INTRODUCTION

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Leptospirosis is an important global human and veterinary health problem caused by spirochetes belonging to the genus Leptospira. Human leptospirosis is a potentially fatal disease which appears to be emerging in both developed and underdeveloped regions of the world (11, 42). In domestic animals, leptospirosis is an important cause of abortion, stillbirth, infertility, decreased milk production, and death (41). Leptospires are ubiquitous in nature, reflecting their ability to adapt to both the ambient environment and the renal tubules of chronically infected reservoir hosts. Cattle and feral rodents are the most important reservoir hosts, although pathogenic Leptospira species have been isolated from essentially every known mammalian species. Leptospirosis control efforts have also been hampered by the fact that commercially available veterinary vaccines, which consist of inactivated whole-cell bacterins, depend largely on serovar-specific leptospiral lipopolysaccharide (LPS) carbohydrate antigens for their efficacy. This approach has been demonstrated to be ineffective in the prevention of disease in cattle (6-8), and its efficacy in other animals has serious limitations (41). For these reasons, there is an urgent need for development of alternative vaccine strategies relying on an improved understanding of leptospiral outer membrane proteins (OMPs).

The focus of our research has been to identify and characterize OMPs which are relevant in the pathogenesis of leptospirosis. For this reason, we have been interested in studying how levels of OMP expression change when cultivated, virulent leptospires are introduced into a mammalian host. The pathogenic Leptospira species L. interrogans and L. kirschneri and other invasive spirochetes express uniquely low levels of transmembrane OMPs (14, 26, 30, 32, 44, 45). Downregulation of OMP expression may be an important mechanism by which spirochetes evade the host immune response (4, 14, 20, 29, 32, 44, 45). Consistent with this hypothesis, there is a correlation between decreased levels of transmembrane OMPs and pathogenicity in both L. kirschneri and Borrelia burgdorferi (14, 31). In the case of L. kirschneri, this observation was a key to the identification of the rare OMP OmpL1, a surface-exposed leptospiral porin (13, 37). A number of proteins have been shown to be subject to differential expression during the life cycle of B. burgdorferi. Expression of the outer surface protein OspA and the outer membrane-associated lipoprotein lp6.6 is downregulated when B. burgdorferi in the tick midgut infects the mammalian host (3, 22). Expression of other B. burgdorferi proteins, including EppA, OspC, OspE, OspF, and pG, is upregulated during mammalian infection (10, 36, 40, 46).

Studies designed to identify OMPs have suggested that some of the most abundant leptospiral proteins are associated with the outer membrane (9, 14, 27, 51). For example, the most prominent protein in the leptospiral total-membrane profile is a 31-kDa protein which is solubilized by extraction of the outer membrane with Triton X-100 (51). These results contrast markedly with the low outer membrane particle density observed by freeze-fracture electron microscopy (14). An explanation for the apparent contradiction between the ultrastructural data and the OMP isolation studies was provided by our subsequent finding that, as in other spirochetes, many of the most abundant leptospiral proteins appear to be lipoproteins which are membrane anchored, not by transmembrane domains, but by fatty acids modifying their amino-terminal cysteine (38). Incubation of L. kirschneri in media containing tritiated palmitate resulted in intrinsic labeling of the 41-kDa protein designated LipL41 and the other major hydrophobic, detergent-extractable membrane proteins. Molecular cloning and sequencing of the gene encoding LipL41 revealed a Leu-X-Y-Cys consensus lipoprotein signal peptidase cleavage site. Furthermore, processing of LipL41 was found to be inhibitable by globomycin, a selective inhibitor of lipoprotein signal peptidase. Consistent with fatty acid modification, native LipL41 partitions exclusively into the Triton X-114 hydrophobic, detergent phase (38).

In this report we describe the gene encoding a second leptospiral lipoprotein, LipL36, which differs from LipL41 in its pattern of protein localization and expression. A previous report presented evidence indicating that LipL41 is exported to the outer leaflet of the outer membrane, while LipL36 is restricted to the periplasmic leaflet of the outer membrane (38). In the present study, we evaluated the outer/cytoplasmic (inner) membrane distribution of LipL36 and LipL41 by comparing rates of Triton X-100 solubilization. We also compared in vivo expression of LipL36 and LipL41, providing evidence for differential OMP expression in the leptospiral life cycle.

(Portions of this work were presented at the 95th and 96th General Meetings of the American Society for Microbiology, in Washington, D.C., 21 to 25 May 1995, and in New Orleans, La., 19 to 23 May 1996, respectively.)

