LruA and LruB, Novel Lipoproteins of Pathogenic Leptospira interrogans Associated with Equine Recurrent Uveitis

Leptospira culture. Leptospira interrogans serovars Pomona type kennewicki (JEN4), Pomona (Pomona) Copenhageni (M 20), Canicola (Hond Utrech IV), Grippotyphosa (Andaman), Hardjo (Hardjoprajitno), and Bratislava (Jez Bratislava) were kindly provided by Mike Donahue (Livestock Disease Diagnostic Center, University of Kentucky, Lexington). Leptospira biflexa serovar Biflexa was obtained from The National Veterinary Services Laboratories, Ames, Iowa. Leptospires were grown in Johnson-Harris bovine serum albumin-Tween 80 medium (Bovuminar PLM-5 Microbiological Media; Intergen, Purchase, NY) at 30°C unless otherwise indicated.

Eye fluids and eye tissue extracts.Eye fluids and companion sera from horses of varied age, breed, and origin were obtained from a commercial horse slaughter plant in North America. Eyes with gross evidence of uveitis were enucleated after slaughter, and aqueous humor was removed with a 10-ml syringe and stored at −20°C. The eyes were placed in 10% formaldehyde for subsequent embedding, sectioning, and staining with hematoxylin and eosin for histologic examination. Eye fluids and sera were assayed for antibodies to serovars Pomona, Canicola, Icterohemorrhagiae, Hardjo, Bratislava, and Grippotyphosa in the microscopic agglutination test (MAT) (Table 1). Extracts were prepared from the ciliary body, cornea, lens, and retina of a normal eye from a young horse serologically negative for Leptospira (38).

Library screening and plasmid rescue.A lambda ZAP II library containing 3- to 5-kb fragments of L. interrogans serovar Pomona type kennewicki DNA (23) was screened to identify phage expressing gene products reactive with pooled eye fluids from uveitic horses. Following propagation on Escherichia coli XL-1 MRF′ (Stratagene, La Jolla, CA) lawns, plaques were transferred in duplicate to IPTG (isopropyl-β-d-thiogalactopyranoside)-saturated nitrocellulose disks and immunoblotted with pooled eye fluids, diluted 1:600, from five uveitic horses (Table 1). The secondary antibody was horseradish peroxidase (HRP)-labeled protein G (Zymed, San Francisco, CA) diluted 1:4,000. Immunopositive plaques were identified by using 4-chloro-1-naphthol as the substrate. Positive plaques on agar plugs were transferred to 500 μl of SM buffer and allowed to elute overnight at 4°C. Reactive plaques were rescreened until clonal. Plasmids containing inserts of leptospiral DNA were rescued from selected reactive phages by using ExAssist helper phage and E. coli SOLR (Stratagene, La Jolla, CA) according to the manufacturer's protocol.

DNA sequencing and analysis.Plasmid DNA was isolated using a QIAprep spin miniprep kit (QIAGEN, Valencia, CA) and sequenced in a commercial sequencing facility (Davis Sequencing LLC, Davis, CA) using T3, T7, and custom-designed primers (Table 2). Sequences were edited and connected using Chromas 1.61 (Technelysium Pty. Ltd., Queensland, Australia) and DNASIS (Hitachi Software Engineering Co., Ltd., San Francisco, CA). The sequences were compared with L. interrogans serovar Lai strain 56601 (41) and L. interrogans serovar Copenhageni Fiocruz L1-130 (36) genomic sequences at http://www.tigr.org/ . Analyses of nucleotide and deduced amino acid sequences were performed using DNASIS, the Genetics Computer Group package of programs (Wisconsin Package version 10.0; Genetics Computer Group, Madison, WI), PSORT (http://psort.nibb.ac.jp/ ), SignalP (4), LipoP (22), TMHMM (http://www.cbs.dtu.dk/ ), and COILS (http://www.ch.embnet.org/index.html ). Homologies were identified by a BLAST search using the National Center for Biotechnology Information server (http://www.ncbi.nlm.nih.gov/BLAST/ ).

