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Research Paper	Pdf file on Synergy omments
Acta Biochim Biophys Sin 2005,37:649-656
doi:10.1111/j.1745-7270.2005.00094.x

Expression and Comparative Analysis of Genes Encoding Outer Membrane Proteins LipL21, LipL32 and OmpL1 in Epidemic Leptospires

Xiang-Yan ZHANG^1#, Yang YU^2,3#, Ping HE¹, Yi-Xuan ZHANG^2,3, Bao-Yu HU¹, Yang YANG¹, Yi-Xin NIE⁴, Xiu-Gao JIANG⁴, Guo-Ping ZHAO^2,3, and Xiao-Kui GUO¹*

¹ Department of Medical Microbiology and Parasitology, Shanghai Jiaotong University, Shanghai 200025, China;

² Research Center of Biotechnology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China;

³ Chinese National Human Genome Center at Shanghai, Zhangjiang High Tech Park, Shanghai 201203, China;

⁴ Institute for Infectious Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China

Received: May 11, 2005

Accepted: June 24, 2005

This work was supported by the grants from the National High Technology Research and Development Program of China (No. 2003AA223030), the National Natural Science Foundation of China and Shanghai Leading Academic Discipline Project (No. T0206)

^# These authors contributed equally to this work

*Corresponding author: Tel/Fax, 86-21-64453285; E-mail, [email protected]

Abstract Leptospiral outer membrane proteins (OMPs) are highly conserved in different species, and play an essential role in the development of new immunoprotection and serodiagnosis strategies. The genes encoding LipL21, LipL32 and OmpL1 were cloned from the complete genome sequence of Leptospira interrogans serovar lai strain Lai and expressed in vitro. Sequence comparison analysis revealed that the three genes were highly conserved among distinct epidemic leptospires, including three major epidemic species Leptospira interrogans, Leptospira borgpetersenii and Leptospira weilii, in China. Immunoblot analysis was further performed to scrutinize 15 epidemic Leptospira reference strains using the antisera of the recombinant OMPs. Both immunoblot assay and reverse transcription-polymerase chain reaction demonstrated that these three OMPs were conservatively expressed in pathogenic L. interrogans strains and other pathogenic leptospires. Additionally, the use of these recombinant OMPs as antigens in enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of leptospirosis was evaluated. The recombinant LipL32 and OmpL1 proteins showed a high degree of ELISA reactivity with sera from patients infected with L. interrogans strain Lai and other pathogenic leptospires. These results may contribute to the identification of candidates for broad-range vaccines and immunodiagnostic antigens in further research.

Key words outer membrane protein; expression; comparative analysis; epidemic leptospire

Leptospirosis is one of the most important zoonoses with worldwide distribution. Protective immunity elicited by leptospiral lipopolysaccharide is generally serovar-specific [1]. The current available whole-cell vaccines can not provide cross-protection against infection with more than 250 different Leptospira serovars known to exist [2,3]. Thus characterization of leptospiral outer membrane proteins (OMPs) has emerged as an important approach.

So far three classes of leptospiral OMPs have been identified: lipoproteins, the most abundant class comprising LipL32, LipL36, LipL41, LipL48, LipL21 [3-7] and the temperature-regulated Qlp42 [8]; transmembrane protein OmpL1 [9]; and peripheral membrane proteins such as LipL45 [10]. Some spirochaetal outer membrane proteins have been characterized by a lipoprotein structure called lipobox [11,12].

The classic Triton X-114 method and the traditional approaches for isolation of the outer membrane protein from Leptospira species have met with some challenges [3,6,13]. On the other hand, the complete genomic DNA sequence of pathogenic Leptospira interrogans serovar lai strain Lai represents a new unexploited field for the design of novel vaccines and development of serodiagnosis [14], as well as for prediction of the immunoreaction of the outer membrane lipoproteins with the host environment.

Three OMP genes from the genome sequence of L. interrogans serovar lai strain Lai, encoding LipL21, LipL32 and transmembrane protein OmpL1 separately, were cloned and expressed in vitro. Primarily, they were identified and annotated by bioinformatics tools on primary structure, transmembrane structure, hydrophobicity, protein domain and protein family [11]. Standard nucleotide sequencing analysis of these corresponding OMP genes of different epidemic Leptospira strains which are of medical importance in China [15,16] were performed. To characterize and validate the potential roles of these recombinant OMPs (rOMPs) as target antigens in the host humoral immune response of leptospirosis [17], and to clarify whether these OMP genes are highly conserved in various epidemic leptospires in China, the three OMP genes were analyzed in detail by both immunoblot and reverse transcription-polymerase chain reaction (RT-PCR). The immunoreaction of purified recombinant OMPs with patients' sera of leptospirosis caused by L. interrogans serovar lai and other pathogenic leptospires were evaluated by enzyme-linked immunosorbent assay (ELISA).