	MATERIALS AND METHODS

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Bacterial strains, media, and plasmids. Virulent and culture-attenuated L. kirschneri RM52 organisms (1, 14) were passaged in Johnson-Harris bovine serum albumin Tween 80 medium (Bovuminar PLM-5 Microbiological Media; Intergen) (19). E. coli DH5 (supE44 lacU169 [80 lacZ M15] hsdR17 recA1 endA1 gyrA96 thi-1 relA1) was used as the host strain for transformations of recombinant DNA. E. coli PLK-F' (recA lac mcrA mcrB hsdR gal supE [F' proAB lacI^qZM15 Tn10 (Tet^r)]) was used as the host strain for infection with the Lambda-Zap II vector (Stratagene). E. coli PLK-F' and the ExAssist helper phage were used for in vivo excision of the pBluescript phagemid (Stratagene). E. coli SOLR (e14[mcrA] [mcrCB-hsdSMR-mrr]171 sbcC recB recJ umuC::Tn5 [Kan^r] uvrC lac gyrA96 relA1 thi-1 endA1 ^r [F' proAB lacI^qZM15] Su [nonsuppressing]) was used as the host strain for replication of the excised pBluescript phagemid from the Lambda-Zap II vector (Stratagene). E. coli JM109 (recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi[lac-proAB] F'[traD36 proAB⁺ lacI^q lacZM15]) was used as the host strain for the pRSET expression vector (Invitrogen). The DE3 lysogen of E. coli JM109 (Promega) was used as the host strain for pET-15b (Novagen). E. coli cells were routinely grown in Luria-Bertani (LB) broth or on LB agar, unless otherwise mentioned (35).

Gel electrophoresis and immunoblotting. Samples for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were solubilized in final sample buffer (FSB) composed of 62.5 mM Tris hydrochloride (pH 6.8), 10% glycerol, 5% 2-mercaptoethanol, and 2% SDS. Proteins were separated on a 12% gel with a discontinuous buffer system (21) and stained with Coomassie brilliant blue, or they were transferred to nitrocellulose filters (Schleicher and Schuell) for immunoblotting. For antigenic detection on immunoblots, the nitrocellulose filter was blocked with 5% nonfat dry milk in PBS (0.1 M phosphate-buffered saline, pH 7.4)-0.1% Tween 20 (PBS-T), incubated for 1 h with antiserum diluted 1:5,000 (unless otherwise noted) in PBS-T, and probed with donkey anti-rabbit antiserum conjugated to horseradish peroxidase (Amersham). Antigen-antibody binding was detected with the enhanced chemiluminescence system (ECL; Amersham). Blots were incubated in ECL reagents for 1 min and then exposed to XAR-5 film (Kodak). Densitometry of immunoblots was performed with an AMBIS imager and QuantProbe software (Scanalytics, Inc., Billerica, Mass.).

Triton X-114 extraction of Leptospira. L. kirschneri was extracted with 0.1% Triton X-114 by a modification of the method described previously (14). In brief, culture-attenuated L. kirschneri organisms were washed in phosphate-buffered saline-5 mM MgCl₂ and extracted in the presence of 0.1% protein grade Triton X-114 (Calbiochem), 10 mM Tris (pH 8), 1 mM phenylmethylsulfonyl fluoride, 1 mM iodoacetamide, and 10 mM EDTA at 4°C. The insoluble material was removed by centrifugation at 17,000 × g for 10 min. The Triton X-114 concentration of the supernatant was increased to 2%. Phase separation was performed by warming the supernatant to 37°C and subjecting it to centrifugation for 10 min at 2,000 × g. The detergent- and aqueous-phase proteins were precipitated with acetone.

Amino acid sequencing of an internal polypeptide fragment. LipL36 was obtained by treatment of L. kirschneri with Triton X-114 (see above). The Triton X-114 detergent-phase proteins were precipitated with acetone and separated by SDS-PAGE. A test strip was stained with Coomassie brilliant blue in order to locate the 36-kDa band, which was cut out of the remainder of the gel and loaded onto a second SDS-PAGE gel in the presence of staphylococcal V8 protease at a concentration of 100 µg ml¹ (Sigma). The proteins were allowed to migrate into the stacking gel by electrophoresis, and the current was disconnected for 45 min, followed by completion of electrophoresis. The polypeptide fragments were subjected to SDS-PAGE, transferred to a Trans-Blot polyvinylidene difluoride protein sequencing membrane (Bio-Rad, Richmond, Calif.), and submitted to the University of CaliforniaLos Angeles (UCLA) Protein Microsequencing Facility. N-terminal amino acid sequence analysis was performed on a Porton 1090-E gas-phase sequenator with on-line detection of phenylthiohydantoin amino acids.