Cloning and expression.Primers a1-1F plus a1-1R and b5-1F plus b5-1R (Table 2), specific for of lruA and lruB, respectively, were designed using Primer 2 (Scientific & Educational Software, 1991). Following PCR amplification of chromosomal DNA of L. interrogans serovar Pomona type kennewicki (JEN4), amplicons were digested with BamHI and XhoI and inserted into pET-15b (Novagen, Madison, WI) predigested with the same restriction endonucleases. Recombinant plasmids were transformed into E. coli BL21(DE3) (Novagen, Madison, WI). Expression of His₆-LruA and His₆-LruB was induced with 1 mM IPTG when cultures reached an optical density of 0.6 at 600 nm, and cells were harvested after 2 to 3 h. Recombinant His-tagged proteins were isolated using Talon metal affinity resin (Clontech Laboratories, Inc.) in buffer containing 8 M urea according to the manufacturer's recommendations. The purity of recombinant proteins was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). His₆-LruA and His₆-LruB were dialyzed against 10 mM Tris (pH 7.5) containing 50 mM NaCl.

[¹⁴C]palmitate radiolabeling and immunoprecipitation of native LruA.A 12-ml culture of L. interrogans Fiocruz L1-130 containing 2 × 10⁸ cells per ml in the log phase of growth was mixed with 50 μCi of [U-¹⁴C]palmitic acid (Amersham, Piscataway, NJ) and incubated at 30°C until the density of the bacteria reached 1.3 × 10⁹/ml; 1.2 × 10¹⁰ bacteria were collected by centrifugation for 3 min at 9,000 × g in a microcentrifuge and frozen at −20°C. The bacteria were lysed by suspending the pellets combined in a single microcentrifuge tube with 1.2 ml boiling lysis buffer (50 mM Tris-Cl, pH 8.0, 100 mM NaCl, 2 mM EDTA, and 0.2% SDS) and boiling for 5 min. The lysate was subjected to centrifugation for 10 min at 4°C at 14,000 × g, and the insoluble material was removed. For immunoprecipitations, 795 μl immunoprecipitation buffer (50 mM Tris-Cl, pH 8.0, 100 mM NaCl, 2 mM EDTA, 0.2% Triton X-100) was mixed with 200 μl lysate (representing 2 × 10⁹ bacteria) and 5 μl anti-LipL41 or anti-LruA rabbit serum and incubated on ice overnight. Twenty-five microliters of EZ View Red protein A affinity gel (Sigma) was then added, and the mixture was mixed for 2 h at 4°C. The antibody-antigen complexes bound to protein A were recovered by centrifugation at 15,000 × g for 8 s, washed twice with 800 μl immunoprecipitation wash buffer (10 mM Tris-Cl, pH 8.0, 0.4 M NaCl, 0.01% Triton X-100), and washed once with 800 μl no-salt wash buffer (10 mM Tris-Cl, pH 8.0, 0.01% Triton X-100). Pellets were resuspended with 100 μl final sample buffer containing 0.25 M phenylmethylsulfonyl fluoride. Ten microliters of each sample was subjected to electrophoresis (12% PAG-SDS gel; Cambrex), and bands were detected by fluorography following a 30-min treatment of the gel with Amplify (Amersham, Piscataway, NJ).

Detection of genes in different Leptospira spp. and serovars.DNAs of L. interrogans serovars Pomona, Canicola, Grippotyphosa, Hardjo, and Bratislava; L. biflexa serovar Biflexa; Leptospira weilii (Sarmin); Leptospira inadai (LT430); and Leptonema illini (Illini 3055) were isolated from 5-ml cultures as previously described (1). Leptospiral DNAs were digested overnight with HindIII at 37°C. Digested DNAs were separated on a 0.8% agarose gel for 4 h at 50 V, transferred to a Hybond-N nylon membrane (Amersham, Piscataway, N.J.), and fixed by UV cross-linking according to the manufacturer's protocol.