Materials and Methods

Database

The complete genomic DNA sequence of L. interrogans serovar lai strain Lai was obtained from GenBank at the National Centre for Biotechnology Information (NCBI) website. Homology searches with the OMP sequences of different epidemic Leptospira species were accomplished using the BLAST program against the GenBank/NCBI nuclear acid sequence database (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=Search&DB=nucleotide).

Leptospira isolates

A panel of pathogenic Leptospira reference strains comprised three main epidemic species in China [15,16] including ten L. interrogans strains, four Leptospira borgpetersenii strains and one Leptospira weilii strain; and the nonpathogenic group composed of saprophytic Leptospira biflexa serovar patoc and serovar monvalerio strains (Table 1). All leptospira isolates were provided by the Institute for Infectious Disease Control and Prevention (Chinese Center for Disease Control and Prevention, Beijing, China). These isolates were cultivated in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium at 30 ºC [18].

Isolation of leptospiral genomic DNA

Stationary-phase culture was harvested. Samples were then extracted with an equal volume of phenol-chloroform-isoamyl alcohol. The aqueous phase was removed and extracted with chloroform-isoamyl alcohol. DNA was precipitated from the aqueous phase with 2.5 volumes of 95% ethanol. DNA pellets were washed in 75% ethanol, recentrifuged, and air-dried before being resuspended in H₂O.

Isolation of total RNA

Mid-logarithmic growth phase Leptospira culture was harvested. Total RNA was isolated by Trizol reagent (Gibco BRL, Gaitherburg, USA). Following the chloroform-isopropyl alcohol process, the RNA pellets were washed with 1 ml of 75% ethanol and air-dried before being resuspended in RNase-free water.

Cloning and expression of OMP genes, and purification of recombinant OMPs

Selected OMP genes were amplified and expressed from pET-28b(+) (Novagen, Madison, USA) in Escherichia coli BL21(DE3) (Novagen). The associated gene features are listed in Table 2. PCR primers were designed as shown in Table 3. The PCR amplification reaction system was as follows: DNA was denatured at 95 ºC for 10 min before 45 cycles at 94 ºC for 0.5 min, 54 ºC for 1 min, and 72 ºC for 1 min. A final extension run for 10 min at 72 ºC concluded the reaction.

Expression of selected OMP genes was induced in mid-logarithmic growth phase E. coli BL21(DE3) with 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG; Sigma, Sydney, Australia). His₆-tagged proteins were purified following the manufacturer's instructions (Novagen).

Antiserum preparation and immunization

Antisera to these OMPs were prepared as described previously [19]. New Zealand white rabbits were immunized with purified His₆-OMP fusion proteins expressed by E. coli BL21(DE3) transformed with the pET-28b(+) plasmid containing the OMP genes. Two hundred micrograms of purified protein was mixed with Freund's complete adjuvant and inoculated subcutaneously into one male New Zealand white rabbit. Additional immunizations with approximately 200 mg of His₆ fusion protein in Freund's incomplete adjuvant were given 4 weeks and 6 weeks after the primary immunization. The rabbits were bled 8 weeks after the primary immunization. Serum samples were collected by centrifuge. The antiserum specificity was examined using ELISA with the purified recombinant OMPs as target antigens.

Nucleotide sequencing and homological analysis

A 50 ml PCR amplification reaction system was prepared by mixing the following reagents: 5 ml 10´DNA polymerase buffer; 1 ml dNTP mixture (10 mM); 25 pmol each of upstream and downstream primers; 0.5 ml pfu DNA polymerase (2.5 U); 50 ng isolated genomic DNA of Leptospira strains evaluated as template; ion-free water to final volume. PCR primers are shown in Table 3.

Primer synthesis and nucleotide sequencing reaction were performed at Shanghai Shenergy Biocolor BioScience and Technology Company, Limited (http://www.biocolors.com). The sequencing procession of PCR amplified production was operated twice.

Homological analysis was performed by BLAST against the nucleotide sequence database on the GenBank/NCBI website (http://www.ncbi.nlm.nih.gov/). Then the sequences of the genes LA0011, LA2637 and LA3138 obtained from the 15 different pathogenic leptospiral strains were submitted to GenBank/NCBI online.

