS, Radolf JD, Norgard MV . Immunogenic integral membrane proteins of Borrelia burgdorferi are lipoproteins . Infect Immun 1990 ; 58 : 983-91 . 21 . Cunningham TM, Thomas DD, Thompson SD, Miller JN, Lovett MA . Identification of Borrelia burgdorferi surface components by Triton X-114 phase partitioning . Ann NY Acad Sci 1988 ; 539 : 376-8 . 22 . Bordier C . Phase separation of integral membrane proteins in Triton X-1 14 solution . J Biol Chem 1981 : 256 :1604 7 23 . Barrett AJ Kembhavi AA, Brown MA et al. L-trans-epoxysuccinyl-leucyl amido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L . Biochem J 1982 ; 201 : 189-98 . 24 . Alexander AD, Smit OH, Hiatt CW, Gleiser CA . Presence of hemolysin in cultures of pathogenic leptospires . Proc Soc Exp Biol Med 1956 ; 91 : 205-11 . 25 . Borg-Petersen C . A thermolabile antigen in the Leptospira strain Ictero No . I . Trop Geogr Med 1971 . 23 : 282-5 . 26 . Uneo K, Yanagawa R, Kida H . Presence of Vi antigen in a virulent strain of Leptospira interrogans serovar pomona and relation of Vi antigens of leptospiras to resistance to leptospiricidal activity mediated by antiserum plus complement . Zbl Bakt Hyg I Abt Orig A 1982 ; 252 : 557-65 . 27 . Bjorck L . Proteinase inhibition, immunoglobulin-binding proteins and a novel antimicrobial principle . Mol Microbiol 1990 ; 4 :1439-42 . 28 . Segers RAM, van der Drift A, de Nijs A, Corcione P, van der Zeijst BAM, Gaastra W . Molecular analysis



322

29 . 30 .

31 . 32 . 33 .

R . L . Zuerner et al of a sphingomyelinase C gene from Leptospira interrogans serovar hardjo . Infect Immun 1990 ; 58 : 2177-85 . Pugsley AP, Schwartz M . Export and secretion of proteins by bacteria . FEMS Microbiol Rev 1985 ; 3 : 3-38. Thiermann AB, Handsaker AL, Moseley SL, Kingscote BF . New method for classification of leptospiral isolates belonging to serogroup Pomona by restriction endonuclease analysis : serovar kennewicki. J Clin Microbiol 1985; 21 : 585-7 . Johnson RC, Harris VG . Differentiation of pathogenic and saprophytic leptospires . I . Growth at low temperatures . J Bacteriol 1967 ; 94 : 27-31 . Yanagihara Y, Kojima T, Mifuchi I . Hemolytic activity of Leptospira interrogans serovar canicola cultured in protein-free medium . Microbiol Immunol 1982 ; 26 : 547-56 . Schagger H, von Jagow G . Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa . Anal Biochem 1987 ; 166 : 368-79 .