Primers a1-1F plus a1-1R and b5-1F plus b5-1R (Table 2), specific for of lruA and lruB, were used in the PCR to amplify the lruA and lruB genes and were labeled with digoxigenin by using the DIG High Prime DNA labeling and detection kit (Roche Applied Science, Indianapolis, IN). Prior to digoxigenin labeling, the lruA PCR amplicon was digested with HindIII and the larger fragment extracted from the gel. The UV-cross-linked nylon membrane was subjected to prehybridization at 42°C for 30 min in DIG Easy Hybridization solution. After denaturation, approximately 25 ng/ml of probe was mixed with DIG Easy Hybridization solution and incubated with the membrane at 42°C with gentle agitation. The next day, the membrane was washed for 15 min at room temperature with three changes of the buffer containing 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and 0.1% SDS and was then washed thrice for 15 min at 65°C in 0.5× SSC with 0.1% SDS, prewarmed to 65°C. After stringency washes, the membrane was treated with anti-digoxigenin-alkaline phosphatase, followed by a chemiluminescent substrate (DIG High Prime DNA labeling and detection kit). Hybridization was detected by exposing the membrane to an X-ray film (Pierce, Rockford, IL).

SDS-polyacrylamide gel electrophoresis and immunoblotting.SDS-PAGE was performed in a 12% acrylamide gel using a discontinuous buffer system as described elsewhere (27). Samples were mixed with an equal volume of 2× sample loading buffer containing 125 mM Tris-Cl, 4% SDS, 2% glycerol, 1% β-mercaptoethanol, and 0.5% bromophenol blue and boiled for 5 min before loading. Electrophoresis was carried out in an X-Cell SureLock minicell (Invitrogen, Carlsbad, CA) for 2 h at 125 V in Tris-glycine running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3). Proteins were transferred to Protran nitrocellulose membranes (0.2-μm pore size; Schleicher & Schuell, Keene, NH) and blocked with 4% nonfat dry milk in Tris-buffered saline (20 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 7.5). Membranes were incubated with protein-specific antiserum, eye fluid, or antiserum to eye antigens, followed by incubation with protein G conjugated to horseradish peroxidase (Zymed, San Francisco, CA). Membranes were developed with the ECL Western blot detection system (Amersham, Piscataway, NJ), and bands were visualized with Hyperfilm (Amersham) or by using 4-chloro-1-naphthol (Sigma, St. Louis, MO).

RT-PCR.Total RNA was extracted from L. interrogans serovar Pomona type kennewicki grown at 30°C and 37°C by using the RNeasy minikit (QIAGEN, Valencia, CA). RT-PCR was performed with gene-specific primers a1rt-F, a1rt-R, b51rt-F, and b51rt-R (Table 2) on total RNA, using the SuperScript one-step RT-PCR kit (Invitrogen, Carlsbad, CA). cDNA synthesis was accomplished in 30 min of incubation at 50°C followed by denaturation at 94°C for 2 min; 35 cycles of 94°C for 30 s, 58°C for 30 s, and 70°C for 1 min; and a final extension step at 72°C for 5 min.

Enzyme-linked immunosorbent assay (ELISA).A checkerboard titration was performed in flexible 96-well flat-bottom polystyrene MicroTest III assay plates (Falcon; Becton Dickinson, Oxnard, CA) with pools of uveitic and normal eye fluids diluted 200-, 400-, 600-, 800-fold and tested against 25, 50, 100, and 200 ng/well of antigen to determine the optimum concentrations of recombinant LruA and LruB. Wells were coated with 100 ng protein, followed by blocking with 4% nonfat dry milk. Eye fluids (1:600) or serum (1:400) was added and left for 1 h at 37°C. Bound immunoglobulin G (IgG) was detected using HRP-protein G (Zymed, San Francisco, CA) and IgA with α-chain-specific monoclonal antibody (BVS-2) followed by HRP conjugated goat anti-mouse. Plates were developed with o-phenylenediamine (Sigma, St. Louis, MO). Analysis of the data was performed using Student's t test and analysis of variance.