Immunoblot

One nonpathogenic and 15 pathogenic leptospire whole protein preparations as samples for sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) were solubilized in final sample buffer 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. After electrophoresis, the gel was stained with Coomassie brilliant blue or transferred to nitrocellulose (Schleicher and Schuell, Keene, USA) for immunoblotting. For antigenic detection on immunoblots, the nitrocellulose was blocked with 10% nonfat dry milk in phosphate-buffered saline (PBS; pH 7.4) containing 0.1% Tween-20 (PBS-T), incubated for 2 h with rabbit antiserum (1:5000) specific to cloned leptospiral OMPs in PBS-T, and probed with goat anti-rabbit IgG-Fc (Sigma) conjugated to alkaline phosphatase to allow colorimetric detection.

RT-PCR

The leptospiral RNA extracted was treated with DNase (Promega, Beijing, China) before reverse transcription. The RNA mixture with RNase-free DNase and reaction buffer was incubated at 37 ºC for 30 min. DNase stop solution was added to terminate the reaction, then the mixture was incubated at 65 ºC for 10 min. Reverse transcription was performed using the AMV reverse transcription system (Promega). The product was resuspended in 100 ml ion-free water. RT-PCR primers are shown in Table 3.

Recombinant OMP-based ELISA

Patients sera were obtained from the Institute for Infectious Disease Control and Prevention. The leptospirosis group consisted of 18 patients with clinical manifestations of leptospirosis due to L. interrogans serovar lai, and defined by a reciprocal microscopic agglutination test [20]; there were four patients with culture-documented leptospirosis due to infection by other pathogenic Leptospira strains. The normal control group comprised 10 blood bank donors.

Immulon microtiter plates (Dynatech, Alexandria, USA) were coated at 37 ºC overnight with purified His₆ fusion proteins, then were blocked with blocking buffer (PBS containing 0.05% Tween-20 and 1% nonfat dried milk) at 37 ºC for 1 h. After washing three times with PBS-T, antiserum was added. Following incubation at 37 ºC for 2 h, the mixture reacted with 1:7500 diluted goat anti-human IgG conjugated to alkaline phosphatase (Promega) at 37 ºC for 1 h. Substrate solution in 20 mM carbonate buffer (pH 9.8) containing 2.5 mM p-nitrophenylphosphate, disodium salt and para-nitrophenyl phosphate (pNPP; Promega) was added. The reaction was terminated by 2 M H₂SO₄. The absorbance value of each well was read at 450 nm with a microplate reader (Tecan Spectra III, Sydney, Australia). Wells without a coating antigen were used as blank control. Sera of healthy individuals were analyzed as negative control. Statistical analysis was performed using GraphPad Prism 4 software.

Results

Sequencing and homological analysis of the OMP genes of different epidemic Leptospira strains

Three OMP genes LipL21, LipL32 and OmpL1 of the 15 pathogenic leptospires were all amplified and sequenced by a standard sequencing process. A BLAST search of the GenBank database revealed high nucleotide sequence identity when compared with the available complete genome sequence database of L. interrogans serovar lai strain Lai [14], L. interrogans serovar copenhageni strain Fiocruz L1-130 [21,22] and the corresponding sequence data of Leptospira kirschneri serovar grippotyphosa strain RM52 [23]. It provided significant evidence for the high conversation of the three OMP components among distinct epidemic leptospires in China. The sequence identity of the three OMP genes were found to be relatively higher in L. interrogans strains than in four L. borgpetersenii strains and one L. weilli strain when compared with the reported sequence data of L. interrogans serovar lai strain Lai [14]. Meanwhile, none of the three genes of two saprophitic L. biflexa strains could be amplified by PCR. Sequences of LipL32 encoding gene in three L. borgpetersenii strains and one L. weilli strain were found to be identical with reference sequences submitted in GenBank by other researchers (accession No. AY609321-AY609325, AY609327-AY609331 and AY609333; data not shown).

The sequences identified in this article were submitted to GenBank with accession No. AY688419-688431, AY634682, AY776292-776294 and AY688396-688409 (http://www.ncbi.nlm.nih.gov/entrez). Homology results with the OMP sequences from different pathogenic Leptospira species using the special BLAST program may be searched online.

Characterization of purified recombinant OMPs

The outer membrane protein components expressed by OMP genes LA0011, LA2637 and LA3138 were described as cpLipL21, cpLipL32 and cpOmpL1 respectively in this article. Lanes of purified proteins were shown in PAGE gel as in Fig. 1, molecular weights of His₆-tagged proteins are 17.2 kDa, 29.5 kDa and 31.9 kDa respectively.