Southern blot analysis. L. kirschneri DNA was prepared by the method of Yelton and Charon (49). Genomic DNA from other leptospiral strains was kindly supplied by C. A. Bolin. Leptospiral DNA was digested with EcoRI and electrophoresed in a 1.0% agarose gel. Following depurination, denaturation, and neutralization, the DNA was transferred to a nylon filter (Zeta-Probe; Bio-Rad) by the method of Southern (35). Filters were prehybridized for 3 h at 37°C in buffer containing 6× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 1× Denhardt's solution, 0.05% sodium PP_i, 0.5% SDS, and 100 µg of denatured salmon sperm DNA per ml. The filters were then hybridized overnight at 37°C with radiolabeled probe.

Cloning and sequencing of the lipL36 gene. Standard recombinant DNA procedures were performed as described elsewhere (35). Restriction endonuclease digests were performed as recommended by the suppliers (New England Biolabs and Promega). EcoRI fragments of L. kirschneri genomic DNA were ligated into the Lambda-Zap II vector (Stratagene). The ligated DNA was packaged with Gigapack II Gold packaging extract (Stratagene) and stored in 0.3% chloroform at 4°C. The plaque titer was determined by infecting E. coli PLK F' (Stratagene). Plaques were plated, transferred to filters in duplicate, and processed as previously described (35). The oligonucleotide probe hybridization and washing conditions described above for Southern hybridization were used. Recombinant pBluescript SK() clones were recovered from phage producing positive plaques by in vivo excision according to the manufacturer's recommendations. After restriction mapping, appropriate DNA fragments were subcloned into pBluescript KS and sequenced at the UCLA Core DNA Sequencing Facility by the dideoxy chain termination method with fluorescein-labeled dideoxy nucleotides (Applied Biosystems).

DNA sequence analysis. DNA sequence information was analyzed with the DNA Strider program (23). Homology searches were performed with the BLAST, FASTA, and Profile Search programs, which are found in the University of Wisconsin Genetics Computer Group (GCG), Inc., package, version 7.0 (12). Secondary-structure predictions were based on analysis with the programs PEPPLOT and PLOTSTRUCTURE, which are also found in the GCG package.

Antisera. Antiserum to leptospiral GroEL was a generous gift of B. Adler (Monash University, Clayton, Victoria, Australia). Murine monoclonal antibody F71C2 against serovar grippotyphosa (16) was a generous gift of Rudy Hartskeerl (Royal Tropical Institute, Amsterdam, The Netherlands). Antisera to OmpL1 and LipL41 were prepared as previously described (13, 38). Briefly, New Zealand White rabbits were immunized with purified His6 fusion proteins expressed by E. coli JM109 (Invitrogen) that had been transformed with the pRSET plasmid (Invitrogen) containing either the ompL1 or the lipL41 gene (13, 38).

[³H]palmitate radiolabeling and immunoprecipitation of native LipL36. A 35-ml culture containing 5 × 10⁷ L. kirschneri organisms in the log phase of growth/ml was intrinsically labeled by addition of [9,10(n)-³H]palmitate (250 µCi; 60 Ci/mmol; Amersham), followed by further incubation in a shaker incubator at 30°C for 48 h until the bacterial concentration reached 10⁹/ml. Organisms were washed in 5 mM MgCl₂ in PBS. A sample for immunoprecipitation containing 8 × 10⁹ L. kirschneri organisms was resuspended in 1.25 ml of a solution containing 10 mM Tris HCl (pH 8.0), 10 mM EDTA, and 1 mM phenylmethysulfonyl fluoride. To this suspension was added 12.5 µl of 10% protein grade Triton X-100 (Calbiochem), followed by gentle agitation for 30 min at 4°C. The insoluble material was removed by centrifugation at 16,000 × g for 10 min. To the supernatant was added 0.2 ml of heat-inactivated (at 56°C for 30 min) LipL36 rabbit antiserum and 0.25 ml of a slurry of staphylococcal protein A-Sepharose CL-4B (Sepharose-SpA) (Sigma). The suspension was gently agitated for 1 h. The Sepharose-SpA-antibody-antigen complexes were washed twice in 0.01% Triton X-100 in 10 mM Tris HCl (pH 8.0) and resuspended in FSB.