Polyclonal antisera.Polyclonal antisera were raised in New Zealand White rabbits by subcutaneous administration of 1 μl of N-acetylmuramyl-l-alanyl-d-isoglutamine (Sigma, St. Louis, MO) and 100 μg of recombinant protein adsorbed to aluminum hydroxide (Alhydrogel; Accurate Chemical & Scientific Corp., Westbury, NY). Booster injections contained 100 μg (subcutaneous) and 5 μg (intravenous) of the antigen and were administered 14 and 28 days after the primary immunization. Serum was obtained 35 days after the primary immunization (20).

Membrane solubilization with Triton X-114.The outer membrane fraction of low (fourth)-passage L. interrogans serovar Copenhageni was extracted by Triton X-114 solubilization and phase partitioning as described previously (14). Briefly, leptospires washed in phosphate-buffered saline containing 5 mM MgCl₂ were extracted in 0.5% protein-grade Triton X-114 (Calbiochem), 150 mM NaCl, 10 mM Tris (pH 8.0), and 1 mM EDTA at 4°C. Insoluble material (protoplasmic cylinder) was pelleted by centrifugation at 10,000 × g for 10 min. Phase separation of the supernatant was performed by warming it to 37°C after the addition of 20 mM CaCl₂, followed by centrifugation for 10 min at 1,000 × g. Proteins in aqueous and detergent phases were precipitated with acetone.

Nucleotide sequence accession numbers.Nucleotide sequences deposited in the GenBank database have accession numbers AY741529 (lruA) and AY741530 (lruB).

Identification and analysis of lruA and lruB.Screening of approximately 10⁵ plaques of a lambda library of L. interrogans serovar Pomona type kennewicki with pooled eye fluids from uveitic horses revealed 14 reactive plaques. Plasmids rescued from these phages were sequenced and compared with L. interrogans serovar Lai strain 56601 (41) and L. interrogans serovar Copenhageni Fiocruz L1-130 (36) genomic sequences. Homologies to eight different regions of the L. interrogans genome were demonstrated. These regions encode the previously described leptospiral proteins LigA/LigB (31, 37) (four phagemids), LigC (one phagemid), GrpE/DnaK/DnaJ (2) (four phagemids), and Qlp42 (30, 35) (two phagemids), plus two novel proteins. Phagemids pA1, pD1, and pB5, which encode these two proteins, were selected for further analysis.

Sequencing of pA1, pD1, and chromosomal DNA of JEN4 using primers T3, T7, a1-1uF, a1-1uR, a1-1dF, and a1-1dR (Table 2) revealed an open reading frame positioned in a 2,243-bp fragment of JEN4 chromosomal DNA (GenBank accession no. AY741529). This open reading frame encoded a protein (designated LruA) of 555 amino acids with a predicted molecular mass of 62 kDa. The amino terminus of LruA consisted of a 22-amino-acid signal sequence with a putative lipobox (FIS↓C) at its carboxy terminus. Several hexanucleotides resembling the −10 region of the σ⁷⁰ bacterial promoter were present upstream of lruA. Downstream of this gene, a stem-and-loop structure (ΔG = −18.6 kcal/mol) followed by a stretch of five thymines resembled a ρ-independent transcriptional terminator. LruA also contained a conserved domain, LysM, between residues 406 and 461. This domain has been found in a variety of microorganisms and is apparently associated with peptidoglycan binding (3, 29). LruA showed 99.6% (two amino acid substitutions) and 99.8% (one amino acid substitution) identity with LIC11003 of L. interrogans serovar Copenhageni (36) and LA3097 of L. interrogans serovar Lai (41), respectively.