Distribution of the corresponding OMP antigens among different Leptospira strains by immunoblot assay

Immunoblot results showed that the antisera to the three OMPs cpLipL21, cpLipL32 and cpOmpL1 had recognized the corresponding antigens in various epidemic leptospiral strains (Fig. 2).

mRNA expression of OMP genes in different Leptospira strains

RT-PCR analysis was performed on a panel of leptospires comprising six pathogenic strains including L. interrogans serovar lai and two nonpathogenic L. biflexa strains (Table 4 and Fig. 3). The expression of the three OMP genes encoding LipL21, LipL32 and OmpL1 in these leptospires differed slightly. These three genes were expressed in tested leptospiral pathogens, but not in the nonpathogenic strains L. biflexa serovar monvalerio or L. biflexa serovar patoc. This is consistent with the immunoblot results in Fig. 2.

Recombinant OMP-based ELISA

ELISA results showed that two recombinant proteins, cpLipL32 and cpOmpL1, reacted significantly with the sera of the 18 patients infected by L. interrogans serovar lai (P<0.01 and P<0.05 respectively), as well as with the sera of four patients infected by the other pathogenic leptospires (both P<0.01), compared with the negative control sera. No significant difference was observed between the reaction of cpLipL21 with patients' sera and with the control individuals' sera (P>0.05) (Fig. 4).

Discussion

Highly conserved OMPs are of special significance in serodiagnosis and vaccine development for leptospirosis. The leptospiral OMPs expressed during mammalian infection may have potential immunoprotective capabilities [24,25]. However, the lack of an effective, widely available laboratory tool remains a major problem [26] and standard serologic tests for case confirmation need to be optimized [27]. Based on bioinformatics and genomic tools, an important approach named "reverse vaccinology" has emerged [28,29]. It has played an essential role in delivering an effective and universal vaccine in the case of serogroup B Neisseria meningitidis [30,31].

The whole genome sequence of two pathogenic leptospires makes it possible that a considerable set of OMPs may be determined through experiments. In our study, nucleotide sequencing results validated that three OMP genes encoding LipL21, LipL32 and OmpL1 were highly conserved among various pathogenic Leptospira strains, which have been identified by epidemic and molecular analysis [16,17], including three pathogenic species L. interrogans, L. borgpetersenii and L. weilii. Both immunoblot assay and RT-PCR results suggest that these recombinant OMPs may play a potential role in developing immunodiagnosis and recombinant vaccine candidates.

The sequencing results revealed that the LipL32 coding gene was highly conserved among all the pathogenic leptospires including three epidemic species. It was demonstrated by immunoblot assay that this gene was expressed conservatively in most cultured epidemic leptospires. Furthermore, mRNA expression of the conserved LipL32 gene was detected in six virulent Leptospira strains tested including five L. interrogans strains and one L. borgpetersenii strain. The results indicated that recombinant LipL32 may act as an optimal antigen molecular candidate in the serodiagnosis of leptospirosis as described [26].

LipL21 gene was found well conserved among pathogenic leptospires including three epidemic species in China, while not found in saprophytic L. biflexa serovar patoc, which is consistent with an earlier report [7]. On the other hand, no statistical significance was detected between the ELISA reaction of the recombinant protein with the sera of leptospirosis patients, and individuals in the control group. As no related reports are available, it remains to be testified whether the recombinant LipL21 protein has potential use in immunodiagnosis.

The transmembrane lipoprotein, OmpL1 [9,32], was reported recently to have sequences with mosaic compositions consistent with horizontal transfer of DNA between related bacterial species [22]. In this study, though less conservative than the other two OMP genes in the epidemic leptospires tested, the gene encoding OmpL1 was found well conserved in all tested pathogenic Leptospira strains including L. interrogans strains, L. borgpetersenii and L. weilii. A strong interaction between the recombinant OmpL1 protein and leptospirosis patients' sera was observed by ELISA. According to the earlier report, OmpL1 and LipL41 together could provide significant protection against homologous challenge in the hamster model of leptospirosis [25]. This reminds us that the combination of recombinant OmpL1 and LipL41 products may be applied to future immunoprotective research and serodiagnosis strategies.

Our study aimed to primarily select and evaluate the leptospiral recombinant OMPs as target antigens by the approach of reverse vaccinology. Obviously more precise and comprehensive explorations need to be done in this field, for example, the monoclonal antibodies should be more optimal as detective probes in the immunoblot assay, and whether the recombinant OMPs could produce effective protection in animal challenges still needs to be confirmed. It is firmly believed that the recombinant leptospiral proteins as antigen molecular candidates should be further examined in the succeeding research.

Acknowledgement

We thank Prof. Jing-Xing LIU (Shanghai Jiaotong University, Shanghai, China) for very valuable guidance.

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