Expression of LipL36 in E. coli. PCR was used to amplify the lipL36 gene. The 5' oligonucleotide contained an NcoI restriction endonuclease site (underlined), followed by nucleotides 3 to 20 of the lipL36 gene: 5'-GT TCT TCC ATG GGG AGA AGA AAC ATA ATG AA-3'. The 3' oligonucleotide contained an XhoI restriction endonuclease site (underlined), followed by the last 18 nucleotides of the lipL36 gene (including the TAA stop codon) in antiparallel: 5'-TTC TAA CTC GAG TTA GTA TCT AGG ATA AGT-3'. L. kirschneri genomic DNA was used as the template. The 1,144-bp amplified lipL36 gene was digested with NcoI and XhoI and ligated into pET15b (Novagen) digested with NcoI and XhoI. The resulting construct, pET15b-LipL36, was transformed into E. coli JM109(DE3) (Promega). Expression of LipL36 was achieved by IPTG induction. After induction, samples were separated by SDS-PAGE and probed with LipL36 antiserum. Processing was inhibited by the addition of globomycin (dissolved in ethanol) at a final concentration of 200 µg/ml immediately prior to the addition of IPTG. The final ethanol concentration was 2%; control experiments without globomycin used ethanol at the same concentration. Globomycin was a generous gift of M. Inukai (Sankyo Company, Tokyo, Japan).

L. kirschneri infection of hamsters. Two groups of Golden Syrian hamsters, consisting of approximately equal numbers of males and females, were infected with L. kirschneri RM52. The first group of hamsters consisted of 21 5-week-old pups inoculated intraperitoneally (i.p.) with serial 10-fold dilutions of virulent L. kirschneri grown in liquid culture. Ten days later, liver tissue from one of the hamsters in the first group was used as a source of host-adapted organisms. The infected liver tissue was divided and incubated for 5 min in 100% normal rabbit serum. The rabbit serum containing host-adapted L. kirschneri was examined by dark-field microscopy, and 0.3 ml of this material was inoculated into a second group of hamsters (four 6-month-old female adults and nine 7-week-old pups). Hamsters from either group surviving 28 days after challenge were euthanized, and serum was harvested for testing in the LipL36 enzyme-linked immunosorbent assay (ELISA).

LipL36 ELISA. Immulon microtiter plates (Dynatech) were coated at 37°C overnight with 125 ng of His6-LipL36 fusion protein in 0.05 M sodium carbonate buffer (pH 9.6). The plates were washed three times with PBS containing 0.05% Tween 20, followed by the addition of 200 µl of blocking buffer (PBS containing 0.05% Tween 20 and 1% nonfat dried milk), and were incubated at 37°C for 1 h. After removal of the blocking buffer, 100 µl of antisera diluted 1:100 with blocking buffer was added, and the plates were incubated at 37°C for 2 h. After three washes, 100 µl of 1:2,000-diluted mouse monoclonal anti-hamster immunoglobulins (Sigma) was added, and the plates were incubated at 37°C for 2 h. After three more washes, 100 µl of 1:5,000-diluted sheep anti-mouse immunoglobulin G conjugated to horseradish peroxidase (Amersham) was added, and the plates were incubated at 37°C for 2 h. After three more washes, 100 µl of 3,3',5,5'-tetramethylbenzidine substrate was added, and the plates were incubated at 37°C for 20 min on an orbital shaker. Then 100 µl of a 1 M solution of sulfuric acid was added to stop the reaction. Absorbance was read at a wavelength of 450 nm with a microplate reader (model 550; Bio-Rad). Each serum sample was tested four separate times. Absorbance readings were normalized by subtracting the value obtained from antigen-negative control wells. Statistical analysis was performed by using Student's t test for two independent means.

Nucleotide sequence accession number. The nucleotide sequence of the lipL36 gene from L. kirschneri RM52 has been deposited in the GenBank database under accession no. AF024626.

	RESULTS

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Design of oligonucleotide probes and cloning of the lipL36 gene. Staphylococcal V8 protease digestion of LipL36 resulted in fragments with molecular masses of 21, 9, and 5 kDa. N-terminal amino acid sequence analysis of the 21-kDa fragment revealed the sequence YFGKTVLVRPSEQAKQKQIVLL. A 23-bp oligonucleotide probe with 256-fold degeneracy, GARCARGCNAARCARAARCARAT, was designed based on the portion of sequence EQAKQKQI. The oligonucleotide probe independently identified a 2.3-kb EcoRI fragment by Southern hybridization of the L. kirschneri genome. The 2.3-kb EcoRI fragment was cloned from a partial Lambda-ZAP II (Stratagene) library of L. kirschneri genomic DNA as described previously (13).

Sequence analysis of the lipL36 gene. Restriction mapping, Southern blot analysis, and DNA sequencing revealed that the entire lipL36 gene is encoded by the 2.3-kb EcoRI fragment (Fig. 1). An intact open reading frame was identified 430 bp downstream from the EcoRI site. The lipL36 structural gene consists of 1,092 bases encoding a protein of 364 amino acids. The locations of E. coli-like 35 (TTGACC) and 10 (TATTAT) promoter regions are shown in Fig. 2. A consensus ribosome-binding site (AAGAGG) is also present upstream of the initiation codon. Between the promoter and the ribosome-binding site there is an inverted repeat which may function as an operator. Another inverted repeat, which may function as a rho-independent transcription terminator, is found 30 bp downstream from the termination codon (Fig. 2).