Phagemid pB5 contained a 1,936-bp insert (GenBank accession no. AY741530) that encoded two proteins, pL13 and LruB. Analysis of the nucleic acid sequence demonstrated ribosomal binding sites for both genes but a putative promoter for pL13 and a transcriptional terminator for lruB only, suggesting translation of pL13 and a 48-kDa protein, LruB, from a polycistronic mRNA. The N-terminal 22 amino acid residues of LruB resembled a signal peptide sequence with a potential lipoprotein signal peptidase II cleavage site, FSN↓C. A conserved domain, IrpA (PSSM-Id 12815), spanning from Q₄₂ to T₃₅₁ was predicted for LruB. Proteins with this conserved domain have been described for several bacteria (24, 29) and are apparently associated with iron metabolism regulation. LruB showed 94.1% (25 amino acid substitutions) and 97.7% (10 amino acid substitutions) identity with LA3469 of Leptospira interrogans serovar Lai (41) and LIC10713 of Leptospira interrogans serovar Copenhageni (36), respectively. These amino acid substitutions were clustered between residues 155 and 176 for LIC10713 of Leptospira interrogans serovar Copenhageni and from amino acid residues 155 to 182 and 216 to 236 for LA3469 of Leptospira interrogans serovar Lai.

Acylation of LruA.Intrinsic labeling of L. interrogans with [¹⁴C]palmitate resulted in acylation of lipopolysaccharide and previously identified lipoproteins, including LipL32 (16), LipL36 (15), and LipL41 (43). Only some of the bands observed by Coomassie blue staining of the gel were labeled with [¹⁴C]palmitate, thus confirming the selectivity of the procedure (data not shown). Because LruA is a leptospiral inner membrane protein, immunoprecipitation of LruA was performed with total membrane lysate rather than with Triton X-100 extract (15, 16, 43). Immunoprecipitation of the total membrane lysate of [¹⁴C]palmitate-labeled L. interrogans by using LruA antiserum confirmed that LruA is acylated by L. interrogans (Fig. 1). Antiserum to LipL41 was included as a positive control for immunoprecipitation.

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FIG. 1.

LruA is acylated by L. interrogans. An autoradiogram of whole L. interrogans and immunoprecipitated LipL41 and LruA after intrinsic labeling with [¹⁴C]palmitate and separation by SDS-PAGE is shown. Lane 1 contains L. interrogans labeled with [¹⁴C]palmitate, including lipopolysaccharide (<27 kDa) and lipoproteins (>27 kDa). Lanes 2 and 3 contain material immunoprecipitated by addition of LipL41 and LruA antisera, respectively, to a total membrane lysate of L. interrogans. Each lane contains material from 2 × 10⁸L. interrogans organisms. Locations of molecular size standards are shown in kilodaltons on the left.

Distribution of lruA and lruB among Leptospira spp.The distribution of lruA and lruB in a number of pathogenic and saprophytic strains of Leptospira was examined by Southern blotting. lruA was found in L. interrogans serogroup Icterohemorrhagiae serovar Copenhageni and serovars Pomona (strains pomona and JEN4), Canicola, Hardjo, Bratislava, and Grippotyphosa and in L. weilii but not in L. biflexa, L. inadai, or Leptonema illini (Fig. 2). lruB was also found to be present in all L. interrogans serovars but not in saprophytic L. biflexa, the intermediate pathogen L. inadai, or the non-L. interrogans pathogenic L. weilii. (Fig. 2). lruA and lruB appear to be restricted to pathogenic Leptospira species, since they were not detected in saprophytic L. biflexa. lruB seems to be present only in L. interrogans and not in other species (Fig. 2).

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FIG. 2.

Detection of lruA and lruB sequences in Leptospira spp. Detection of lruA and lruB sequences by Southern blotting in pathogenic serovars of L. interrogans or other Leptospira spp. is shown; Leptonema illini and L. interrogans serovar Pomona JEN4 were included as controls. The arrowhead indicates the lruA sequence in L. weilii.