View larger version (6K): [in this window] [in a new window]   FIG. 1.   Partial restriction map of the 2.3-kb EcoRI fragment containing the lipL36 gene and strategy for determining the nucleotide sequence. The lipL36 gene is 1,092 bp; its location is indicated by the shaded region. The arrows below the map indicate the direction and extent of sequence analysis. Letters above the map represent the following restriction enzymes: EcoRI (E), BamHI (B), DraI (D), EcoRV (Ev), HindIII (Hd), HincII (Hc), PvuII (P), and Sau3A (S).

View larger version (61K): [in this window] [in a new window]   FIG. 2.   Nucleotide sequence and deduced amino acid sequence of lipL36. Putative sigma 70 35 and 10 promoter regions and ribosome-binding site (RBS) are shown. The putative lipoprotein signal peptidase cleavage site is indicated by an arrow. The amino acid sequence obtained from the staphylococcal V8 protease digestion of the native protein is underlined. Asterisk, location of the TAA stop codon. Horizontal dashed arrows, inverted repeats. The inverted repeat downstream of the termination codon may function as a rho-independent transcription terminator.

View larger version (41K): [in this window] [in a new window]   FIG. 3.   Amino acid similarities between the amino-terminal sequences of LipL36 and 12 other spirochetal lipoproteins. The LipL36 sequence contains the L-X-Y-C consensus sequence for lipid modification. The amino terminus of LipL36 has a cluster of six consecutive aspartate residues located from position +4 to +9 of the mature polypeptide sequence. Acidic amino acids (underlined) occur commonly near the amino termini of the spirochetal outer membrane-associated lipoproteins listed here. This is especially true in the region from position +7 to +9, where 56% (22 of 39) of the residues are either aspartate or glutamate.

Conservation of the lipL36 gene in Leptospira spp. To address the frequency and distribution of the lipL36 gene, we performed Southern hybridization analysis with a fragment of the lipL36 gene. Figure 4 shows the results of probing genomic digests from representative strains from most of the known pathogenic and nonpathogenic Leptospira spp. The lipL36 gene appeared to be present in nine strains, representing all six of the pathogenic Leptospira species tested. The only two pathogenic strains that appeared not to contain the lipL36 gene were members of the species L. kirschneri. This is surprising, considering that the lipL36 gene was isolated from L. kirschneri serovar grippotyphosa strain RM52. Leptospira species assignments are based on DNA-DNA hybridization studies (34). This is consistent with the earlier finding that Southern blots with the ompL1 probe of L. kirschneri RM52 show better binding to other L. kirschneri strains than to other leptospiral species (13). For these reasons, it is likely that the lack of binding of the lipL36 probe represents absence of the gene rather than poor hybridization. The lipL36 gene appears to be present in a single copy; the only strain in which more than one band was present was L. noguchii serovar Proechymis (LT 796), a finding which could be explained by an EcoRI site within the hybridization region. The gene was not detected in L. biflexa, L. wolbachii, or L. inadai, three nonpathogenic Leptospira species, or in the related nonpathogen Leptonema illini.

View larger version (60K): [in this window] [in a new window]   FIG. 4.   Southern blot analysis of EcoRI-restricted genomic DNA from selected strains of Leptospira spp. A fragment of the lipL36 gene was used to probe EcoRI genomic digests of 11 pathogenic and 4 nonpathogenic leptospiral strains. The lipL36 gene was present in all six tested pathogenic Leptospira species, and in all tested strains of these species except for two of three L. kirschneri strains. The gene was not present in nonpathogenic strains of Leptospira. The locations of DNA size standards (in kilobases) are indicated on the left.

Behavior of LipL36 during Triton X-114 extraction and phase partitioning. We analyzed the behavior of LipL36 in the nonionic detergent Triton X-114 and tested the specificity of the LipL36 antiserum. Triton X-114 extraction of L. kirschneri solubilizes the leptospiral outer membrane, including the LPS, the porin OmpL1, and the surface-exposed lipoprotein LipL41 (13, 14, 38). LipL36 antiserum was used to probe Triton X-114 fractions of L. kirschneri, revealing a single band in both the whole-organism and the detergent phase. No material was detected in the aqueous phase, indicating selective partitioning of LipL36 into the Triton X-114 detergent phase (Fig. 5). Lipoproteins characteristically partition into the Triton X-114 detergent phase because of the hydrophobicity of the fatty acids. Unlike LipL41 (38), LipL36 was completely extracted in 0.1% Triton X-114, as demonstrated by complete removal from the detergent-insoluble pellet (Fig. 5).

View larger version (37K): [in this window] [in a new window]   FIG. 5.   Behavior of LipL36 in Triton X-114 and specificity of LipL36 antiserum. Triton X-114 fractions of L. kirschneri organisms were separated by SDS-PAGE and probed with LipL36 antiserum. Fractions analyzed were the whole organism (W) and Triton X-114-insoluble pellet (P), and aqueous-phase (A) and detergent-phase (D) material. The number of cell equivalents was the same in all four lanes. Locations of molecular size standards are shown (in kilodaltons) on the left. LipL36 is completely solubilized by Triton X-114 and partitions exclusively into the detergent phase.

L. kirschneri acylates LipL36. Intrinsic labeling of culture-attenuated L. kirschneri with [³H]palmitate resulted in the incorporation of label in leptospiral LPS, which appears diffusely at the bottom of the whole-organism lane in Fig. 6, as well as in at least 10 proteins which form discrete bands in the whole-organism lane. Immunoprecipitation experiments with anti-LipL36 antiserum (Fig. 6) confirm that LipL36 is the second-smallest lipoprotein identified in this autoradiograph.

View larger version (39K): [in this window] [in a new window]   FIG. 6.   LipL36 is selectively acylated by L. kirschneri. An autoradiogram of L. kirschneri proteins intrinsically labeled by [3H]palmitate and separated by SDS-PAGE is shown. Lane 1, total L. kirschneri proteins; lane 2, material immunoprecipitated by addition of LipL36 antiserum to a Triton X-100 extract of L. kirschneri. Locations of molecular size standards are shown (in kilodaltons) on the left.

Globomycin inhibits processing of LipL36. In order to demonstrate that LipL36 is processed by lipoprotein signal peptidase, E. coli JM109 containing pET-15b-LipL36 was treated with IPTG with or without globomycin, a selective inhibitor of lipoprotein signal peptidase. The majority of LipL36 expressed was unprocessed, with an apparent molecular mass about 2 kDa greater than that of the processed form (Fig. 7). The apparent molecular mass of the processed form of LipL36 was identical to that of the native protein (data not shown). Induction in the presence of globomycin inhibited processing of LipL36, resulting in a decrease of the processed form. These data indicate that the LipL36 signal peptide is processed by E. coli lipoprotein signal peptidase. The observation that processing was incomplete suggests that the LipL36 signal peptide is processed more efficiently in L. kirschneri than in E. coli, a finding also made in the case of LipL41 (38).

View larger version (44K): [in this window] [in a new window]   FIG. 7.   Globomycin inhibition of LipL36 processing in E. coli. E. coli JM109(DE3) containing pET15b-LipL36 was treated with 0.1 mM IPTG with (+) or without () globomycin and was probed with LipL36 antiserum. The locations of unprocessed (pre36) and processed (36) LipL36 are shown on the right. The location of a molecular size standard is shown (in kilodaltons) on the left.

Complete solubilization of LipL36 and LPS in 0.1% Triton X-100. Leptospira species produce a glycolipid LPS that is similar in structure to gram-negative LPS. By virtue of its surface exposure and by analogy with gram-negative bacteria, we concluded that leptospiral LPS is a useful marker for the outer membrane. We have shown previously, using periodate silver stain, that leptospiral LPS solubilization is essentially complete in 0.1% Triton X-114 (14). In the present report we show that 0.1% Triton X-100 has a similar pattern of LPS solubilization by probing an immunoblot with the LPS monoclonal antibody (Fig. 8, left panel). As expected, the protoplasmic cylinder marker GroEL was found to remain with the Triton X-100-insoluble fraction (Fig. 8, middle panel). Like LPS, LipL36 is completely solubilized by Triton X-100 (Fig. 8, right panel). In contrast, at least half the LipL41 was found to be insoluble in Triton X-100 (Fig. 8, right panel). These differences in Triton X-100 solubility may indicate that LipL36 is localized exclusively in the outer membrane, while LipL41 is present in both the inner and outer membranes.

View larger version (29K): [in this window] [in a new window]   FIG. 8.   Fate of L. kirschneri antigens after extraction with 0.1% Triton X-100. L. kirschneri antigens in the Triton X-100-soluble fraction (S) or insoluble pellet (P) were separated by SDS-PAGE and visualized by probing with a monoclonal antibody specific for serovar grippotyphosa LPS (left panel), with antisera to leptospiral GroEL (middle panel), or with a mixture of antisera to LipL36 and LipL41 (right panel). Locations of molecular size standards are shown (in kilodaltons).