Expression and cellular localization of LruA and LruB.Since culture temperature can affect expression of several L. interrogans genes, transcription of lruA and lruB in L. interrogans serovar Pomona grown at 30°C or 37°C was examined by RT-PCR. lruA and lruB transcripts were clearly detectable in cultures grown at 30°C or 37°C (data not shown). Reactions without reverse transcriptase yielded no product, indicating purity of the RNA preparations. Immunoblotting of whole-cell lysates from cultures grown at 30°C or 37°C with LruA- and LruB-specific rabbit antisera showed equivalent expression levels of each protein (data not shown).

Triton X-114 was used to separate hydrophobic outer membrane proteins, hydrophilic periplasmic proteins, and the protoplasmic cylinder (Fig. 3), using antisera to known outer and inner membrane proteins LipL32 and LipL31, respectively (16, 18). Triton X-114 fractions immunoblotted with antisera specific for LruA and LruB revealed a pattern of fractionation similar to that of LipL31 in both cases. This indicated that LruA and LruB are found largely, if not exclusively, in the leptospiral inner membrane.

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FIG. 3.

Cellular localization of LruA and LruB. Immunoblots of Triton X-114 extracts of L. interrogans show the cellular localization of LruA and LruB. Blot 1 contains whole cells (Wh), Triton X-114-insoluble material (PC), aqueous phase (AQ), and detergent phase (D) and was probed with rabbit antisera to LipL32 (1:20,000) and LruA (1:1,000). Blot 2 was probed with rabbit antisera to LruB (1:500) and LipL31 (1:10,000). Antisera to LipL32 and LipL31 were included as controls for proteins of detergent and protoplasmic cylinder fractions, respectively.

LruA- and LruB-specific antibody levels in sera and eye fluids of uveitic horses.Eye fluids and sera from uveitic horses and sera from aborted mares were tested by ELISA, using LruA and LruB as antigens (Fig. 4). High levels of LruA- and LruB-specific IgG and IgA antibodies were detected in uveitic eye fluids but not in companion sera (Fig. 4). Aborted mares with high serum MAT titers showed only moderate serum levels of LruA- and LruB-specific IgG. IgA specific for LruA and LruB was detected only in uveitic eye fluids. Normal eye fluids did not contain detectable levels of LruA- and LruB-specific IgG and IgA antibodies (not shown).

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FIG. 4.

LruA- and LruB-specific IgG (left panels) and IgA (right panels) levels in eye fluids and companion sera of 12 uveitic horses and in sera of 10 recently aborted mares. Error bars indicate standard deviations. OD₄₉₀, optical density at 490 nm.

Cross-reactivity of LruA and LruB with equine ocular tissue.Possible roles of LruA and LruB in autoimmunity were investigated by immunoblotting extracts of ciliary body, lens, and retina of a normal equine eye with specific antisera. LruA antiserum reacted with a ∼22-kDa band in lens extract and a ∼65-kDa band in ciliary body extract (Fig. 5A and B, respectively). Antiserum to LruB reacted very strongly with a ∼30-kDa band of equine retinal extract (Fig. 5C). Preimmunization rabbit sera served as controls for nonspecific reactivity. Antiserum to Lk73.5 (1), an immunoreactive, host-inducible sphingomyelinase of L. interrogans, showed no reactivity with eye tissue extracts (not shown).

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FIG. 5.

Cross-reactivity of LruA and LruB with equine ocular tissue. (A and B) Immunoblots showing reactivity of rabbit antiserum to LruA with extracts of normal equine lens and ciliary body. (C) Reactivity of LruB antiserum with extracts of normal equine retina. Preimmunization sera from rabbits were used as controls for specificity of binding. The arrows and asterisk indicate cross-reactive bands to LruA- or LruB-antiserum in normal equine ocular tissues.