Changes in LipL36 levels during growth in culture. Initial studies revealed that leptospiral membrane protein preparations differed in LipL36 representation. For this reason, we explored the possibility that LipL36 levels were affected by the growth phase of the culture from which the membrane proteins were prepared. Whole-cell samples were obtained at various times after inoculation of culture medium with L. kirschneri. Immunoblot analysis of these samples with LipL36 and LipL41 antisera indicated that LipL41 content remained constant, while LipL36 content decreased markedly as cell density increased (Fig. 9). These growth phase-dependent changes in LipL36 expression were found to be independent of passage number (data not shown).

View larger version (18K): [in this window] [in a new window]   FIG. 9.   Changes in LipL36 levels during growth in culture. (A) Immunoblot of L. kirschneri proteins separated by SDS-PAGE and probed with antisera to LipL36 and LipL41. Each lane contains 2.5 × 107 L. kirschneri cells. Cells were obtained 2.0 days (lane 1), 2.5 days (lane 2), 3.5 days (lane 3), 5.5 days (lane 4), and 6.5 days, (lane 5) after inoculation of the culture. Locations of molecular size standards are shown (in kilodaltons) on the left. (B) Plot of the time after culture inoculation versus the cell density and ratio of LipL36 levels to LipL41 levels as determined by densitometric analysis of panel A. While LipL41 levels remain constant during growth of cultivated L. kirschneri, LipL36 levels are highest during early-log phase.

Humoral immune response to leptospiral proteins during infection with virulent L. kirschneri. We used the hamster model of leptospirosis to compare the humoral immune response to infection with culture-adapted versus host-adapted L. kirschneri. Hamsters are extremely sensitive to infection with virulent Leptospira species. In the first group, only 4 of 21 (19%) hamsters survived i.p. challenge with virulent L. kirschneri grown in culture. In the second group, three of four adults and one of nine 7-week-old hamsters survived to day 28 after infection with host-adapted L. kirschneri. An attempt was made to determine the concentration of host-adapted organisms by dark-field microscopy. No organisms were seen in 10 high-power fields, indicating that the concentration of host-adapted organisms was below the sensitivity of dark-field microscopy (<10⁵/ml). Sera from all survivors in both groups were tested by LipL36 ELISA. The mean LipL36 ELISA reading of sera from animals surviving challenge with culture-adapted L. kirschneri (0.575) was significantly higher (P < 0.001) than that of sera from animals surviving challenge with host-adapted L. kirschneri (0.320).

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

LipL36 is a 36-kDa leptospiral outer membrane lipoprotein. Several lines of evidence support the conclusion that LipL36 is lipid modified at its amino-terminal cysteine residue. First of all, LipL36 was found to be blocked to N-terminal amino acid sequencing until subjected to staphylococcal V8 protease digestion. Analysis of its deduced amino acid sequence reveals a signal peptide followed by an L-X-Y-C consensus lipoprotein signal peptidase cleavage site, a pattern homologous to that of known bacterial lipoproteins (43). LipL36 is labeled by [9,10(n)-³H]palmitate intrinsic labeling of L. kirschneri. Like other spirochetal lipoproteins, LipL36 selectively partitions into the Triton X-114 hydrophobic, detergent phase. Finally, processing of LipL36 is inhibitable by globomycin, a selective inhibitor of lipoprotein signal peptidase. The combination of the sequence information, palmitate labeling, and globomycin inhibition satisfies three of Wu's criteria for definition of a lipoprotein (15). Although Leptospira species metabolize fatty acids by beta-oxidation, intrinsic labeling of L. kirschneri with [9,10(n)-³H]palmitate appeared to selectively label LPS and several other proteins, including LipL36 (Fig. 6) and LipL41 (38). It should also be noted that, as we have discussed previously, incorporation of the tritium label of [9,10(n)-³H]palmitate into amino acids would be extremely inefficient relative to modification of lipoproteins by one or more molecules of [9,10(n)-³H]palmitate (38).

Very little is known about secretory pathways in spirochetes. Comparison of sequences and membrane destination of lipoproteins such as LipL36 and LipL41 may provide insights into how leptospiral membrane proteins are localized. In this regard, there are several unusual, and potentially relevant, features of the deduced amino acid sequence of LipL36. The first is a series of six consecutive aspartate residues located from position +4 to +9 of the mature polypeptide sequence. The hydrophilicity of the aspartate cluster is reflected by a deep negative inflection in the Kyte-Doolittle plot occurring immediately after the signal peptide peak (data not shown). Figure 3 demonstrates that acidic amino acids occur commonly near the amino terminus of spirochetal outer membrane-associated lipoproteins. Acidic residues in this region may be important in guiding spirochetal lipoproteins to the correct secretory pathway (28). Substitution of serine for aspartate near the amino terminus of the E. coli murein lipoprotein alters outer membrane export efficiency (48). Experimental confirmation that the amino-terminal region of lipoproteins contains secretory information will require the development of tools for genetic manipulation of these bacteria.

Another unusual feature of the LipL36 sequence is the abundance of alanine residues. Almost 16% (55 of 344) of the residues in the mature LipL36 protein are alanines, and 25 of these alanine residues are arranged in pairs or triplets. Experiments involving model polypeptides have shown that alanine-containing polypeptides form unusually stable alpha-helices (25). Roughly 38% of the LipL36 sequence is predicted to be alpha-helical by Chou-Fasman analysis, and 30% (39 of 131) of the residues in the alpha-helical regions are alanines. The TmpB proteins of Treponema phagedenis and Treponema pallidum are rich in alanine (50). Several lipoproteins of pathogenic Neisseria species have also been found to be rich in alanine (9a, 16a, 17). The alpha-helical conformation of LipL36 could be stabilized by the presence of 14 potential salt bridges conforming to the N + 4 rule (24).

Previous studies have suggested that the nonionic Triton detergents selectively solubilize the leptospiral outer membrane (14, 51). Although LipL36 and LipL41 are both outer membrane lipoproteins, they differ in their pattern of Triton X-100 solubilization (Fig. 8), indicating that LipL36 and LipL41 differ in their outer/cytoplasmic (inner) membrane distribution. Controls used in this experiment were LPS, an outer membrane marker, and GroEL, a cytoplasmic cylinder marker. Like LPS, LipL36 was completely solubilized by 0.1% Triton X-100, implying that LipL36 is found exclusively in the outer membrane. In contrast, LipL41 appears to be located in both leptospiral membranes, a pattern of distribution found for other spirochetal lipoproteins (5, 33, 39). Previous surface immunoprecipitation studies indicate that LipL41 is surface exposed, while LipL36 is not (38). In combination, these data suggest that LipL36 is efficiently exported to the periplasmic leaflet of the outer membrane but is unable to gain access to the outer leaflet of the outer membrane. While LipL41 does contain the signal(s) required for export to the leptospiral surface, its export to the outer membrane appears to be incomplete. An alternative interpretation of the findings presented in Fig. 8 is that differences in Triton X-100 solubility are innate properties of LipL36 and LipL41 that do not reflect cellular localization. Ultimately, ultrastructural studies and improved outer membrane isolation techniques will be necessary to more firmly establish the membrane distribution of leptospiral antigens.

The evidence presented here indicates that LipL36 is differentially expressed during growth in culture. LipL36 is present in large amounts in cultivated L. kirschneri during early-log-phase growth but is found to decrease beginning in mid-log phase (Fig. 9). One interpretation of these data is that LipL36 expression is downregulated during late-log-phase growth. Another possible interpretation is that LipL36 is subject to digestion by an endogenous protease and that this process becomes more active during late-log-phase growth. However, proteolytic digestion of LipL36 is unlikely to explain the changes in LipL36 levels for the following reasons: (i) organisms isolated throughout this experiment retained gross structural integrity and >99% motility, (ii) samples were handled at 4°C and denatured in FSB immediately after isolation, and (iii) other than changes in the LipL36 level, SDS-PAGE protein profiles of the samples analyzed in Fig. 9 were essentially identical (data not shown). We did not examine the effects of cell density on LipL36 expression. Cell-density-dependent expression of the B. burgdorferi outer membrane lipoprotein P35 has been described previously (18). However, in contrast to LipL36, P35 expression is upregulated when B. burgdorferi enters the stationary phase of growth in culture.

A universal property of pathogenic spirochetes is the ability to cause chronic infections. One proposed mechanism by which spirochetes are able to persist in the host is evasion of the host immune response by downregulation of OMP expression (4, 14, 20, 29, 32, 44, 45). Analysis of the humoral immune response to LipL36 during L. kirschneri infection suggests that LipL36 expression is downregulated during mammalian infection. The antibody response to LipL36 was significantly stronger in hamsters infected with culture-adapted L. kirschneri than in those infected with host-adapted L. kirschneri. We acknowledge that measurement of antibody levels is an indirect approach to studying in vivo OMP expression. Alternative interpretations of these data could involve effects of challenge inoculum and hamster age on the humoral immune response. However, downregulation of LipL36 expression in vivo has recently been confirmed by more direct evidence involving immunohistochemistry (2). Based on these findings, LipL36 should be a useful tool for studying the adaptation of pathogenic Leptospira species to the host environment. Northern blot studies are needed to determine whether or not regulation of LipL36 expression occurs at the transcriptional level. It may be possible to apply molecular strategies for identification of LipL36 regulatory proteins, a key step towards an understanding of the mechanisms by which pathogenic Leptospira species control their response to the diverse environments encountered during their life cycle.